WO2017036600A1

WO2017036600A1 - Fluorescent light detection system and microscopy system

Info

Publication number: WO2017036600A1
Application number: PCT/EP2016/001478
Authority: WO
Inventors: Christoph Nieten; Enrico Geissler; Kai Wicker; Alois Regensburger; Helge Jess
Original assignee: Carl Zeiss Meditec Ag
Priority date: 2015-09-01
Filing date: 2016-09-01
Publication date: 2017-03-09
Also published as: DE102015011441A1

Abstract

The aim of the invention is the detection of fluorescent light of a plurality of different fluorescent dyes. This is achieved by a fluorescent light detection system (1) which comprises a beam splitter system (35), a spatially resolving first camera (21), and a spatially resolving second camera (23) which differs from the first camera (21). The beam splitter system (35) is configured to transmit light (35) entering the beam splitter system to the first camera (21) according to a first transmission spectrum and to the second camera (23) according to a second transmission spectrum which differs from the first transmission spectrum. The first transmission spectrum has an average transmittance of at least 10%, in particular at least 20% or 50%, in a red spectral range, and the second transmission spectrum has an average transmittance of at least 90% in each of a blue and a green spectral range.

Description

FLUORESCENT LIGHT DETECTION SYSTEM AND MICROSCOPY SYSTEM

The present invention relates to a fluorescent light detection system for detecting observation and fluorescent light, and to a microscopy system comprising a fluorescent light detection system. In particular, the present invention relates to fluorescent light detection systems for detecting fluorescence of the fluorescent dyes protoporphyrin IX (PpIX), fluorescein and indocyanine green (ICG).

The fluorescent dyes PpIX, fluorescein and ICG are used in the medical field, in particular for coloring biological material, for example blood cells, tumors or other tissue. When the tissue stained with a fluorescent dye is illuminated with light of a wavelength which is in the absorption spectrum of the fluorescent dye, the fluorescent dye emits fluorescent light. This fluorescent light can in turn be detected, whereby the area of a particular tissue dyed with the fluorescent dye can be made visible and distinguishable from the surrounding biological material. Frequently, several different fluorescent dyes are used in an operation to color different biological materials. On the one hand, it may be advantageous for the surgeon to simultaneously observe different biological material, each colored with different fluorescent dyes. On the other hand, it may also be advantageous to selectively visualize certain biological materials by fluorescent light, so as not to be simultaneously distracted from another fluorescence.

In the field of surgical applications, fluorescence observation systems are usually integrated with or provided by conventional surgical microscopes. The fluorescence observation system is therefore usually arranged over the area to be operated and should therefore, in order not to hinder the surgeon, be as compact as possible, ie occupy little space. Conventional fluorescence observation systems are designed, for example, as a stereomicroscope, whereby the surgeon can take an ocular view of the surgical area. The observation beam path of the stereomicroscope can with the help of another optical system on a Display overlaid fluorescence image are overlaid, so that the surgeon can see the surgical area as well as the fluorescence image simultaneously.

Conventional fluorescent light detection systems use a variety of illumination and detection filters, each suitable for excitation and detection of only one fluorescence of a fluorescent dye. In order to excite and detect the fluorescence of several fluorescent dyes, such fluorescent light detection systems include filter changers comprising a plurality of filters. However, this requires a larger space, which hinders the surgeon.

Other conventional fluorescent light detection systems comprise a beam splitter, which splits light coming from an object, which may comprise fluorescent light but also illumination light, into two radiation beams. Usually, light of the visible spectral range, for example from 400 nm to 750 nm, is guided in the first ray bundle, whereas the second ray bundle carries infrared light. Furthermore, such conventional fluorescent light detection systems comprise two cameras, wherein the first beam is supplied to the first camera and the second beam is supplied to the second camera. While the first camera is configured to detect an image of visible light, the second camera is configured to detect an image of infrared light. This configuration is particularly useful in fluorescent light detection systems used to detect indocyanine green. In this configuration, detectors with RGB Bayer matrix or RGGB Bayer matrix are usually used for the first camera. In this case, the camera is divided into regularly repeating color filter cells, each color filter cell consisting of four pixels, one of which can transmit the red spectral range, two the green spectral range and one blue spectral range, so that these spectral ranges can each be selectively detected. In contrast, the second camera is configured to detect infrared light.

This configuration provides high sensitivity to infrared light, ie, a high number of pixels for detecting infrared light, but relatively few pixels are used to detect other spectral regions (red, green, blue). provided. Therefore, this configuration is suitable only for the fluorescent dye indocyanine green, but not for other fluorescent dyes whose fluorescence is outside the infrared spectral range.

It is therefore an object of the present invention to provide a fluorescent light detection system and a microscopy system, with which a plurality of different fluorescent dyes can be used and whose fluorescent light can be detected with a high sensitivity. In particular, it is an object of the present invention to provide a fluorescent light detection system which can do without interchangeable components, can detect various fluorescent dyes and requires a small installation space.

As shown schematically in FIG. 1A, the fluorescent dye protoporphyrin IX (PpIX) has an absorption spectrum which has a normalized absorption intensity of more than 0.2 between 350 nm and 430 nm. The normalized absorption intensity is normalized to the maximum absorption intensity, i. H. the normalized absorption spectrum has only values between 0 and 1. In the range from 350nm to 430nm, the fluorescent dye PpIX can therefore be efficiently excited. The maximum of the absorption, the fluorescent dye at about 405nm. The fluorescent dye PpIX emits fluorescent light in a spectral range of about 600nm to 750nm, with a major peak of emission intensity at 635nm and a minor peak at about 705nm.

As shown schematically in Figure 1B, the fluorescent dye fluorescein has a normalized absorption intensity of greater than 0.2 between about 450nm and 530nm. In this area, the fluorescent dye fluorescein can therefore be well stimulated. The absorption spectrum of fluorescein peaks at about 495nm. The fluorescent dye fluorescein emits emission light in the range of about 490nm to 650nm. The maximum of the emission spectrum is around 520nm.

As shown schematically in Figure 1C, the fluorescent dye indocyanine green (ICG) has a normalized absorption intensity of greater than 0.2 between 700nm and about 840nm. Indocyanine green can therefore be well stimulated in this area. The maximum of the absorption spectrum of indocyanine green is about 800nm. Of the Fluorescent dye indocyanine green emits emission light in the range of about 750nm to 100nm. The maximum of the emission spectrum is around 835nm.

According to one embodiment, a fluorescent light detection system for detecting observation and fluorescent light comprises a beam splitting system, a spatially resolving first camera and a second resolving second camera different from the first camera, the beam splitter system being configured to transmit light entering the beam splitter system according to a first transmission spectrum to the first Camera and according to a different from the first transmission spectrum second transmission spectrum to the second camera to transmit; wherein the first transmission spectrum in a red spectral range has an average transmittance of at least 10%, in particular at least 20% or 50%, and wherein the second transmission spectrum in a blue and green spectral range in each case has an average transmittance of at least 90%.

The light entering the beam splitter system may, for example, be light emanating from an object or object region and may comprise light in a wavelength range from about 400 nm to 1000 nm. The light entering the beam splitter system may further include a portion which is illumination light reflected at an object. Further, the light entering the beam splitter system may include fluorescent light generated by one or more fluorescent dyes excited in an object. Usually, the intensity of the fluorescent light is much smaller than the intensity of the reflected illumination light, the latter being used to produce an overview image.

The beam splitter system is used to spectrally separate the light entering the beam splitter system, ie the light entering the beam splitter system is transmitted to a first output of the beam splitter system according to the first transmission spectrum and to a second output of the beam splitter system according to the second transmission spectrum. The light emerging at the first output of the beam splitter system is supplied to the first camera and the light emerging at the second output of the beam splitter system is supplied to the second camera. The Supplying the light emerging from the beam splitter system may be via one or more optical components and / or systems.

Herein, the transmission spectrum refers to the wavelength-dependent transmittance through the beam splitter system. The degree of transmission of the beam splitter system accordingly denotes the ratio of light of a wavelength λ entering the beam splitter system from the beam splitter system at one of the outputs of light of wavelength λ exiting. Since the beam splitting system inevitably absorbs light or may include specially provided absorption filters, the first transmission spectrum and the second transmission spectrum are not necessarily complementary to each other.

The first and second cameras are spatially resolved, d. H. these are configured to take pictures. In particular, the cameras can each be configured to detect images of a specific spectral range, for example a blue image which is generated exclusively from detected light of the blue spectral range.

The first and second cameras preferably have the same sensor format, i. H. the spatial resolution or number of pixels of the two cameras is either the same or one of the two cameras has an integer multiple of the pixel number of the other camera in at least one spatial direction. The camera with the smaller number of pixels then has a correspondingly larger light-sensitive detection area. As a result, the same imaging optics between the beam splitter system and the cameras can be used. Furthermore, the camera on which less light intensity is expected to be specially adapted for low light conditions. Alternatively or additionally, by electrical interconnection / summing ("binning") of several pixels, the signal-to-noise ratio for the camera, to which less light hits, can be improved. The two cameras can be adapted in their design to the wavelength ranges to be detected by you, for example, in CMOS sensors by adjusting the thickness of the photosensitive sensor layer.

The first transmission spectrum has a mean transmittance of at least 10 ° / o, 20% or 50% for a red spectral range, ie at least 10%, 20% or 50% of the light entering the beam splitter system of the red spectral range is transmitted to the first output of the beam splitter system and supplied to the first camera. As previously explained, this does not necessarily mean that the second transmission spectrum has the complementary proportion of 90%, 80% or 50%. The second transmission spectrum has in each case a mean transmittance of at least 90% in a blue spectral range and in a green spectral range. This means that 90% of the light entering the beam splitter system of the blue or green spectral range is transmitted to the second output of the beam splitter system and the second camera can be supplied.

As a result, light of the red spectral range can be transmitted to the first camera and detected by it. Furthermore, as a result, light of the blue and green spectral range is transmitted to the second camera and can be detected by it.

Here, the blue, green, red and infrared spectral range can be defined as follows: The blue spectral range can be between 350nm and 480nm, the green spectral range can be between 480nm and 580nm, the red spectral range can be between 580nm and 750nm, especially between 580nm and 680nm or between 630nm and 680nm, and the infrared spectral range can be between 750nm and 100nm. Furthermore, the blue, green, red and infrared spectral ranges can each have a spectral width of at least 50 nm, in particular 70 nm.

Herein, the average transmittance T denotes an average of the transmittances Τ (λ) within a limited wavelength range. For example, the average transmittance T may be defined as:

According to an exemplary embodiment, the first transmission spectrum in the blue and / or green spectral range has an average transmittance of at most 10%. Furthermore, the second transmission spectrum in the red spectral range has an average transmittance of at most 50%, in particular not more than 80% or 90%. In this embodiment, only a small proportion of light of the blue and / or green spectral range is transmitted to the first camera, so that the light supplied to the first camera of other spectral ranges is only slightly superimposed with light of the blue and / or green spectral range. In this way it can be prevented that light of the blue and / or green spectral range is detected as light of another spectral range, whereby the detection of the other spectral range is improved.

According to another exemplary embodiment, the first camera is configured to detect light of the red spectral region and the second camera is configured to selectively detect light of the blue and green spectral regions, respectively.

In this embodiment, the first camera is configured to detect the light of the red spectral range transmitted to the first camera. In particular, the first camera can be configured to detect a spatially resolved intensity distribution of light of the red spectral range supplied by the first camera. For example, the first camera may have detection surfaces that are configured to exclusively or predominantly detect light of the red spectral range and to generate a value that corresponds to the intensity of the light of this spectral range. In an analogous manner, the second camera may be configured to detect light of the blue spectral range and light of the green spectral range. In particular, the second camera can be configured to detect a spatially resolved intensity distribution of light of the blue spectral range or of light of the green spectral range. For this purpose, the second camera may, for example, have detection surfaces which exclusively or predominantly detect light of the blue spectral range and have detection surfaces which exclusively or predominantly detect light of the green spectral range.

It means predominantly that less than 10%, in particular less than 1% or less than 0.1%, of an intensity value which represents the intensity of light of a specific spectral range is caused by light outside the specific spectral range. According to an exemplary embodiment, a camera of the first and second cameras is configured to detect a spatially resolved first intensity distribution of light of a first spectral range; wherein a controller of the fluorescent light detection system is configured to determine, based on the first intensity distribution and a spatially resolved second intensity distribution, a spatially resolved third intensity distribution representing the intensity distribution of light of a third spectral region, the second intensity distribution comprising a spatially resolved intensity distribution of or detected by the controller Represents light of a second spectral range and wherein the first, second and third spectral range are different from each other.

In this embodiment, the fluorescent light detection system comprises a controller which determines a spatially resolved third intensity distribution based on a spatially resolved first and second intensity distribution. The first intensity distribution represents the spatially resolved intensity distribution of light of a first spectral range, the light being detected by the first or second camera. By way of example, the first camera can be configured to detect light of the red spectral range, such that the first intensity distribution corresponds to an intensity distribution of light of the red spectral range. The second intensity distribution may be an intensity distribution detected by one of the cameras or an intensity distribution determined by the controller. For example, the second intensity distribution corresponds to the intensity distribution of light of the red and infrared spectral ranges, which are detected jointly by the first camera. The controller may now be configured to determine the third intensity distribution, for example the intensity distribution of light of the infrared spectral range, based on the first intensity distribution (light of the red spectral range) and the second intensity distribution (light of the red and infrared spectral range). In the present example, the third intensity distribution can be obtained by subtracting the first intensity distribution from the second intensity distribution. Various mathematical operations may be used to determine the third intensity distribution, such as addition, subtraction, multiplication and division. Furthermore, even more complex formulas or Algorithms can be used to determine the third intensity distribution implemented by the controller.

In the present embodiment, two spectral ranges are different from each other when they include different spectral ranges, i. h., Two spectral ranges are different from each other when the two spectral ranges do not overlap or at most partially overlap or one spectral range completely covers the other spectral range, but also includes more spectral ranges.

According to an exemplary embodiment herein, the second intensity distribution is based on a spatially resolved intensity distribution detected by those of the first and second cameras other than the camera that detects the first intensity distribution. For example, when the first camera detects the first intensity distribution, the second intensity distribution is based on a spatially resolved intensity distribution detected by the second camera. Otherwise, if the first intensity distribution is detected by the second camera, the second intensity distribution is based on a spatially resolved intensity distribution detected by the first camera. The second intensity distribution may be equal to or determined based on the spatially resolved intensity distribution.

In this embodiment, the third intensity distribution is determined based on the first and second intensity distributions, wherein the first and second intensity distributions are based on intensity distributions detected by different cameras.

For determining the third intensity distribution, the controller may use characteristics of the first and / or second transmission spectrum.

According to an exemplary embodiment, the first transmission spectrum for an infrared spectral range has an average transmittance of at least 90% and the second transmission spectrum for the infrared spectral range has an average transmittance of at most 10%. Alternatively, the second transmission spectrum for the infrared spectral range may have an average transmittance of at least 90% and the first Transmission spectrum for the infrared spectral range have an average transmittance of at most 10%.

In this embodiment, light of the infrared spectral range, for example, fluorescent light of indocyanine green, is transmitted predominantly to the first camera or predominantly to the second camera. In addition, the first or second camera may be configured to detect light of the infrared spectral range. In particular, the first or second camera may be configured to detect a spatially resolved intensity distribution of light of the infrared spectral range. This makes it possible to detect fluorescent light of indocyanine green either by the first camera or by the second camera.

According to an exemplary embodiment, a controller of the fluorescent light detection system is configured to provide a spatially resolved infrared intensity distribution representing a spatially resolved intensity distribution of light of the infrared spectral range detected or determined by the controller, a spatially resolved red intensity distribution that is detected or determined by the controller represents spatially resolved intensity distribution of red spectral region light, a spatially resolved green intensity distribution representing a detected or controlled spatial resolution intensity distribution of green spectral region light, and a spatially resolved blue intensity distribution comprising detected or determined by the controller, spatially resolved intensity distribution of light of the blue spectral range represents.

The infrared, red, green and blue intensity distributions provided by the controller can then be used to display an overview image and a fluorescent light image. An intensity distribution of a specific spectral range may correspond to the intensity distribution detected by a camera configured to detect the particular spectral range. By way of example, the infrared intensity distribution may correspond to the spatially resolved intensity distribution of light of the infrared spectral range detected by a camera. Alternatively, the intensity distribution of a specific spectral range may also be made up of a plurality of spatially resolved ones Intensity distributions of different spectral ranges are determined by the controller.

According to an exemplary embodiment, the beam splitter system comprises a beam splitter and at least one optical filter. In this embodiment, the beam splitter and the at least one optical filter provide the first and / or second transmission spectrum. That at least one optical filter can be advantageous in particular if the beam splitter can not provide the spectral separation of the light striking the beam splitter system with sufficient accuracy.

According to an exemplary embodiment, the beam splitter is configured to spectrally and / or spatially and / or polarize the light entering the beam splitter system. In particular, the beam splitter may be configured to separate the light entering the beam splitter system into mutually orthogonal polarizations.

According to an exemplary embodiment herein, the first transmission spectrum furthermore has an average transmittance of at least 90% in the red and infrared spectral range and an average transmittance of at most 10% in the blue and green spectral range. In addition, the second transmission spectrum also has an average transmittance of at most 10% in the red and infrared spectral ranges. A controller of the fluorescent light detection system is configured to determine a spatially resolved intensity distribution of light of the red spectral region and a spatially resolved intensity distribution of light of the infrared spectral region based on intensity distributions detected by the first camera. Furthermore, the controller is configured to determine a spatially resolved intensity distribution of light of the blue spectral range and a spatially resolved intensity distribution of light of the green spectral range based on intensity distributions detected by the second camera.

In this embodiment, light of the blue spectral range and light of the green spectral range are transmitted substantially exclusively to the second camera. On the other hand, there is essentially no light of the red and infrared Spectral range transmitted to the second camera. Light of the red spectral range and light of the infrared spectral range is transmitted substantially exclusively to the first camera. On the other hand, essentially no light of the blue and green spectral range is transmitted to the first camera.

Furthermore, the first and second cameras are each configured to detect spatially resolved intensity distributions. The two cameras can be configured to detect multiple intensity distributions of light of different spectral ranges. From the intensity distributions detected by the first camera, the controller of the fluorescent light detection system determines a spatially resolved intensity distribution of light of the red spectral region and a spatially resolved intensity distribution of light of the infrared spectral region. This means that, based on the intensity distributions detected by the first camera, which need not necessarily correspond to the red spectral range and the infrared spectral range, a spatially resolved intensity distribution is determined, which represents the spatially resolved intensity distribution of light of the red spectral range incident on the first camera. In the same way, based on the intensity distributions detected by the first camera, a spatially resolved intensity distribution is determined by the controller, which represents an intensity distribution of light of the infrared spectral range incident on the first camera.

From the intensity distributions detected by the second camera, the controller of the fluorescent light detection system determines a spatially resolved intensity distribution of light of the blue spectral region and a spatially resolved intensity distribution of light of the green spectral region. This means that, based on the intensity distributions detected by the second camera, which need not necessarily correspond to the blue spectral range and the green spectral range, a spatially resolved intensity distribution is determined which represents the spatially resolved intensity distribution of light of the blue spectral range impinging on the second camera. In the same way, based on the intensity distributions detected by the second camera, a spatially resolved intensity distribution is determined by the controller, which represents an intensity distribution of light of the green spectral range incident on the second camera. According to an exemplary embodiment herein, the first camera comprises a spatially resolving detector having a first Bayer matrix, wherein the first Bayer matrix comprises a plurality of non-overlapping, regularly arranged color filter cells, each color filter cell of the first Bayer matrix having a plurality of different non-overlapping ones A color filter, wherein at least a first color filter of the color filter of each color filter cell of the first Bayer matrix is a filter of a filter group, wherein at least a second color filter of the color filter of each color filter cell of the first Bayer matrix one of the at least one first color filter different filter of the filter group , wherein the filter group comprises a filter which predominantly transmits the red spectral range, a filter which predominantly transmits the infrared spectral range, and a filter which predominantly transmits the red and infrared spectral range.

In this embodiment, the first camera is equipped with a spatially resolving detector and a first Bayer matrix. The spatially resolving detector is configured to output a signal representing the spatially resolved intensity distribution of light striking the detector. Bayer matrices are optical filters which comprise a plurality of non-overlapping, regularly arranged color filter cells. Usually, the color filter cells are arranged in a square grid, i. H. Color filter cells are arranged side by side and one above the other and adjacent to each other. However, the color filter cells can also be arranged in other regular grating structures, for example a hexagonal grating. A detection surface of the detector superposed by a color filter cell is commonly referred to as a pixel. Each color filter cell includes a plurality of, typically different, spatially non-overlapping color filters that are configured to transmit different spectral regions. The individual color filters can be designed, for example, as absorption filters.

In the present embodiment, each of the color filter cells has at least a first and at least a second color filter of a filter group, wherein the at least one first color filter and the at least one second color filter are different from each other, ie configured to transmit different spectral regions. Spectral ranges are different if they are not identical. That is, an example first color filter may be configured to include a first color filter Transmit spectral range and the second color filter may be configured to transmit a second spectral range, wherein the first and second spectral range can not overlap or partially. Furthermore, for example, the first spectral range may comprise the second spectral range and comprises at least one further spectral range.

In the present embodiment, the filter group comprises a filter that predominantly transmits the red spectral region, a filter that predominantly transmits the infrared spectral region, and a filter that predominantly transmits the red and infrared spectral regions. The first Bayer matrix may therefore have at least the following combinations for the at least one first color filter and the at least one second color filter: (R, IR); (R, R + IR) and (IR, R + IR). The first combination means that the first or second color filter is a filter that predominantly transmits the red spectral range, and the other of the two color filters is a filter that predominantly transmits the infrared spectral range. The second combination means that the first or second color filter is a filter that predominantly transmits the red spectral region, and the other of the two color filters is a filter that predominantly transmits the red and infrared spectral regions. The third combination means that the first or second color filter is a filter that predominantly transmits the infrared spectral range, and the other of the two color filters is a filter that predominantly transmits the red and infrared spectral range.

The sensitivity for detecting a specific spectral range, here the red or infrared spectral range, can be adjusted by a suitable configuration of the first Bayer matrix. For example, if a high sensitivity is required to detect the infrared spectral range, a configuration can be chosen in which all or many color filters of each color filter cell can transmit the infrared spectral range. Such a configuration allows, for example, a high sensitivity for detecting the fluorescent light of indocyanine green. Alternatively, if the sensitivity to fluorescent light of the fluorescent dye PpIX is to be increased because the fluorescence of this fluorescent dye is usually weak, all or many color filters of each color filter cell may be configured to transmit the red spectral region. According to an exemplary embodiment herein, half of the color filters of each color filter cell of the first Bayer matrix are configured to predominantly transmit the red spectral region or predominantly the infrared spectral region, and half of the color filters of each color filter cell of the first Bayer matrix are configured predominantly to transmit the red and infrared spectral range.

In this embodiment, each color filter cell of the first Bayer matrix comprises only color filters of the second and third above-mentioned combination. Accordingly, the first camera is exclusively configured to detect a spatially resolved intensity distribution of light of the red spectral region or a spatially resolved intensity distribution of light of the infrared spectral region next to a spatially resolved intensity distribution of light of the red and infrared spectral regions. In a PpIX preferred embodiment for detecting fluorescent light, half of the color filters of each color filter cell of the first Bayer matrix are configured to transmit predominantly the red spectral region and half of the color filters of each color filter cell of the first Bayer matrix are configured predominantly to transmit the red and infrared spectral range. Each color filter of the first Bayer matrix is therefore configured to transmit the fluorescent light from PpIX in the red spectral region to the detector.

According to another exemplary embodiment herein, the first camera is configured to detect a spatially resolved first intensity distribution of the light transmitted through the first color filters and incident on the detector and configured to have a spatially resolved second intensity distribution of the transmitted through the second color filters and incident on the detector To detect light. Further, the controller is configured to determine the spatially resolved intensity distribution of light of the infrared spectral range based on the first and second detected intensity distribution and / or to determine the spatially resolved intensity distribution of light of the red spectral range based on the first and second detected intensity distribution. For example, if half of the color filters of each color filter cell of the first Bayer matrix predominantly transmits the red spectral region and half of the color filters of each color filter cell of the first Bayer matrix predominantly transmits the red and infrared spectral regions, the first intensity distribution on the detector represents the incident light of Red spectral range and the second intensity distribution represents the light striking the detector of the red and infrared spectral range. The controller now determines the spatially resolved intensity distribution of light of the infrared spectral range from the first and second intensity distribution, for example by subtracting intensity values of an (R) subpixel from an (R + IR) subpixel. In this way, both a spatially resolved intensity distribution of light of the red spectral range and a spatially resolved intensity distribution of light of the infrared spectral range can be provided and at the same time a high sensitivity for light of the red spectral range can be made possible.

According to a further exemplary embodiment, the second camera comprises a spatially resolving detector having a second Bayer matrix, wherein the second Bayer matrix comprises a plurality of non-overlapping, regularly arranged color filter cells, each color filter cell of the second Bayer matrix being several different ones includes overlapping color filters.

The second Bayer matrix can be designed as RGGB matrix, i. H. One quarter of the color filters of each color filter cell transmits predominantly the blue spectral range, one quarter of the color filters of each color filter cell transmits predominantly the red spectral range and half of the color filters of each color filter cell transmits predominantly the green spectral range.

Alternatively, the second Bayer matrix may be formed as a BGGB matrix, ie half of the color filters of each color filter cell transmits predominantly the blue spectral range and half of the color filters of each color filter cell transmits predominantly the green spectral range. In the present embodiment, since substantially no light of the red spectral region is transmitted to the second camera, the (R) subpixel can be replaced by a (B) subpixel, thereby improving the sensitivity to light of the blue spectral region. Alternatively, the second Bayer matrix may be formed as a GGGB matrix, ie one fourth of the color filters of each color filter cell transmits predominantly the blue spectral range and three quarters of the color filters of each color filter cell predominantly transmit the green spectral range. Since none of the fluorescence dyes emits PpIX, fluorescein and ICG in the blue spectral range, the sensitivity to the green spectral range, in which, for example, fluorescein emits, can be improved at the expense of the sensitivity to the blue spectral range.

According to a further exemplary embodiment, the first transmission spectrum further has an average transmittance of at least 90% in the red spectral range and an average transmittance of at most 10% in the blue, green and infrared spectral ranges, and the second transmission spectrum also has an average transmittance in the infrared spectral range of at least 90% and in the red spectral region an average transmittance of at most 10%. Further, a controller of the fluorescent light detection system is configured to determine a spatially resolved intensity distribution of red spectral region light based on intensity distributions detected by the first camera, and a spatially resolved intensity distribution of blue spectral region light, a spatially resolved intensity distribution of green spectral region light, and a spatially resolved intensity distribution of To determine light of the infrared spectral range based on intensity distributions detected by the second camera.

In this embodiment, light of the red spectral range is transmitted substantially exclusively to the first camera and light of the blue, green and infrared spectral range is transmitted substantially exclusively to the second camera. Therefore, the first camera can be optimized for detecting light of the red spectral region, whereby, for example, fluorescent light of PpIXX can be detected with high sensitivity. As explained above in connection with the first camera, the second camera may be configured to detect spatially resolved intensity distributions of different spectral ranges. The controller can then determine the spatially resolved intensity distribution of light of the blue, green and infrared spectral range on the basis of the detected intensity distributions. According to an exemplary embodiment herein, the second camera comprises a spatially resolving detector having a second Bayer matrix, wherein the second Bayer matrix comprises a plurality of non-overlapping, regularly arranged color filter cells, each color filter cell of the second Bayer matrix being several different ones includes overlapping color filters.

The second Bayer matrix may be configured according to the pattern (RGGB) + IR, i. H. One quarter of the color filters of each color filter cell transmits predominantly the blue and infrared spectral range, one quarter of the color filter of each color filter cell transmits predominantly the red and infrared spectral range and half of the color filter of each color filter cell transmits predominantly the green and infrared spectral range.

According to an exemplary embodiment herein, the second camera is configured to detect a spatially resolved first intensity distribution of the light transmitted through the red and infrared spectral regions and incident on the detector, and the controller is configured to provide the spatially resolved intensity distribution of infrared spectral region light based on the first intensity distribution.

Since essentially no light of the red spectral range is transmitted to the second camera due to the configuration of the beam splitter system, the (R + IR) subpixel of the spatially resolving detector essentially strikes exclusively the light of the infrared spectral range. The intensity detected by these subpixels can therefore be used as a spatially resolved intensity distribution of light of the infrared spectral range. Alternatively, using the knowledge about the transmission spectra of the beam splitter system and the intensity distribution of light of the red spectral range detected by the first camera, the intensity of the light of the red spectral range incident on the second camera can be compensated.

Further, after the controller is configured to determine the spatially resolved intensity distribution of light of the infrared spectral range, the controller may be configured to provide the spatially resolved intensity distribution of light of the blue To determine spectral range and the spatially resolved intensity of light of the green spectral range based on the intensity distributions detected by the second camera and the spatially resolved intensity distribution of light of the infrared spectral range.

For this purpose, the second camera can be configured to detect a spatially resolved second intensity distribution of the transmitted through the blue and infrared spectral range color filter and incident on the detector light and a spatially resolved third intensity distribution of the transmitted through the green and infrared spectral color filter and on to detect the detector striking the light. Further, the controller may be configured to determine the spatially resolved intensity distribution of blue spectral region light based on the second intensity distribution and the intensity distribution of infrared spectral region light and / or the spatially resolved intensity distribution of green spectral region light based on the third intensity distribution and the intensity distribution of light of the infrared spectral range. In particular, the controller may determine the spatially resolved intensity distribution of light of the blue or green spectral range by subtracting the intensity distribution of light of the infrared spectral range from the second and third intensity distribution, respectively.

In this embodiment, four subpixels per color filter cell are available for detecting light of the infrared spectral range, one subpixel per color filter cell for detecting light of the blue spectral range and two subpixels per color filter cell for detecting light of the green spectral range.

As an alternative to the configuration of the second Bayer matrix explained above, the second Bayer matrix can be formed as a G (G + IR) (G + IR) (B + IR) matrix, ie one fourth of the color filters of each color filter cell predominantly transmits the blue and infrared spectral range, a quarter of the color filter of each color filter cell transmits predominantly the green spectral range and half of the color filter of each color filter cell transmits predominantly the green and infrared spectral range. According to an exemplary embodiment herein, the second camera is configured to detect a spatially resolved first intensity distribution of the light transmitted through the green and infrared spectral regions and incident on the detector, and the controller is configured to provide the spatially resolved intensity distribution of infrared spectral region light based on the first intensity distribution and the intensity distribution of light of the green spectral range to determine.

In this embodiment, since a sub-pixel is configured to detect only light of the green spectral range, the spatially resolved intensity distribution of light of the infrared spectral range can be determined by subtracting the intensity distribution of light of the green spectral range from the first intensity distribution.

Furthermore, the second camera may be configured to detect a spatially resolved second intensity distribution of the light transmitted through the blue and infrared spectral regions and incident on the detector, and the controller may be configured to base the spatially resolved intensity distribution of blue spectral region light on the second intensity distribution and the intensity distribution of light of the infrared spectral range. Herein, the intensity distribution of light of the infrared spectral range previously determined by the controller is used to determine the spatially resolved intensity distribution of light of the blue spectral range, for example by subtracting the intensity distribution of light of the infrared spectral range from the second intensity distribution.

In this embodiment, three subpixels per color filter cell are available for detecting light of the green spectral range, three subpixels per color filter cell for detecting light of the infrared spectral range and one subpixel per color filter cell for detecting light of the blue spectral range.

According to a further embodiment herein, the first transmission spectrum furthermore has an average transmittance of at least 90% in the infrared spectral range, an average transmittance of between 10% and 90%, in particular between 25% and 75%, in the red spectral range. or between 40% and 60%, and in the blue and green spectral range in each case an average transmittance of at most 10%. In addition, the second transmission spectrum also has an average transmittance in the red spectral range of between 10% and 90%, in particular between 25% and 75% or between 40% and 60%, and in the infrared spectral range an average transmittance of at most 10%. Further, a controller of the fluorescent light detection system is configured to determine a spatially resolved intensity distribution of light of the infrared spectral region and a spatially resolved intensity distribution of light of the red spectral region based on intensity distributions detected by the first and second cameras and a spatially resolved intensity distribution of light of the blue spectral region and a spatially resolved one Determine intensity distribution of light of the green spectral range based on detected by the second camera intensity distributions.

In this embodiment, light of the blue spectral range and light of the green spectral range are transmitted substantially exclusively to the second camera and light of the infrared spectral range is transmitted substantially exclusively to the first camera. Light of the red spectral range is transmitted to both the first and the second camera. The first and second cameras may be configured to detect intensity distributions of different spectral regions which are used by the controller to determine the spatially resolved intensity distribution of infrared spectral region light and the spatially resolved red-light intensity intensity distribution of light. Furthermore, the controller may determine the spatially resolved intensity distribution of light of the blue spectral range and the spatially resolved intensity distribution of light of the green spectral range from intensity distributions of light of different spectral ranges detected by the second camera.

In particular, the first camera may be configured to detect a spatially resolved first intensity distribution of light of the red and infrared spectral ranges. This means that the pixels of the first camera together detect both light of the red spectral range and light of the infrared spectral range and output a corresponding intensity value. According to an exemplary embodiment herein, the second camera comprises a spatially resolving detector having a second Bayer matrix, the second Bayer matrix comprising a plurality of non-overlapping, regularly arranged color filter cells, each color filter cell of the second Bayer matrix having a plurality of different non-overlapping ones Includes color filter. In particular, the second Bayer matrix can be embodied as an RGGB matrix, ie one fourth of the color filters of each color filter cell transmits predominantly the blue spectral range, one quarter of the color filters of each color filter cell transmits predominantly the red spectral range and half the color filters of each color filter cell transmit predominantly the green spectral range.

In this embodiment, the spatially resolved intensity distribution of light of the blue spectral range and the spatially resolved intensity distribution of light of the green spectral range can be detected directly by the second camera. Furthermore, the second camera detects a spatially resolved intensity distribution of light of the red spectral range, which can be used to determine a spatially resolved intensity distribution of light of the infrared spectral range, which is detected by the first camera. This can be implemented as follows.

The controller may be configured to calculate the spatially resolved intensity distribution of light of the infrared spectral range based on the first intensity distribution of light of the red and infrared spectral range detected by the first camera and an intensity distribution of the color filter transmitted through the red spectral range and detected by the second camera to determine the light striking the detector. Furthermore or alternatively, the controller may be configured to determine the spatially resolved intensity distribution of light of the red spectral region based on the first intensity distribution detected by the first camera and the intensity distribution of the color filter transmitted through the red spectral region and incident on the detector detected by the second camera Determine the light.

For example, the intensity distribution of light of the infrared spectral range can be determined by the intensity distribution of the light detected by the second camera of the red spectral range from that of the first Camera detected first intensity distribution of light of the red and infrared spectral range is subtracted.

Alternatively, the second camera may comprise a second Bayer matrix which is an (R + G) GGB matrix, i. H. One quarter of the color filters of each color filter cell transmits predominantly the blue spectral range, one quarter of the color filters of each color filter cell transmits predominantly the red and green spectral range and half of the color filters of each color filter cell transmits predominantly the green spectral range.

In an exemplary embodiment thereof, the second camera is configured to detect a spatially resolved second intensity distribution of the light transmitted through the green and red spectral region and incident to the detector, and the controller is configured to provide a spatially resolved third intensity distribution of red light Spectral range based on the second intensity distribution and an intensity distribution of light of the green spectral range to determine.

In this embodiment, the intensity distribution of light of the blue spectral range and the intensity distribution of light of the green spectral range can be detected directly by the second camera. Furthermore, the second camera detects the second intensity distribution which represents the intensity distribution of the light of the light transmitted through the color filters transmitting the green and red spectral ranges. The third intensity distribution, which represents an intensity distribution of light of the red spectral range incident on the second camera, can be determined by the controller, for example, by subtracting the intensity distribution of light of the green spectral range from the second intensity distribution.

The third intensity distribution can also be used to determine the intensity distribution of light of the infrared spectral range. For this purpose, the controller may be configured to determine the spatially resolved intensity distribution of light of the infrared spectral range based on the first intensity distribution detected by the first camera, which represents light of the red and infrared spectral range, and the third intensity distribution, for example, by subtracting these intensity distributions. Furthermore or alternatively, the controller may be configured to determine the spatially resolved intensity distribution of light of the red spectral region based on the first intensity distribution detected by the first camera and the third intensity distribution. In this case, the intensity distributions can be added, for example.

According to an exemplary embodiment, for the wavelength-dependent transmittance Τ (λ) of the first and / or second transmission spectrum within the red spectral range, the following applies:

T ₀ - ΔΤ <Τ (λ) <T ₀ + ΔΤ; 15% <T ₀ <85%; 0 <ΔΤ <5%.

In this embodiment, the light of the red spectral region incident on the beam splitter system is transmitted to both the first and the second camera. Within the red spectral range, the transmittance Τ (λ) of the first and second transaction spectrum varies by a maximum of 2ΔΤ by a value of T ₀ . Within the red spectral range, the transmittance has an approximately constant value. For the value T ₀ , a different value can be selected for the first transmission spectrum than for the value T _{0 of} the second transmission spectrum. In particular, it may be approximated that the value T _{0 of} the first transmission spectrum and the value T _{0 of} the second transmission spectrum add up to approximately 1 in total. In particular, T ₀ can be equal to 50%.

According to a further embodiment, a first spectral range of the second transmission spectrum within the red spectral range exclusively comprises wavelengths which are smaller than a cut-off wavelength λ o, and has an average transmittance T _t . Furthermore, a second spectral range of the second transmission spectrum within the red spectral range exclusively comprises wavelengths that are greater than the limit wavelength λ o, and has an average transmittance T ₂ . Furthermore:

Τ _Ί > 70%, preferably T _a > 80% or Τ>90%; and

T ₂ <30%, preferably T ₂ <20% or T ₂ <10%. In this embodiment, the red spectral range of the second transmission spectrum (and, correspondingly, also the red spectral range of the first transmission spectrum) is divided into a first spectral range below the cut-off wavelength λο and a second spectral range above the cut-off wavelength λο. The first and second spectral range each have significantly different average transmittances Ύ and T ₂ , which is why in the transitional region of the two spectral ranges around the cutoff wavelength λο, the transmittance of the second transmission spectrum T2 drops sharply or the degree of transmittance of the first transmission spectrum Tl increases sharply.

For example, the cut-off wavelength λo may be chosen to be below the maximum of the emission spectrum of PpIX, for example: 600nm <λο ^ 635nm. Alternatively, the cut-off wavelength λ o can be chosen to be between the main maximum of the emission spectrum of PpIX at 635 nm and the secondary maximum at 705 nm. For example: 640nm <λο ^ 680nm.

In the former case (600nm <λο ^ 635nm), the fluorescent light from PpIX is transmitted substantially exclusively to the first camera. This can then be optimized especially for the detection of the fluorescence light of PpIX. In the latter case (640nm <λο ί 680nm), the main peak of the emission spectrum is transmitted from PpIX to the second camera and the sub-maximum is transmitted to the first camera.

Another embodiment provides a microscopy system that includes at least one light source, a lens system, and a fluorescent light detection system. The light source is configured to generate illumination light and excitation light to excite fluorescent dyes and direct it to an object area. The illumination light serves to illuminate the object area and to generate an overview image of the object area from the light reflected thereby. The excitation light serves to excite at least one fluorescent dye. The fluorescent light detection system may be, for example, one of the fluorescent light detection systems described herein. The objective system and the fluorescent light detection system are arranged such that at least a section of the object area is imaged by the objective system and the fluorescence light detection system onto the first and second camera of the fluorescence light detection system.

The microscopy system may further comprise a display system for displaying an image, wherein the image may be generated based on intensity distributions provided by a controller of the fluorescent light detection system. In particular, the image may be a superposition of an overview image and a fluorescent light image. For improved representation, the spectral ranges used for the display do not have to correspond to the actually detected spectral ranges. In particular, the fluorescent light of indocyanine green, which lies in the infrared spectral range, can be represented in any desired color or by another marking. In particular, the image can be generated from the spatially resolved intensity distributions of the red, infrared, green and blue spectral regions or the infrared, red, green and blue intensity distributions provided by the controller.

Furthermore, a detection filter can be arranged in the beam path between the object area and the fluorescence light detection system, which is configured to suppress at least a part of the excitation light. The suppression can take place both in terms of intensity and in terms of the spectral range.

Figure 1A shows the normalized absorption and emission spectrum of

Fluorescent dye PpIX.

Figure 1B shows the normalized absorption and emission spectrum of

Fluorescent dye fluorescein.

Figure IC shows the normalized absorption and emission spectrum of

Fluorescent dye indocyanine green.

FIG. 2 shows an exemplary embodiment of a microscopy system. FIG. 3 shows an exemplary embodiment of a fluorescence light detection system.

FIG. 4 shows a further exemplary embodiment of a fluorescence light detection system.

FIG. 5 shows a further exemplary embodiment of a fluorescence light detection system.

FIG. 6 shows a further exemplary embodiment of a fluorescence light detection system.

FIG. 2 shows an exemplary embodiment of a microscopy system 1. The microscopy system 1 comprises at least one light source 3 which directs illumination and excitation light 5 onto an object region 7. The excitation light is suitable for excitation of fluorescent dyes contained in an object 9, for example PpLX and / or fluorescein and / or ICG. Furthermore, the microscopy system has an illumination optical system 11, which directs the light generated by the at least one light source 3 onto the object region 7. In the beam path 13 from the at least one light source 3 to the object region 7, an illumination filter 15 is arranged, which can form the illumination and excitation light 5 with respect to its intensity spectrum.

The microscope system 1 further includes an objective system 17 and a fluorescence detection system 19. The objective system 17 and the fluorescence detection system 19 are configured and arranged so that at least a portion of the subject area 7 is imaged on a first camera 21 and a second camera 23 of the fluorescent light detection system 19 is indicated by the detection beam 25.

The objective system 17 may comprise a plurality of optical components, for example an objective 27 and a zoom lens system consisting of the optical elements 29 and 31. Within the detection beam path 25 may be arranged a detection filter 33 which is configured to excite light for excitation of To suppress fluorescent dyes and to transmit illumination light to produce an overview image.

The fluorescent light detection system 19 comprises, in addition to the first camera 21 and the second camera 23, a beam splitter system 35, which transmits the light entering the beam splitter system at its input 37 to two outputs 41.

The fluorescent light detection system 19 further includes a controller 43. The controller 43 is connected to the at least one light source 3 through a connection 45. Through this connection 45, the controller 43 may control the at least one light source and, in general, a light generating system including the at least one light source 3. Furthermore, the controller 43 is connected via connections 47 to the first camera 21 and the second camera 23. Via these connections 47, the controller can receive signals from the cameras, which can represent, for example, spatially resolved intensity distributions of light of different spectral ranges. The controller 43 is further connected to a display device 49 of the microscope system 1. By means of the display device 49, the controller can display images of the object area.

FIG. 3 shows an exemplary embodiment of a fluorescence light detection system 19A, as can be used, for example, in the microscopy system shown in FIG. The fluorescent light detection system 19A includes a first camera 21A, a second camera 23A, and a beam splitter system 35A.

By the configuration of the fluorescent light detection system 19A explained below, it is configured to detect fluorescent light of the fluorescent dyes PpIX, fluorescein and ICG, without having to make individual components of the fluorescent light detection system interchangeable or exchangeable. Instead, the fluorescent light of the three fluorescent dyes as well as an overview image can be detected by the configuration explained below.

The beam splitting system 35A is configured to display the light 51 entering the beam splitting system, which is represented by four arrows in FIGS. 3 to 6, according to a first transmission spectrum T1 to the first camera 21A and to a second transmission spectrum T2 different from the first transmission spectrum T1 to the second camera 23A. The representation 53 A shows the first transmission spectrum Tl as a function of the wavelength λ. The representation 55 A shows the transmission spectrum T2 as a function of the wavelength λ.

In the representations of the first transmission spectrum Tl and the second transmission spectrum T2 in FIGS. 3 to 6, a "B" shown in a circle represents a blue spectral range, a "G" shown in a circle represents a green spectral range, a "R" shown in a circle "a red spectral region and in a circle represented 'IR' an infrared spectral range.

The red spectral range covers the emission range of the fluorescent dye PpIX, d. H. the spectral range within which the fluorescent dye PpIX emits fluorescent light. The spectral range IR comprises the emission range of the fluorescent dye ICG. The emission region of the fluorescent dye fluorescein is partially covered by the green spectral region and partly by the red spectral region.

As shown in FIG. 53A, the first transmission spectrum T 1 has a low average transmittance in the blue spectral range and in the green spectral range. In particular, the average transmittance of the blue and green spectral range is less than 10% in each case. Furthermore, the first transmission spectrum Tl in the red and infrared spectral range in each case has a high average transmittance, which is for example at least 90%. Light of the red spectral region represented by long-dashed line arrows and infrared spectral region light represented by solid line arrows are therefore transmitted to the first camera 21A. On the other hand, essentially no light of the blue and green spectral range is transmitted to the first camera 21A.

As shown in illustration 55A, the second transmission spectrum T2 in the blue and green spectral range in each case has a high average transmittance, which may be in particular at least 90%. On the other hand, the second transmission spectrum T2 knows in the red and infrared Spectral range in each case only low average transmittances, which are for example at most 10%. Accordingly, light of the blue spectral range represented by short dashed line arrows and green spectral range light represented by dashed line arrows are transmitted to the second camera 23A. For ease of understanding, the shape of the line of arrows, in Figures 53A and 55A, corresponds to the shape of the circle of the respective spectral range. Accordingly, the blue spectral region "B" is represented by a short-dashed circle, the green spectral region "G" by a dot-dashed circle, the red spectral region "R" by a long-dashed circle, and the infrared spectral region "IR" by a solid-line circle.

The first camera 21A comprises a spatially resolving detector 59A with a first Bayer matrix 61A. The first Bayer matrix 61A comprises a plurality of non-overlapping, regularly arranged color filter cells, each color filter cell of the first Bayer matrix 61A comprising a plurality of different non-overlapping color filters.

An exemplary color filter cell 63A of the first Bayer matrix 61A consists of four non-overlapping color filters represented by labeled squares. The regular non-overlapping arrangement of a plurality of color filter cells 63A is indicated by the projecting edges 65 of the color filter cell. The color filter cell 63A comprises color filters "R", which predominantly transmit the red spectral range. Furthermore, the color filter cell 63A comprises color filters "R + IR", which predominantly transmit the red and infrared spectral range.

A detection area of the detector 59A corresponding to the size of a color filter cell is called a pixel. A detection area of the detector 59A corresponding to the area of a color filter of a color filter cell is called a subpixel. When using the color filter cell 63A, four subpixels per color filter cell are therefore available for detecting the red spectral range. Two subpixels per color filter cell are available for the detection of the infrared spectral range. As a result, the first camera 21A for fluorescent light from PpIX is particularly sensitive in the red spectral range and can also detect the fluorescent light from ICG. A spatially resolved intensity distribution of light of the infrared spectral range may be determined by the controller 43. For this purpose, for example, the intensity of light transmitted through the color filters "R" can be subtracted from the intensity of the light transmitted through the color filters "R + IR".

The second camera 23A comprises a spatially resolving detector 67A with a second Bayer matrix 69A. The second Bayer matrix 69A, like the first Bayer matrix, comprises a multiplicity of non-overlapping, regularly arranged color filter lines, each color ilter cell of the second Bayer matrix comprising a plurality of different, non-overlapping color filters. An exemplary color filter cell 71 A of the second Bayer matrix comprises four color filters, wherein a color filter denoted by "R" predominantly transmits the red spectral range, a color filter denoted by "G" predominantly transmits the green spectral range and a color filter designated "B" predominantly transmits the color spectrum transmitted blue spectral range. In this way, the second camera 23A is configured to detect a spatially resolved intensity distribution of light of the red spectral region, a spatially resolved intensity distribution of light of the green spectral region and a spatially resolved intensity distribution of light of the blue spectral region.

In this configuration of the fluorescent light detection system 19A, fluorescent light of the fluorescent dye fluorescein is detected by both the first camera 21A and the second camera 23A. In particular, the proportion in the yellow spectral range of the fluorescent light is detected by the green subpixels of the second camera 23A, d. H. is detected by the subpixels preceded by the color filters "G", and the proportion in the red spectral region of the fluorescent light is detected by the red subpixels of the first camera 21A, i. H. the subpixels, which are preceded by the color filter "R" detected.

Another exemplary color filter cell 73A of the second Bayer matrix 69A has equal proportions of color filter "B" and color filter "G". Since no light of the red spectral region is supplied to the second camera 23A by the configuration of the beam splitting system, the red sub-pixel may be dispensed with in favor of a blue sub-pixel. Another exemplary color filter cell 75A of the second Bayer matrix includes a color filter "B" and three color filters "G". Accordingly, the second green spectrum spectral range camera 23A provides more subpixels, thereby also improving the sensitivity to yellow spectral region light, and thus fluorescein fluorescence light.

FIG. 4 shows a further embodiment of a fluorescence light detection system 19B. The beam splitting system 35B is configured to transmit the light 51 entering the beam splitting system 35B to the first camera 21B according to the first transmission spectrum Tl shown in the illustration 53B and to transmit it to the second camera 23B according to the second transmission spectrum shown in the illustration 55B. The beam splitter system 35B is configured to transmit light of the red spectral range substantially exclusively to the first camera 21B and to transmit light of the blue, green, and infrared spectral ranges, respectively, substantially exclusively to the second camera 23B. For this purpose, the first transmission spectrum Tl in the red spectral range have an average transmittance of at least 90% and in the blue, green and infrared spectral range each have an average transmittance of at most 10%. Furthermore, the second transmission spectrum in the blue, green and infrared spectral range can each have an average transmittance of at least 90% and in the red spectral range an average transmittance of at most 10%.

The first camera 21B is configured to detect light of the red spectral region, in particular it is configured to detect a spatially resolved intensity distribution of light of the red spectral region. The first camera 21B may comprise a symbolically indicated filter 63, which is configured to transmit exclusively or predominantly light of the red spectral range. Since the first camera 21B is provided exclusively for detecting light of the red spectral region, the fluorescent light detection system 19B for detecting fluorescent light of PpIX provides high sensitivity.

The second camera 23B comprises a spatially resolving detector 67B having a second Bayer matrix 69B. An exemplary color filter cell 71B of the second Bayer matrix 69B includes four color filters, one of which transmits predominantly the blue and infrared spectral regions, one transmits the red and infrared spectral regions, and two transmits the green and infrared spectral regions.

Since according to the second transmission spectrum T2 substantially no light of the red spectral region strikes the second camera 23B, the subpixel "R + IR" detects only light of the infrared spectral range. With knowledge of the illumination light spectrum, of the first transmission spectrum T 1 and of the second transmission spectrum T 2, the spatially resolved intensity distribution of light of the red spectral range detected by the first camera 21 B can also be used to compensate for the light of the red spectral range incident on the second camera 23B. This compensation can be performed by the controller 43.

According to this configuration, the second camera 23B is configured to detect a spatially resolved intensity distribution of the light transmitted through the color filters transmitting the blue and infrared spectral regions and incident on the detector 67B. The spatially resolved intensity distribution of light of the blue spectral range can be determined from this intensity distribution and the intensity distribution of light of the infrared spectral range, for example by subtracting the intensities of these intensity distributions. Analogously thereto, the intensity distribution of light of the green spectral range can also be determined from the intensity distribution of light of the infrared spectral range and the intensity distribution of light of the light transmitted through the color filter transmitting the green and infrared spectral range and detected by the detector.

Another exemplary color filter cell 73B of the second Bayer matrix comprises four color filters, one of which predominantly transmits the green spectral range, one predominantly transmits the blue and infrared spectral range and two transmits predominantly the green and infrared spectral range. In this configuration, the intensity distribution of light of the infrared spectral range can be determined, for example, by subtracting the intensity distribution detected by the "G" sub-pixel from the intensity distribution detected by the "G + IR" sub-pixels. Further, by using the intensity distribution of light of the infrared spectral range determined in this way, an intensity distribution of light of the blue spectral range can be determined.

In the embodiment illustrated in FIG. 4, the entire detection area of the first camera 21B is provided for the detection of fluorescent light from PpIX. For the detection of fluorescent light of ICG, three subpixels per color filter cell of the second camera 23B are respectively provided. Fluorescence light of the fluorescent dye fluorescein is detected partly by the first camera 21B and partly by the second camera 23B. For the proportion in the red spectral range of the fluorescent light, the entire detection area of the first camera 21B is available and for detection of the proportion in the yellow spectral range, two or three subpixels of the second camera 23B detecting the green spectral range are available.

FIG. 5 shows a further embodiment of a fluorescence light detection system 19C. In this embodiment, the beam splitter system 35C is configured to transmit light of the red spectral range to both the first camera 2 IC and the second camera 23C. The first transmission spectrum T 1 has an average transmittance of at most 10% for the blue and green spectral range and an average transmittance of at least 90% for the infrared spectral range. In the red spectral range, which may for example comprise the spectral range from 580 nm to a wavelength between 680 nm and 800 nm, the first transmission spectrum T1 has an average transmittance of about 40%. In particular, for the transmittance Τ (λ) of the first transmission spectrum Tl within the red spectral range, T ₀ -ΔΤ <Τ (λ) <T ₀ + ΔΤ, wherein T _{0 is} about 40% and ΔΤ is about 5%.

Accordingly, the transmittance Τ (λ) of the first transmission spectrum Tl within the red spectral range has a substantially constant value T ₀ of about 40%. For this purpose, the second transmission spectrum T2 for the blue and green spectral range in each case has an average transmittance of at least 90% and for the infrared spectral range an average transmittance of at most 10%. Within the red spectral range, the transmittance of the second transmission spectrum T2 has a relatively constant value of about 60%.

With this configuration, fluorescent light of PpIX and fluorescein is transmitted to both the first camera 2 IC and the second camera 23C. The fluorescent light of ICG is transmitted substantially exclusively to the first camera 2 IC.

The first camera 2 IC is configured to detect light of the red and infrared spectral regions and for this purpose has a corresponding filter, which is represented by the color filter cell 63C. The second camera 23C includes a spatially resolving detector 67C and a second Bayer matrix 69C. An exemplary color filter cell 71C of the second Bayer matrix 69C may have the previously discussed RGGB pattern.

In this configuration, the spatially resolved intensity distribution of light of the blue spectral range and the spatially resolved intensity distribution of light of the green spectral range can be detected directly by the second camera 23C. The spatially resolved intensity distribution of light of the red spectral range detected by the second camera 23C can be used to determine the spatially resolved intensity distribution of light of the infrared spectral range, for example by subtracting the intensity distribution of light of the red spectral range detected by the second camera 23C from the intensity distribution detected by the first camera 21C. Further, the intensity distribution detected by the first camera 21C may be added to the intensity distribution of light of the red spectral region detected by the second camera 23C to improve the sensitivity for detecting light of the red spectral region.

For improving the sensitivity to the light of the green spectral range, as shown in the example color filter cell 73C, the color filter "R" of the color filter cell 7 IC are replaced by the color filter "R + G", which predominantly transmits the red and green spectral range. According to this configuration, the second camera 23C is configured to detect a spatially resolved first intensity distribution of the light transmitted through the color filters transmitting the red and green spectral regions and incident on the detector 67C. The spatially resolved intensity distribution of light of the green spectral range detected by the second camera 23C can be used to determine the spatially resolved intensity distribution of red light incident on the second camera 23C, for example by subtracting the detected intensity distribution from light of the green Spectral range from the first intensity distribution.

FIG. 6 shows another embodiment of a fluorescent light detection system 19D. Similarly to the fluorescent light detection system 19C shown in FIG. 5, the fluorescent light detection system 19D is also configured to transmit red-spectrum light to both the first camera 21D and the second camera 23D. In contrast to the embodiment shown in FIG. 5, however, the red spectral range is not divided according to intensities, but separated into spectral ranges, i. That is, light of the red spectral region having a wavelength smaller than a cut-off wavelength λ o is substantially not transmitted to the first camera 21 D according to the first transmission spectrum T 1 and transmitted substantially exclusively to the second camera 23 D according to the second transmission spectrum T 2. On the other hand, light of the red spectral region having a wavelength larger than the cut-off wavelength λ o is substantially exclusively transmitted to the first camera 21 D according to the first transmission spectrum T 1 and substantially not transmitted to the second camera 23 D according to the second transmission spectrum T 2. The cut-off wavelength λο can be, for example, in the range between 600 nm and 630 nm. In this configuration, the fluorescent light from PpIX is transmitted substantially exclusively to the first camera 21D. Alternatively, the cut-off wavelength λο may be in a range of 640nm to 680nm. In this configuration, emission light from PpIX is transmitted partly to the first camera 21D and partly to the second camera 23D.

The first camera 21D may be the same as the first camera 2 IC shown in FIG. Further, the second camera 23D may be the second camera 23C shown in FIG be equal. In particular, the exemplary color filter cells of the second Bayer matrix 71C and 73C shown in FIG. 5 may also be used as the color filter cell of the second Bayer matrix 69D of the second camera 23D.

In this embodiment, the fluorescent light of indocyanine green is detected substantially exclusively by the first camera 21D, and the fluorescence light of fluorescein is detected substantially exclusively by the second camera 23D.

The intensity distributions of the red and infrared spectral range can be determined by the controller according to the schemes already explained above.

Claims

claims

Fluorescent light detection system for the detection of observation and fluorescent light, comprising:

a beam splitting system, a spatially resolving first camera and a second resolution camera other than the first camera,

wherein the beam splitter system is configured to transmit light entering the beam splitter system to the first camera according to a first transmission spectrum and to the second camera according to a second transmission spectrum different from the first transmission spectrum;

wherein the first transmission spectrum in a red spectral range has an average transmittance of at least 10%, in particular at least 20% or 50%,

wherein the second transmission spectrum in a blue and green spectral range each have an average transmittance of at least 90%;

wherein a predetermined camera of the first and second cameras is configured to detect a spatially resolved first intensity distribution of light of a first spectral range;

wherein a controller of the fluorescent light detection system is configured to determine a spatially resolved third intensity distribution representing the intensity distribution of light of a third spectral range based on the first intensity distribution and a spatially resolved second intensity distribution,

wherein the second intensity distribution represents a spatially resolved intensity distribution of light of a second spectral range detected or controlled by the controller, and

wherein the first, second and third spectral range are different from each other. Fluorescent light detection system according to claim 1,

wherein the second intensity distribution is based on a spatially resolved intensity distribution detected by the camera of the first and second cameras other than the predetermined camera.

3. Fluorescence light detection system according to claim 1 or 2,

wherein the controller uses the first and / or second transmission spectrum to determine the third intensity distribution.

4. Fluorescence light detection system according to one of claims 1 to 3,

wherein the first transmission spectrum in the blue and / or green spectral range has an average transmittance of at most 10%; and

wherein the second transmission spectrum in the red spectral range has an average transmittance of at most 50%, in particular at most 80% or 90%.

The fluorescent light detection system according to claim 1, wherein the first camera is configured to detect light of the red spectral region; and

wherein the second camera is configured to selectively detect light of the blue and green spectral ranges, respectively.

6. Fluorescent detection system according to one of claims 1 to 5,

wherein the first transmission spectrum for an infrared spectral range has an average transmittance of at least 90% and the second transmission spectrum for the infrared spectral range has an average transmittance of at most 10%; or

wherein the second transmission spectrum for the infrared spectral range has an average transmittance of at least 90% and the first transmission spectrum for the infrared spectral range has an average transmittance of at most 10%.

7. Fluorescence light detection system according to one of claims 1 to 6,

wherein the first or second camera is configured to detect light of the infrared spectral range.

8. Fluorescence light detection system according to one of claims 1 to 7,

wherein a controller of the fluorescent light detection system is configured to a spatially resolved infrared intensity distribution, which represents a spatially resolved intensity distribution of light of the infrared spectral range detected or determined by the control,

a spatially resolved red intensity distribution which represents a spatially resolved intensity distribution of light of the red spectral range detected or determined by the control,

a spatially resolved green intensity distribution, which represents a detected or determined by the control, spatially resolved intensity distribution of light of the green spectral range, and

a spatially resolved blue intensity distribution, which represents a detected or determined by the control, spatially resolved intensity distribution of light of the blue spectral range to provide.

9. Fluorescence light detection system according to one of claims 1 to 8,

wherein the beam splitter system comprises a beam splitter and at least one optical filter.

10. Fluorescence light detection system according to claim 9,

wherein the beam splitter is configured to separate the light entering the beam splitter system spectrally and / or spatially and / or polarizations, in particular mutually orthogonal polarizations.

11. Fluorescence light detection system according to one of claims 1 to 10,

wherein the first transmission spectrum in the red and infrared spectral range in each case has an average transmittance of at least 90% and in the blue and green spectral range in each case an average transmittance of at most 10%,

the second transmission spectrum in the red and infrared spectral ranges each having an average transmittance of at most 10%,

wherein a controller of the fluorescent light detection system is configured to

a spatially resolved intensity distribution of light of the red spectral range and a spatially resolved intensity distribution of light of the determine infrared spectral range based on intensity distributions detected by the first camera, and

determine a spatially resolved intensity distribution of light of the blue spectral range and a spatially resolved intensity distribution of light of the green spectral range based on intensity distributions detected by the second camera.

12. fluorescence detection system according to claim 11,

wherein the first camera comprises a location-resolving detector having a first Bayer matrix, the first Bayer matrix comprising a plurality of non-overlapping, regularly arranged color filter cells, each color filter cell of the first Bayer matrix comprising a plurality of different non-overlapping color filters,

wherein at least a first color filter of the color filters of each color filter cell of the first Bayer matrix is a filter of a filter group,

wherein at least a second color filter of the color filters of each color filter cell of the first Bayer matrix is a filter of the filter group different from the at least one first color filter,

wherein the filter group comprises a filter that predominantly transmits the red spectral region, a filter that predominantly transmits the infrared spectral region, and a filter that predominantly transmits the red and infrared spectral regions.

13. Fluorescence light detection system according to claim 14,

wherein half of the color filters of each color filter cell of the first Bayer matrix predominantly transmits the red spectral range or predominantly the infrared spectral range and half of the color filters of each color filter cell of the first Bayer matrix predominantly transmits the red and infrared spectral range.

14. Fluorescent light detection system according to claim 12 or 13,

wherein the first camera is configured to detect a spatially resolved first intensity distribution of the light transmitted through the first color filters and incident on the detector, wherein the first camera is configured to detect a spatially resolved second intensity distribution of the light transmitted through the second color filters and incident on the detector; and

wherein the controller is configured to determine the spatially resolved intensity distribution of light of the infrared spectral range based on the first and second intensity distribution, and / or

wherein the controller is configured to determine the spatially resolved intensity distribution of red spectral region light based on the first and second intensity distributions.

15. Fluorescent light detection system according to one of claims 11 to 14,

the second camera comprising a position-resolving detector having a second Bayer matrix, the second Bayer matrix comprising a plurality of non-overlapping, regularly arranged color filter cells, each color filter cell of the second Bayer matrix comprising a plurality of different non-overlapping color filters,

wherein a quarter of the color filters of each color filter cell of the second Bayer matrix predominantly transmits the blue spectral range, one quarter of the color filters of each color filter cell of the second Bayer matrix predominantly transmits the red spectral range and half of the color filters of each color filter cell of the second Bayer matrix predominantly transmits the green spectral range; or

wherein half of the color filters of each color filter cell of the second Bayer matrix predominantly transmits the blue spectral range and half of the color filters of each color filter cell of the second Bayer matrix predominantly transmits the green spectral range; or

wherein a quarter of the color filters of each color filter cell of the second Bayer matrix predominantly transmits the blue spectral range and three quarters of the color filters of each color filter cell of the second Bayer matrix predominantly transmit the green spectral range.

16. Fluorescent light detection system according to one of claims 1 to 10,

wherein the first transmission spectrum in the red spectral range has an average transmittance of at least 90% and in the blue, green and infrared Spectral range has an average transmittance of at most 10%,

wherein the second transmission spectrum has an average transmittance of at least 90% in the infrared spectral range and an average transmittance of at most 10% in the red spectral range,

wherein a controller of the fluorescent light detection system is configured to

to determine a spatially resolved intensity distribution of light of the red spectral region based on intensity distributions detected by the first camera, and

a spatially resolved intensity distribution of light of the blue spectral range, a spatially resolved intensity distribution of light of the green spectral range, and a spatially resolved intensity distribution of light of the infrared spectral range based on intensity distributions detected by the second camera.

17. Fluorescent detection system according to claim 16,

wherein the second camera comprises a position-resolving detector having a second Bayer matrix, the second Bayer matrix comprising a plurality of non-overlapping, regularly arranged color filter lines, each color filter cell of the second Bayer matrix having a plurality of different non-overlapping color filters includes,

wherein a quarter of the color filters of each color filter cell of the second Bayer matrix predominantly transmits the blue and infrared spectral range, one fourth of the color filters of each color filter cell of the second Bayer matrix predominantly transmits the red and infrared spectral range and half of the color filters of each color filter cell of the second Bayer matrix predominantly transmits the green and infrared spectral range.

18. Fluorescence light detection system according to claim 17,

wherein the second camera is configured to detect a spatially resolved first intensity distribution of the light transmitted through the color filters transmitting the red and infrared spectral regions and incident on the detector, wherein the controller is configured to determine the spatially resolved intensity distribution of light of the infrared spectral range based on the first intensity distribution.

19. Fluorescence light detection system according to claim 18,

wherein the second camera is configured to detect a spatially resolved second intensity distribution of the light transmitted through the color filters transmitting the blue and infrared spectral regions and incident on the detector,

wherein the controller is configured to determine the spatially resolved intensity distribution of light of the blue spectral range based on the second intensity distribution and the intensity distribution of light of the infrared spectral range; and or

wherein the second camera is configured to detect a spatially resolved third intensity distribution of the light transmitted through the color filters transmitting the green and infrared spectral regions and incident on the detector,

wherein the controller is configured to determine the spatially resolved intensity distribution of light of the green spectral range based on the third intensity distribution and the intensity distribution of light of the infrared spectral range.

20. Fluorescence light detection system according to claim 16,

wherein a quarter of the color filters of each color filter cell of the second Bayer matrix predominantly transmits the blue and infrared spectral range, one fourth of the color filters of each color filter cell of the second Bayer matrix predominantly transmits the green spectral range and half of the color filters of each color filter cell of the second Bayer Matrix predominantly transmits the green and infrared spectral range.

21. Fluorescence light detection system according to claim 20,

wherein the second camera is configured to detect a spatially resolved first intensity distribution of the light transmitted through the color filters transmitting the green and infrared spectral regions and incident on the detector,

wherein the controller is configured to determine the spatially resolved intensity distribution of light of the infrared spectral range based on the first intensity distribution and the intensity distribution of light of the green spectral range.

22. Fluorescence light detection system according to claim 21,

wherein the controller is configured to determine the spatially resolved intensity distribution of light of the blue spectral range based on the second intensity distribution and the intensity distribution of light of the infrared spectral range.

23. Fluorescence light detection system according to one of claims 1 to 10,

wherein the first transmission spectrum in the infrared spectral range has an average transmittance of at least 90 ° / o, in the red spectral range an average transmittance between 10% and 90%, in particular between 25% and 75% or between 40% and 60%, and in the blue and green Spectral range has an average transmittance of at most 10%,

wherein the second transmission spectrum in the red spectral range has an average transmittance between 10% and 90%, in particular between

25% and 75% or between 40% and 60%, and in the infrared spectral range has an average transmittance of at most 10%,

wherein a controller of the fluorescent light detection system is configured to

a spatially resolved intensity distribution of light of the infrared spectral range and a spatially resolved intensity distribution of light of the red spectral range based on intensity distributions detected by the first and second cameras,

to determine a spatially resolved intensity distribution of light of the blue spectral range and a spatially resolved intensity distribution of light of the green spectral range based on intensity distributions detected by the second camera.

24. Fluorescence light detection system according to claim 23,

wherein the first camera is configured to detect a spatially resolved first intensity distribution of light of the red and infrared spectral regions.

25. Fluorescent light detection system according to claim 24,

wherein a quarter of the color filters of each color filter cell of the second Bayer matrix predominantly transmits the blue spectral range, one quarter of the color filters of each color filter cell of the second Bayer matrix predominantly transmits the red spectral range and half of the color filters of each color filter cell of the second Bayer matrix predominantly transmitted the green spectral range.

26. Fluorescence light detection system according to claim 25,

wherein the controller is configured to

determine the spatially resolved intensity distribution of light of the infrared spectral range based on the first intensity distribution detected by the first camera and an intensity distribution of the light transmitted through the color filter transmitting the red spectral range and detected by the second camera; and or

the spatially resolved intensity distribution of light of the red spectral region based on the first detected by the first camera Determine intensity distribution and detected by the second camera intensity distribution of the transmitted through the red spectral region color filter transmitted and incident on the detector light.

27. Fluorescence light detection system according to claim 24,

wherein one fourth of the color filters of each color filter cell of the second Bayer matrix predominantly transmits the blue spectral range, one fourth of the color filters of each color filter cell of the second Bayer matrix predominantly transmits the red and green spectral range and half of the color filters of each color filter cell of the second Bayer Matrix predominantly transmits the green spectral range.

28. Fluorescence light detection system according to claim 27,

wherein the second camera is configured to detect a spatially resolved second intensity distribution of the light transmitted through the color filters transmitting the green and red spectral regions and incident on the detector,

wherein the controller is configured to determine a spatially resolved third intensity distribution of light of the red spectral region based on the second intensity distribution and an intensity distribution of light of the green spectral region.

29. Fluorescent light detection system according to claim 28,

wherein the controller is configured to

determine the spatially resolved intensity distribution of light of the infrared spectral range based on the first intensity distribution detected by the first camera and the third intensity distribution; and or determine the spatially resolved intensity distribution of light of the red spectral region based on the first intensity distribution detected by the first camera and the third intensity distribution.

30. Fluorescent light detection system according to one of claims 23 to 29,

wherein the following applies to the wavelength-dependent transmittance Τ (λ) of the first and / or second transmission spectrum within the red spectral range:

T ₀ - ΔΤ <Τ (λ) <To + ΔΤ; 15% <T ₀ <85%; 0 <ΔΤ <5%.

31. Fluorescent light detection system according to one of claims 23 to 29,

wherein a first spectral range of the second transmission spectrum within the red spectral range comprises only wavelengths which are smaller than a cut-off wavelength λ o, and has an average transmittance T _x , and

wherein a second spectral range of the second transmission spectrum within the red spectral range comprises only wavelengths which are greater than the cut-off wavelength λ o and has an average transmittance T ₂ , where:

i> 70%, preferably T _x > 80% or>90%; and

T ₂ <30%, preferably T ₂ <20% or T ₂ <10%.

Fluorescent light detection system according to claim 31,

where:

600nm <<635nm or

640nm <λο ^ 680nm.

33. Fluorescence light detection system according to one of claims 1 to 32,

wherein the blue spectral range is between 350nm and 480nm and / or wherein the green spectral range is between 480nm and 580nm and / or wherein the red spectral range is between 580nm and 750nm, more preferably between 580nm and 680nm or between 630nm and 680nm and / or the infrared Spectral range is between 750nm and 100nm.

34. Microscopy system comprising:

at least one light source configured to generate illumination light and excitation light to excite fluorescent dyes and direct it to an object area,

an objective system and a fluorescence detection system according to one of claims 1 to 33,

wherein the objective system and the fluorescent light detection system are arranged so that at least a section of the object area is imaged by the objective system and the fluorescent light detection system on the first and second camera.

35. The microscopy system of claim 34, further comprising:

a display system for displaying an image, wherein the image is generated based on intensity distributions provided by a controller of the fluorescent light detection system.

36. The microscopy system according to claim 34 or 35, further comprising:

a detection filter disposed in the optical path between the object region and the fluorescent light detection system, which is configured to suppress at least a part of the excitation light.