WO2008010120A2

WO2008010120A2 - Employing beam scanning for optical detection

Info

Publication number: WO2008010120A2
Application number: PCT/IB2007/052499
Authority: WO
Inventors: Erik M. H. P. Van Dijk; Marius I. Boamfa; Marcello L. M. Balistreri; Reinhold Wimberger-Friedl
Original assignee: Koninklijke Philips Electronics N.V.
Priority date: 2006-07-17
Filing date: 2007-06-27
Publication date: 2008-01-24
Also published as: WO2008010120A3

Abstract

A detection system is described allowing scanning of an excitation irradiation beam over a substrate (8) for detecting luminescence sites. The scanning thereby is performed by using an optical deflector (5) for varying an angle of incidence of the excitation irradiation beam on a single focusing element (7) on an irradiation path between the deflector (5) and the substrate (8). By varying the angle of incidence on the single focusing element (7), the focus point of the excitation irradiation beam is shifted in the focal plane of the focusing element (7), where the substrate (8) is positioned. The latter allows fast scanning of the excitation irradiation beam. In a preferred embodiment, an additional scanning motion in a substantially different direction is furthermore superimposed, resulting in the possibility for scanning an appropriate area of the substrate (8). The additional scanning motion may be obtained using a split optics system, whereby substantially only the single focusing element (7) and the optical deflector (5) are moved with respect to the substrate (8), whereas other optical components typically are fixed with respect to the substrate (8) during the scanning.

Description

Employing beam scanning for optical detection

FIELD OF THE INVENTION

The present invention relates to the field of optical detection. More particularly, the present invention relates to methods and systems for detecting luminescent signals, e.g. as used in qualitative or quantitative detection of biological, chemical or bio- chemical particles, and to means for improving such detection methods and systems.

BACKGROUND OF THE INVENTION

In general, molecular diagnostics of a bio-sample, usually a liquid analyte mixture, typically comprises screening of the bio-sample for detection of certain biological components, referred to as target particles, such as genes or proteins. This is done by first allowing selective bindings to be formed between target particles and capture probes that typically are attached to a solid surface. Such selective binding is also known as hybridisation. The hybridisation step is then typically followed by a washing step, where all unbounded target particles are flushed away. Finally a detection step is performed for detecting the presence of target particles captured by the capture probes. Taking into account the probability for capturing the target particle to the capture probe, a quantitative analysis of the concentration of target particles present in the bio-sample can be determined. The detection step is typically based on detection of fluorescent labels attached to the target molecules. In order to cope with the small concentration of target molecules typically present in the bio-samples, it is important that the fluorescent detection is very sensitive, ideally close to the ultimate detection limit of single fluorescent label sensitivity. To reach this high sensitivity, the detection step is typically time consuming.

At present, there are generally two different methods to detect fluorescence arising from a sample. The first method uses statically irradiating a large sample area and spatially detecting a fluorescence response, e.g. by imaging the whole sample with a camera in parallel. To image an area of 1 x 1 cm² with a sufficiently high resolution to obtain single fluorescent label sensitivity, e.g. a resolution of 0.5 μm, requires a camera with 400 megapixels and a lens that has an appropriately large field of view, putting severe requirements on the components used. The second method is to scan a small spot over the sample in order to detect fluorescence arising from different positions within that sample and to build up an image in time. In order to scan an area of e.g. 1 x 1 cm² with a spot size of about 0.5μm within a reasonable time (e.g. 60 seconds) a linear velocity of approximately 3.3 m/s is required, which puts stringent requirements on the operation of the detection system. A number of biosensors based on scanning rotating disks are known, allowing for fast scanning of large areas, as e.g. described in US 6685885. However, for a large number of applications, the latter is not very efficient as typically only a small area, e.g. 1x1 cm², is required for an adequate number of experiments to be run simultaneously. In addition, systems based on a rotating disk typically result in a relative large form factor for the substrate (that holds the sample) and the reader. Another typical problem for biosensors based on rotating systems is to enable interfacing with the outside world, e.g. to allow fluid handling or heating.

A large number of commercial confocal scanners use some form of beam scanning to build up an image. Here the beam is scanned back and forth through the field of view of the lens. Nevertheless, such systems are in general rather bulky and use large and complex optics . Furthermore, most of these scanners have a maximum range of some hundreds of microns limited by the field of view of the lens. When a larger area is to be scanned the sample typically is moved mechanically after each 2D area is scanned.

SUMMARY OF THE INVENTION

An object of the present invention is to obtain good methods and systems for detecting biological, chemical and/or bio-chemical particles. It is an advantage of embodiments of the present invention that the measurement time for optical scanning of samples can be reduced. The present invention relates to a detection system for detecting luminescence sites on a substrate, the detection system comprising an irradiation unit for generating at least one excitation irradiation beam for exciting luminescence sites on the substrate, a first optical scanning means comprising an optical deflector for deflecting the at least one excitation irradiation beam, and a single focusing element on the irradiation path between the optical deflector and the substrate, the single focusing element having an optical axis and being adapted for receiving the deflected at least one excitation irradiation beam and for focusing the deflected at least one excitation irradiation beam on the substrate, the optical deflector adapted for varying an angle of incidence of the at least one excitation irradiation beam on the single focusing element with respect to an optical axis of the single focusing element, the varying providing a first scanning motion Z' of the at least one excitation irradiation beam over the substrate. Varying an angle of incidence may be substantially continuously varying an angle of incidence with respect to the optical axis of the focusing element.

The optical deflector may be moveable with respect to the single focusing element.

The detection system furthermore may comprise a second scanning means adapted for providing a relative motion between the single focusing element and the substrate thus providing a second scanning motion of the at least one excitation irradiation beam over the substrate simultaneously with the first scanning motion Z'. The second scanning motion may be substantially perpendicular to the first scanning motion.

The second scanning means may comprise a slider whereon the focusing element and the optical deflector are mounted, the slider being moveable with respect to the substrate and other components of the detection system for generating the second scanning motion Z". The slider and the focusing element and the optical deflector may be moveable with respect to a beam splitter for splitting an excitation irradiation beam from a luminescence irradiation beam.

The first optical scanning motion may be substantially faster than the second scanning motion. The first optical scanning motion may be at least twice as fast, preferably at least ten times at fast as the second scanning motion. The slider may be part of a sliding means which is moveable with respect to a substrate and other components of the detection system according to a third scanning motion Z"' in a direction that is substantially parallel to the first scanning motion. It is an advantage of particular embodiments of the present invention that an appropriate detection area of the substrate can be scanned, suitable for performing molecular diagnostic tests. The third scanning motion Z'" may be a discontinuous scanning motion.

The angle of incidence CC may be smaller than 1°. It is an advantage of particular embodiments of the present invention that the diameter of the beam spot and the aberrations generated by the shift off the focal axis can be kept small.

The distance between the substrate and the focusing element can be changed or may be changeable in order to focus the at least one excitation irradiation beam. It is an advantage of particular embodiments of the present invention that proper focusing on the substrate can be ensured.

The detection system furthermore may comprise a detection unit having at least a detector element and the detection system may be adapted for guiding luminescence irradiation beams via the focusing element and the optical deflector to the at least a detector element. It is an advantage of particular embodiments of the present invention that only a limited number of optical elements is required.

The optical deflector may be a reflective device being any of a plane mirror, a spherical mirror, or an aspherical mirror.

The optical deflector may be a reflective device being a rotating mirror.

It is an advantage of particular embodiments of the invention that a rotating mirror may be used rotating with a variable angular speed CO in an alternatingly clockwise and counter-clockwise direction between the angles of rotation β and -β, respectively, thereby providing angles of incidence of the defected excitation irradiation beam with respect to the focusing element between α and -α, respectively.

The optical deflector may be a reflective device translated with a variable linear velocity v in an alternating forward and backward direction.

The present invention also relates to a control system for controlling a detection system for detecting luminescence sites on a substrate, the control system comprising an optical deflector driver for controlling movement of an optical deflector adapted for deflecting at least one excitation irradiation beam thus varying an angle of incidence of the at least one excitation irradiation beam on a single focusing element on an irradiation path between the optical deflector and the substrate, the varying providing a first scanning motion of the at least one excitation irradiation beam over the substrate.

The present invention furthermore relates to a method for detecting luminescence sites on a substrate, the method comprising generating at least one excitation irradiation beam for exciting luminescence sites on the substrate, deflecting the at least one excitation irradiation beam with a deflector on a single focusing element on the irradiation path between a deflector and the substrate and focusing the at least one excitation irradiation beam on the substrate, the deflecting comprising varying an angle of incidence of the at least one excitation irradiation beam on the focusing element with respect to an optical axis of the focusing element, the varying providing a first scanning motion Z' of the at least one excitation irradiation beam over the substrate. It is an advantage of particular embodiments of the present invention that a detection system and method allowing fast optical scanning of samples is obtained, wherein furthermore the amount of aberrations induced, e.g. generated by a shift off the focal axis, can be kept small. It is an advantage of particular embodiments of the present invention that a detection system and method with fast optical scanning of samples is obtained, wherein the diameter of the beam spot can be small allowing relatively high detection resolution and wherein an appropriate focusing on the substrate is ensured.

It is an advantage of particular embodiments of the present invention that parts of the biosensor can be based on existing mass-produced, low-cost components allowing to obtain detection systems in a cost effective manner.

It is an advantage of particular embodiments of the present invention that the form factor for the substrate and the detection system is kept small thereby contributing to cost-effective practice.

It is an advantage of particular embodiments of the present invention that a relatively cheap and robust detection system can be obtained.

It is an advantage of particular embodiments of the present invention that the interfacing with external, i.e. the outside world can be easily enabled thereby making provision for fluid handling and e.g. heating.

It is an advantage of particular embodiments of the present invention that the excitation irradiation beam(s) can be directed in complex patterns enabled by the multiple degrees of freedom of the optical deflector, thereby increasing the number of potential applications.

The above objective at least some of the advantages is accomplished by a device, a system, and a method according to the present invention. Particular and preferred aspects of the invention are set out in the accompanying independent and dependent claims. Features from the dependent claims may be combined with features of the independent claims and with features of other dependent claims as appropriate and not merely as explicitly set out in the claims.

The teachings of the present invention permit the design of improved methods and apparatus for detecting chemical, biological and/or biochemical particles. The above and other characteristics, features and advantages of the present invention will become apparent from the following detailed description, taken in conjunction with the accompanying drawings, which illustrate, by way of example, the principles of the invention. This description is given for the sake of example only, without limiting the scope of the invention. The reference figures quoted below refer to the attached drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 is a schematic illustration of a detection system according to embodiments of the first aspect of the present invention. Fig. 2 illustrates the principle of beam scanning used in detection systems according to embodiments of the first aspect of the present invention, indicating that changing the angle CC under which the beam enters the single focusing element results in a shift in the focal position. Fig. 3 is a detailed view of a shift in focal position for a given deflection angle α, for deflection as e.g. illustrated in Fig. 2.

Fig. 4 is a graph of light intensity versus position showing the change in spot size for different deflection angles cc, for beam deflection as e.g. illustrated in Fig. 2 Fig. 5 is a schematic illustration of the light path in a detection system according to a first embodiment of the first aspect of the present invention.

Fig. 6a and Fig. 6b show a schematic top view and side view of an optical scanning system for a detection system according to a second embodiment of the first aspect of the present invention.

Fig. 7 is a schematic illustration of the route followed by the irradiation beam as it is scanned over the substrate in a detection system according to a second embodiment of the first aspect of the present invention.

Fig. 8 is a schematic illustration of an electronic control system for providing controlling signals to an optical scanning system according to the third aspect of the present invention. In the different Figures, the same reference signs refer to the same or analogous elements.

DETAILED DESCRIPTION OF EMBODIMENTS

The present invention will be described with respect to particular embodiments and with reference to certain drawings but the invention is not limited thereto but only by the claims. Any reference signs in the claims shall not be construed as limiting the scope. The drawings described are only schematic and are non-limiting. In the drawings, the size of some of the elements may be exaggerated and not drawn on scale for illustrative purposes. Where the term "comprising" is used in the present description and claims, it does not exclude other elements or steps. Where an indefinite or definite article is used when referring to a singular noun e.g. "a" or "an", "the", this includes a plural of that noun unless something else is specifically stated.

Furthermore, the terms first, second, third and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein. Moreover, the terms forward and backward are interchangeable under appropriate circumstances and embodiments of the present invention described herein are capable of operation in other orientations than described or illustrated herein.

The following terms or definitions are provided solely to aid in the understanding of the invention. These definitions should not be construed to have a scope less than understood by a person of ordinary skill in the art.

With the term "irradiation" and "luminescence" typically UV, visible or infrared irradiation may be meant although the invention is not limited thereto and other types of electromagnetic irradiation could also be used. The wavelength of the irradiation beam referred to may be the average wavelength of the irradiation beam or the wavelength at which the maximum emission is obtained.

The term "substrate", as used herein, relates to the area on which the irradiation beam has to be focused and from where luminescence is collected. Luminescence sites of which detection is envisaged according to the present invention may be distinct sites such as lines or spots on a substrate that emit at least one luminescence beam having at least one luminescence beam wavelength. Any luminescent signal such as reflection, scattering, fluorescence, chemi-luminescence, bio luminescence, or other luminescence may be envisaged. Luminescence sites may also emanate from structural features of a surface that scatter or reflect light. Luminescence sites may relate to occupied sites on a substrate, for example occupied by luminescent labeled target particles. Luminescence emanating from a substrate may include irradiation that is transmitted through the substrate, or may be created by elements placed on the substrate, for example, fluorescent labels that create fluorescent light within a micro array pursuant to excitation with an appropriate wavelength of light, or may be created by structural features of the surface of the substrate such as light-scattering gratings. The substrate may e.g. be a glass slide, a micro array, a semiconductor chip such as e.g. a silicon chip, a membrane e.g. a nylon membrane, a filter e.g. a nylon filter, a micro fluidic device, a roughened metal substrate, a gel e.g. an agarose gel containing stained DNA or proteins, or any other device having a suitable surface for providing luminescence sites. The substrate also may be a cartridge e.g. a disposable cartridge, containing any of the above cited materials. The term "sample", as used herein, relates to a composition comprising analyte(s) of interest. The term "analyte", as used herein, refers to a substance to be detected by the methods of the present invention. The analyte may be optically dead, may be an inherent luminescence provider or may be labeled with a label to emanate luminescence or may bind to something else that generates a light change, e.g. a light output or change in wavelength. The term "label", as used herein, refers to a molecule or material capable of generating a detectable signal, e.g. any change in an measurable signal such as light output, extinguishing of light, change in brightness, change in wavelength, etc. The labels may be attached directly to the analyte or through a linker moiety, e.g. a labeled probe. These probes, intended to either specifically bind to the analyte, may be obtained by linking a compound capable of specifically binding to the analyte or corresponding to at least (a specific) part of the analyte, to a label. The nature of the analyte-specific probe will be determined by the nature of the analyte to be detected. Most commonly, the probe may be developed based on a specific interaction with the analyte such as, but not limited to, antigen-antibody binding, complementary nucleotide sequences, carbohydrate-lectin, complementary peptide sequences, ligand-receptor, coenzyme-enzyme, enzyme inhibitors-enzyme, etc. The label need not be bound to the analyte but may be placed on the substrate.

According to a first aspect, the present invention provides a detection system for detecting luminescence sites on a substrate. Such a detection system may be for example a detection system for detecting chemical, biological or bio-chemical particles, or molecules, the invention not being limited thereto. The detection system typically comprises an irradiation unit for generating at least one excitation irradiation beam for irradiating a substrate. The at least one excitation irradiation beam, according to the present invention, is scanned over the substrate, either by moving the beam with respect to the substrate, moving the substrate with respect to the beam or a combination of the two. Application of the excitation beam may typically result in excitation of the luminescence sites at the substrate. Detection typically may be enabled by detecting the luminescence response, e.g. at least one luminescence signal or a change in a signal, generated by the luminescence sites on the substrate. The detection system typically comprises a first single focusing element adapted for receiving at least one excitation irradiation beam to be focused on the substrate using the first focusing element. In other words, the first focusing element is adapted for focusing the at least one excitation irradiation beam on the substrate. In order to obtain a first scanning motion of the at least one excitation beam over the substrate, the present invention typically also comprises a first optical scanning means comprising an optical deflector for deflecting the at least one excitation beam to the first focusing element. The optical deflector, the first focusing element and the substrate thereby are positioned relatively with respect to each other such that the first single focusing element is the only focusing element on the irradiation path between the optical deflector and the substrate, i.e. a single focusing element. The latter allows to obtain a simple scanning detection device. The optical deflector thereby is adapted for varying an angle of incidence of the at least one excitation irradiation beam on the single focusing element with respect to its optical axis of the single focusing element, whereby varying the angle provides a scanning motion of the at least one excitation irradiation beam over the substrate. In other words, the optical deflector is (part of) a first optical scanning means allowing to provide a first scanning motion of the at least one excitation irradiation beam over the substrate. The detection system typically also may comprise at least one second scanning means for scanning the excitation irradiation beam over the substrate. Such second scanning means may provide a scanning motion in another direction and/or in the same direction as the first scanning motion. Typically detection systems according to the present embodiments allow detection within a limited measurement time of an appropriate substrate area. A schematic overview of a detection system 100 comprising essential and optional components is shown by way of illustration in Fig. 1. The detection system 100 is suitable for detecting light emission sites on a substrate 8. As shown in Fig. 1, the detection system 100 comprises an irradiation unit 102, a focusing element 7 adapted for receiving at least one excitation irradiation beam and an optical deflector 5 typically being part of a first optical scanning means 202, and adapted for providing scanning of the excitation irradiation beam over the substrate 8 for detecting light emission sites on the substrate 8. The optical deflector 5 typically is adapted for varying the angle of incidence of the excitation irradiation beam on the focusing element 7 thus shifting the focus position around in the focal plane of the focusing element 7. Preferably, the detection system thereby is adapted such that the full field of view of the focusing element 7 is used. The above components and additional or optional components of the exemplary detection system as shown in Fig. 1 will be further described in more detail below.

As set out above, the detection system 100 typically comprises an irradiation unit 102 comprising at least one irradiation source. The at least one irradiation source may be any irradiation source suitable for use in a detection system, such as e.g. a light source. Typically, the irradiation source is adapted for generating at least one excitation irradiation beam having an at least one predetermined wavelength λ. The at least one irradiation source may be an illumination array comprising irradiation sources, such as lasers, emitting excitation irradiation beams having radiation at wavelengths λ_{l s} λ₂, λ₃, ..., X_n, or emitting radiation in predetermined wavelength ranges, for irradiating the substrate. The irradiation source may also comprise a white light source which may be filtered to several irradiation beams having radiation at a specific wavelength or in a specific wavelength range. The irradiation source may also comprise one or more monochromatic optical sources such as lasers. The light source may comprise argon lasers, diode lasers, helium lasers, dye lasers, titanium sapphire lasers, Nd: YAG lasers or others. The irradiation unit 102 may for example comprise a tuneable irradiation source, such as e.g. a tuneable semiconductor laser, for consecutively supplying at least one irradiation beam, or at least one semiconductor laser for simultaneously or consecutively supplying at least one radiation beam. The at least one irradiation source may thus be a plurality of irradiation sources e.g. two or three irradiation sources. The latter typically allows multiplexing. The at least one irradiation source may be adapted for emitting radiation, e.g. light, at a predetermined wavelength or a predetermined wavelength range, suitable for exciting or irradiating luminescence sites, e.g. luminescence sites like fluorescence sites. Such sites may e.g. comprise optically variable particles, present in the sample. For example, in the case where the generated radiation is fluorescence radiation, the optical wavelength of the excitation radiation typically may e.g. be in the range from 200 nm to 2000 nm, or e.g. in the range from 400 nm to 1100 nm, the invention not being limited thereto. The excitation field of the at least one excitation irradiation beam may be a single spot, although the invention is not limited thereto.

The detection system 100 furthermore comprises a first focusing element 7 that is positioned such that, in operation, it may receive at least one excitation irradiation beam. The first focusing element 7 may also be positioned such that it receives, besides at least one excitation irradiation beam, a luminescence signal such as a luminescence beam collected from luminescence sites on the substrate 8, generated in response to the at least one excitation irradiation beam. In other words, the focusing element 7 may be used both for focusing the excitation on the substrate 8 and/or for collecting the generated luminescence signal from the substrate 8. Such first focusing element 7 may be a conventional or standard focusing element. The first focusing element 7 typically may be the objective lens in the detection system used for focusing the excitation irradiation beam(s) on the substrate 8. The focusing element 7 however also may be a parabolic mirror, as well as any other dioptric or catoptic imaging means, including a prism. As described above, the first focusing element 7 alternatively or in addition thereto may be used both for focusing the excitation irradiation beam(s) on the substrate and for collecting the luminescence irradiation beam(s), if present, from the substrate 8.

The detection system 100 in one embodiment comprises a first optical scanning means 202 comprising an optical deflector 5 for varying the angle of incidence of the excitation irradiation beam on the first focusing element 7. The latter typically results in a shift of the focus position of the excitation irradiation in the focal plane of the lens, resulting in a scanning motion of the excitation irradiation beam on the substrate 8. In other words, the optical scanning means 202 is adapted for scanning the excitation irradiation beam(s) over the substrate 8. The optical deflector 5 may be a mirror e.g. a plane, spherical or aspherical mirror. The optical deflector 5 may be interconnected to one or more control devices 104 for adjusting the orientation of the optical deflector 5 relative to the irradiation unit 102. Such a control device may comprise a mechanical part such as e.g. a servo system, and may comprise an electronic part for driving the servo system. The electronic part furthermore may provide information to a detection system in order to synchronize excitation and detection in the detection system.

Fig. 2 illustrates the principle of beam deflection used in a detection system according to an embodiment of the present invention. Fig. 2 shows two different states of the deflector 5 with their corresponding beam path, i.e. a first one shown with full lines, a second one shown with dotted lines. It can be seen that for varying incidence angle CC of the beam on the first focusing element 7, e.g. an objective lens, the focus will remain in the same plane P but is shifted away from the optical axis A of the focusing element. Changing the angle CC under which the beam enters the focusing element 7 thus results in a shift in the focal position on the substrate 8. The change in the position of the focus for a given change in angle depends on the focal length f of the focusing element 7. Fig. 3 shows the change in the position of the focus when the beam is deflected over an angle cc with respect to the optical axis A of the focusing element 7. The resulting shift in the focal position Δ is given by:

Δ = f tan(cc), where f is the focal length of the focusing element 7. For example, for a conventional lens with a focal length of about 2.75 mm a change in the incoming angle of 1° will result in a shift of the focus over 50 μm. This means that only minimal deflections are needed to scan the beam over 100 μm, which typically may be an appropriate range for performing beam scanning, especially when used in combination with at least one other beam scanning motion. For the above example variations of less than 1° are sufficient. Typically, in order to obtain a scanning motion of the excitation beam on the substrate 8, the angle of deflection of the excitation beam is varied during operation. The latter can be performed stepwise or substantially continuously. A varying angle of incidence on the focusing element 7 may be obtained by providing a relative movement between the deflector 5 and the focusing element 7. The deflector 5 may be moved with respect to the focusing element 7, the focusing element 7 may be moved with respect to the deflector 5 or both elements may be moved whereby the angle of an incident beam on the focusing element 7 is varied. Providing beam deflection that is variable in time may be performed using any suitable way. The scanning may be performed by rotating or translating a mirror resulting in a variation of the deflection angle for an excitation irradiation beam incident on the optical deflector 5. In a preferred embodiment, scanning may be performed by rotating a mirror or deflector. For example, one may use a deflector, e.g. polygon mirror, that rotates at high speed, as is e.g. used in optical tape systems. Polygon mirrors can be very fast but usually have a deflection range of more than 20° which makes them less suitable if a deflection range of not more than 1° is aimed for. Another example is to use a small curved mirror that is translated back and forth. The curvature introduced over the cross-section of the beam may then e.g. compensated for, e.g. using a phase plate. Scanners are available that can operate at a frequency of up to 16 kHz. Since we would be scanning both on the forward as well as on the backward stroke of 100 μm this would mean that the achieved scan speed would be 3.2 m/s, adequate for the envisioned application.

For realistic lenses a shift away from the optical axis will result in a reduction of the quality of the focus and a shift of the focal plane. Fig. 4 shows how the cross-section of the spot changes when the beam enters a conventional lens, in the present example an SD6 1 lens, under an angle. For the particular example provided, it can be seen that the width of the spot increases, however for angles below 0.7° the width only increases from 0.44 μm to 0.55 μm, this should be adequate for most applications. For this lens an angle of 0.7° corresponds to a shift of the focus by approximately 50 μm. This shows that scanning the beam over a total of approximately 100 μm, i.e. approximately 50 μm in both the forward and the backward direction, is feasible with current conventional lenses. It is however preferred that under all angles the whole lens is filled such that the full NA is used. Since the angles needed are rather small (0.7°) it is enough to just place the optical deflector close to the objective (< 10 mm) in that case the change in the angle only results in a minor shift (0.1 mm) of the beam. As long as the beam diameter is slightly larger than the diameter of the objective the whole NA will be available for all the different angles. Irradiation deflection patterns on the substrate 8 may be periodic, harmonic or of other complex structure. The first optical scanning means 202 within the detection system 100 utilizes an optical deflector 5 that may be controllably rotated, translated, tilted or oscillated. The optical deflector 5 may be rotated about an axis of rotation to any angular position between 0° and 360°. However, the optical deflector 5 is preferably rotated over an angle of rotation of not more than 3°, preferably not more than 2°, even more preferably not more than 1°. The optical deflector 5 may be tilted in any one of a number of planes passing through the intersection point of the axis of oscillation and axis of rotation. Furthermore, the optical deflector 5 may be oscillated between first and second angular positions or tilted to a fixed angle relative the axis of oscillation, in conjunction with controlled movement around the axis of rotation. In this regard, an optical scanning means 202 is provided wherein the optical deflector 5 has multiple degrees of freedom allowing for a plurality of beam deflection applications. The scanning motion of the irradiation beam may in principle be according to any or a combination of any direction in the 2-dimensional plane of the substrate 8. In a preferred embodiment, the beam deflection such that the deflection generates scanning motion of the excitation irradiation beam in one direction. It is to be noted that the above mentioned beam deflection can also be provided by the controlled movement of the focusing element 7 with respect to the optical deflector 5.

The scanning motion of the irradiation beam caused by deflection of the excitation irradiation beam with respect to the optical axis of the focusing element 7 may be performed at relatively high speed. Typically a scanning speed of e.g. about 4 m/s may be obtained. It is an advantage of detection systems according to the present invention that simple optics may be used. In embodiments according to the present invention, typically the first focusing element 7 is the only focusing element on the irradiation path between the optical deflector 5 and the substrate 8. In other words, only a single focusing element 7 may be present on the irradiation path between the optical deflector 5 and the substrate 8. As mentioned above, in particular embodiments, the present invention furthermore comprises at least one second scanning means 204 for providing at least a second scanning motion of the excitation irradiation beam with respect to the sample. Whereas the scanning by beam deflection varying the angle of incidence with respect to the focusing element 7 may e.g. result in a small strip of approximately 100 x 0.5 μm², the use of a second scanning motion of the excitation beam allows measuring a larger area. Typically, the latter requires that in the second scanning motion of the excitation irradiation beam, the position of the substrate 8 is varied with respect to the focusing element 7. The latter can be obtained in a number of ways. E.g. in a particular embodiment, the whole substrate can be moved back and forth. This embodiment, however, might not always be preferred, especially when there are connections between the detection system/substrate and the fixed world e.g. when fluidic or electronic functions are incorporated in the detection system/substrate. Another particular embodiment enables the incorporation of all the optics in a single package and the movement of this package with respect to the substrate 8. This might however not always be feasible since the optics needed to detect the fluorescence can be rather bulky. Another example is to use a split beam path, where most of the optics remains on the fixed world and only part of the optics, e.g. the focusing element 7 and the deflector 5 are moved with respect to the substrate 8. A particular example of a split optics embodiment according to the present invention will be described in more below. In order to scan a typical appropriate area for molecular diagnostics detection purposes, e.g. an 1x1 cm² area, the focusing element 7 typically is to be moved in two distinct directions. In conclusion, for the at least one second scanning motion either the substrate may be moved with respect to the system, the system or part thereof may be moved with respect to the substrate or the substrate and the system may move relatively with respect to each other.

The overall scanning may preferably occur along Cartesian coordinates, e.g. along line segments, e.g. finite line segments, or a combination of line segments, e.g. finite line segments, in a Cartesian coordinate system. Such scanning typically includes for example XY-scanning, raster scanning, XY-scanning along line segments, e.g. finite line segments, with stochastically chosen directions. Although not excluded from the present invention, it is thus less preferable to perform scanning in a relative rotational movement, as the latter typically results in the need for scanning very large areas. However other scanning techniques may be used then rastering or other linear motions. Scanning may be done by following a space filling curve of which rastering is on example. In a space filling curve each point in an array is traversed once. An example of such a curve is a Peano curve. Also partial scanning of the surface may be performed.

Typically the detection system additionally may comprise a detection unit 108 for detecting and quantifying luminescence responses, obtained by collecting luminescence irradiation beams from the substrate 8. Such a detection unit 108 may comprise at least one detector 106, such as a photodetector, a charged coupled device (CCD), a charged injection device (CID), a complementary metal-oxide semi-conductor (CMOS), a photomultiplier tube, an avalanche photodiode, a solid state optical detection device, or a video camera. The at least one detector 106 may be a number of detectors, adapted for detecting different luminescence irradiation beams collected from the substrate 8. The at least one detector 106 may be a pixelated detector or a line of multiple single-pixel detectors. Such a detector 106 may e.g. be a charge coupled device (CCD) detector or a CID, a row of photon tube multipliers, a row of avalanche photodiodes or any other irradiation detector that comprises an array of individual detection pixels. The width of the at least one detector 106 or, in case pixelated detectors are used, of the detector elements of the at least one detector typically preferably may be such that detection may occur for spatially distinctive areas on the substrate, whereby the spatially distinctive areas are such that approximately always maximally one occupied binding site is present within the area detected by a single pixel during examination. The latter allows a way of digital detection, i.e. allowing to detect whether or not a given binding site is occupied or not. A typical area detected by a single pixel may be sized between 0.01 μm² and 100 μm², preferably between 0.1 μm² and 25 μm², such as e.g. 1 μm².

It is to be noticed that when the focus of the excitation beam is off center, the luminescence irradiation beam, e.g. fluorescence, that is generated as response still may be collected by the focusing element 7. The luminescence centers can be seen as point sources emitting in all directions thus generated luminescence will reach the focusing element 7. The resulting luminescence beam typically will make the same angle with respect to the optical axis of the focusing element 7. The latter is counteracted when the luminescence beam is deflected by the same deflector 5 as used to shift the excitation beam. In a preferred embodiment, the luminescence irradiation beam thus will be deflected using the same deflector 5 resulting in a luminescence irradiation beam that is substantially parallel with the excitation irradiation beam before deflection. When confocal detection is used, the detected fluorescence is focused on a small pinhole to remove out-of-focus contributions, but the beam orientation is similar as without confocal detection.

In a particular embodiment, an evaluation unit 111 may be provided for determining a concentration or distribution of luminescence sites and/or for statistical processing of the obtained detection results, e.g. to correlate two different measurements for checking whether or not lightly bounded luminescence particles have influenced the detection. Such an evaluation unit 111 may comprise a processing means 109, such as e.g. a microprocessor, and/or a memory component for storing the obtained and/or processed evaluation information. Furthermore typical input/output means may be present. The evaluation unit 111 may be controlled using appropriate software or dedicated hardware processing means for executing the evaluation steps. The evaluation means 111 thus may be implemented in any suitable manner, e.g. dedicated hardware or a suitably programmed computer, microcontroller or embedded processor such as a microprocessor, programmable gate array such as a PAL, PLA or FPGA, or similar. The results may be displayed on any suitable output means 110 such as a visual display unit, plotter, printer, etc. The evaluation means 111 may also have a connection to a local area or wide area network for transmission of the results to a remote location.

Other optional components of the detection system 100 may be a focus controlling means 112, e.g. a focusing servo system, and a tracking controlling means 113, e.g. a tracking servo system, for controlling the focusing of the excitation irradiation beam and for controlling the position of the excitation irradiation beam e.g. on specific tracks. The focus controlling means 112 may be based on different focusing methods, such as for example, but not limited to Foucault wedge focusing. Typically part of the excitation irradiation has to be reflected by the substrate 8 in order to be able to generate a useful signal to provide the auto-focus function. This part of the excitation irradiation reflected by the substrate 8 will leave the focusing element running parallel to the incoming excitation irradiation. In a preferred embodiment of the present invention it is ensured that the part of the excitation irradiation reflected by the substrate 8 is again reflected by the deflector 5, thus avoiding a substantial shift of the irradiation used for auto-focus and keeping the focus position on an auxiliary detector of the part of the excitation irradiation reflected by the substrate 8 constant while the excitation irradiation beam itself shifts over the substrate 8.

Small shifts of the part of the excitation irradiation beam reflected by the substrate 8 and used for auto-focus may be negligible, especially if the optics used have a large enough diameter to accommodate such small shifts. A tracking controlling means 113 typically may be used for controlling tracking, which is needed for obtaining accurate spatial detection. Such systems may comprise actuators. The detection system 100 furthermore may comprise high frequency controlling means and an auxiliary detector such as e.g. a charge coupled device (CCD), which may be used for optimizing the tracking and focusing functions. The tracking controlling means 113 may furthermore comprise a position correcting means adapted for correcting the position the excitation irradiation beam. Such a position correcting means may comprise means for altering the position of the irradiation unit itself or may provide correction signals to provide a correction to the position of the irradiation unit 102 or e.g. to the deflector 5 or corresponding first optical scanning means 202 or to another optical scanning means 204. The correction signals then typically allow the means for altering the position of the detection system 100 to redirect the irradiation beam to the appropriate region of the substrate 8, i.e. the region to be scanned.

The detection system 100 may furthermore comprise, besides the first focusing element 7 which typically is the objective element used for focusing the excitation irradiation beam on the substrate 8, other optical elements 6 such as e.g. a beam splitters such as polarization selective or dichroic beam splitters, a polarizer, a retarder, dichroic filters, lenses and/or mirrors for directing light from the irradiation unit 102 towards and from the substrate 8, etc. Such additional components especially are introduced if a number of irradiation sources are present adapted for each providing an excitation irradiation beam having a different predetermined wavelength or wavelength range and/or if a number of different luminescence irradiation beams are to be detected using different detectors. A dichroic filter or a dichroic beam splitter may be used for blocking unwanted excitation radiation to be incident on the at least one detector element.

Although a disc shaped substrate may be used, in a preferred embodiment the detection system is adapted for using substrates having a limited substrate area, e.g. of around 1x1 cm² as this typically suffices for the most applications. The substrate may e.g. be credit card format shaped. The first aspect of the present invention will now be illustrated by a number of particular embodiments and examples, the invention not being limited thereto. A first particular embodiment according to the first aspect describes a detection system as described above for the first aspect, wherein the light path of the irradiation beams travels as depicted in Fig. 5. The excitation irradiation beam from an irradiation unit, in the present example comprising a laser diode 1, i.e. laser light, typically may be collimated and shaped by a focusing element 2. The polarization of the excitation irradiation beam may be such that it will be reflected by the polarizing beam splitter 3. The excitation irradiation beam may be further reflected by a dichroic beam splitter 4 that is reflective for the excitation irradiation beam e.g. laser light but transparent for the luminescence irradiation beam e.g. fluorescent light. The excitation irradiation beam then impinges on the optical deflector e.g. a scanning mirror 5 that can be rotated over a small angle CC to shift the excitation irradiation beam. The deflected excitation irradiation beam then may pass through a quarter wavelength plate 6 to make the laser light circularly polarized. The focusing element 7 is used to focus the excitation irradiation beam on the substrate 8 containing e.g. fluorescent molecules. Part of the excitation light will be reflected backwards and again be collimated by the focusing element 7 and may pass again through the quarter wavelength plate 6 making the light polarized perpendicular to the original excitation irradiation. The optical deflector 5 and the dichroic beam splitter 4 both reflect the excitation irradiation. The polarizing beam splitter 3, however, transmits the rotated polarization such that it can be focused by a lens 14 through a wedge prism 15 onto a split detector 16. The luminescence irradiation generated e.g. by molecules present on the substrate 8 typically may be collected by the focusing element 7 and collimated. If the optical deflector 5 had induced an angle in the incoming beam that resulted in a shift of the position of the focus, the collected luminescence irradiation will leave the focusing element 7 under the same angle. The optical deflector will correct this angle and the resulting luminescence irradiation beam will, after leaving the optical deflector, run parallel to the incoming excitation irradiation beam. The dichroic mirror 4 typically may transmit the luminescence irradiation beam and a long pass filter 9 can be used to reject any remaining excitation light. A lens 12 may be used to focus the luminescence irradiation on a pinhole 11 placed in front of the detector 13 to ensure confocal detection. Since a change in the angle of the optical deflector will not result in a shift of the luminescence irradiation the lens 12 will always focus the luminescence irradiation on the pinhole 11 while the excitation irradiation beam is scanned over the substrate 8.

In a second particular embodiment, the present invention relates to a detection system as described above, possibly, but not necessarily, according to the first particular embodiment, wherein at least a second scanning means 204 is present, introduced based on split optics. In other words, according to the second particular embodiment, the scanning of a substrate area is obtained with a detection system using split optics. The latter has the advantage that connections between the substrate and the fixed world can be relatively easy made and that a less bulky system may be obtained. In this embodiment using split optics, most of the optics remains on the fixed world, i.e. fixed with respect to the substrate, and only a small part of the optics, e.g. the single focusing element and the optical deflector are moved with respect to the substrate. The single focusing element and the optical deflector therefore may be moveable with respect to the major part of the optical system. In other words, the single focusing element and the optical deflector may be introduced on at least a second scanning means 204, not comprising the remaining components of the system, such as other optical components like beam splitters for splitting the excitation irradiation and the luminescence irradiation. Typically, in order to scan an appropriate area of a substrate, e.g. about 1x1 cm², a scanning motion of the single focusing element may be needed in two directions. Part of a detection system having split optics is depicted in Fig. 6a and Fig. 6b. By way of example, the detection system may be a detection system as described in the first particular embodiment, wherein the light path as described in Fig. 5 is split after the dichroic mirror 4. The latter may be obtained by positioning the optical deflector 5, e.g. a scanning mirror, and the focusing element 7, e.g. an objective, on a sliding means 20 comprising a slider 22 and a slider holder 24. Fig. 6a and Fig. 6B depict a top view respectively side view of one possible implementation of split optics for moving the focusing element 7 with respect to the substrate 8. In the example shown in Fig. 6a and Fig. 6b, the optical deflector 5 and the focusing element 7 are introduced on the slider 22 which is moveable with respect to the slider holder 24 in a first direction, whereas the sliding means 20, comprising the slider 22 and the slider holder 24, may be moveable in a second direction. In other words, the slider 22 which is moveable with respect to the slider holder 24 may be part of or form the second scanning means 204 and the sliding means 20 being moveable with respect to other components may be part of or form a third scanning means. In operation, an excitation irradiation beam is guided by the fixed optics towards the sliding means 20 in a first direction. The latter may be received by a receiving optical element on the sliding means 20. In the present exemplary scanning system 200 shown in Fig. 6a, the embodiment not being limited thereto, an excitation irradiation beam from the dichroic mirror (not shown) runs parallel to the second direction, e.g. the Y-axis of a reference system indicated in Fig. 6a. A receiving optical element, e.g. a mirror 21, that is placed on the sliding means 20, sends the light towards the optical deflector 5. The optical deflector 5 and the focusing element 7 are incorporated on the slider 22 and both components move together with the slider 22 in the first direction, e.g. the X direction of a reference system indicated in Fig. 6a. The slider 22 thereby moves relative to the slider holder 24. The optical deflector 5 itself furthermore is subjected to an additional movement, it is a tilting, shifting or rotational movement, with respect to the focusing element 7, thus providing variation of the angle of incidence of the excitation irradiation beam on the focusing element 7. The latter is described in more detail above and provides movement of the focus point in the focal plane of the focusing element 7, i.e. on the substrate, in the first direction. Once a region of the substrate is scanned based on the combined motion of the optical deflector 5 with respect to the focusing element 7 and of the optical deflector 5 and focusing element 7 with respect to the slider holder, a neighboring region may be scanned by shifting the sliding means 20 incorporating these components to the neighboring region. This shifting movement allows to increase the scanned area of the substrate and typically may be performed as the width of the scanned area would otherwise be limited to the scanning width obtained by varying the angle of incidence of the excitation irradiation beam on the focusing element 7. A further movement which may be performed is the movement of the focusing element 7 with respect to the substrate in a third direction e.g. substantially perpendicular to the substrate such as e.g. the Z direction of a reference system indicated in Fig. 6b, to ensure proper focusing on the substrate (not shown). The reflected excitation irradiation and the luminescence irradiation follow the same irradiation path back to the fixed world.

In conclusion, in the present embodiment a plurality of motions may be performed using different actuators. In the present example, a first optical scanning means provides a Z' motion by actuating the optical deflector 5, thus providing a relative movement of the optical deflector with respect to the focusing element 7. The resulting scanning motion, as described in more detail above, may be a continuous motion and may be a fast motion, as it is based on optical deflection of the beam with respect to the angle of incidence on the focusing element 7. An optional second scanning means may provide a second Z" motion of the excitation irradiation beam by actuating the slider 22 thus moving the optical deflector 5 and the single focusing element 7 with respect to the slider holder 24. The resulting second scanning motion Z" typically may be a motion which is substantially perpendicular to the Z' motion. The Z" motion may be a continuous motion. The Z" motion furthermore may be substantially slower than the Z' motion. An optional third scanning means may provide a third Z"' motion of the excitation irradiation beam by providing a movement of the sliding means 20 comprising the slider 22 and slider holder 24, thus providing a movement of the optical deflector 5 and the single focusing element 7 positioned thereon. The resulting third Z'" scanning motion typically may be a discontinuous motion of the slider holder to start scanning of a new band. Fig. 7 depicts the total scanning pattern used by combining the above cited three motions Z', Z", and Z'". The velocities of the different scanning motions may be tuned such that the whole sample is scanned. The present embodiment in this way provides detection with a reduced measurement time by providing bi-directional scanning, whereby both interconnecting the substrate with the fixed world are still relatively easy and the manufacturing of the detection system is easily feasible.

According to a second aspect, the present invention also relates to a control system for controlling a scanning motion in a detection system for detecting luminescence sites on a substrate. The control system may be adapted for controlling a detection system for detecting luminescence sites on a substrate. The control system may be especially suitable for controlling a detection system as described in the first aspect. The control system typically comprises means for controlling the movement of a excitation beam with respect to a substrate. The motion may be that of a space filling curve such as a raster scan or a Peano curve scan, as an example. The motion also may be such that only part of the surface is scanned. In an embodiment of the present invention the controller may include an optical deflector driver for controlling movement of an optical deflector of the detection system, i.e. for controlling the first optical scanning means, adapted for deflecting at least one excitation irradiation beam. In this way, the angle of incidence of the at least one excitation irradiation beam on a single focusing element (7) on an irradiation path between the optical deflector and the substrate is varied. This variation provides a first scanning motion of the at least one excitation irradiation beam over the substrate. Typically such a control system 300, may be an electronic control system of at least one optical scanning system 200 in the detection system. The control system 300 furthermore may control the irradiation unit for controlling generation of the at least one excitation irradiation beam. Depending on the number of scanning systems provided in the detection system, the control system 300 may provide drive signals for movement of the optical deflector with respect to the focusing element 7, and if present, drive signals for movement of a slider and/or sliding means for movement of the optical deflector and the focusing element with respect to the substrate. Furthermore the control system 300 also may provide drive signals for movement of the focusing element 7 with respect to the substrate in a direction perpendicular to the substrate. Furthermore the control system 300 also may provide drive signals for movement of the substrate with respect to the excitation beam, either in addition or alternatively. The relative movement of the substrate and the excitation beam may be sequentially or simultaneously in more than one direction.

Fig. 8 is a block diagram of the electronic control system 300 for a scanning system 200, which is one example of a control system for use with an optical deflector 5 in accordance with the present invention. The control system 300 typically may comprise a deflector driver 51 for providing control signals to a deflector drive motor 52 for driving the conveying mechanism of the optical deflector 5. The control system optionally may comprise a slider driver 61 for providing control signals to a slider drive motor 62 for driving the conveying mechanism of the slider 22, i.e. thus creating movement of the slider 22 with respect to the slider holder 24. In addition, the control system optionally also may comprise a sliding means driver 71 for providing control signals to a sliding means driver motor 72 for driving the conveying mechanism of the sliding means 20, thus creating movement of the sliding means 20 with respect to the substrate. A focusing element driver 81 for a focusing element drive motor 72 may optionally be present for driving the conveying mechanism of the focusing element 7 with respect to the substrate for optimizing the distance between the focusing element and the substrate, thus providing proper focusing. The control system typically may comprise a data store 48 for storing parameters for controlling the scanning operation. The data store 48 may comprise any suitable device for storing digital data as known to the skilled person, e.g. a register or set of registers, a memory device such as RAM, EPROM or solid state memory. The control system may be a computing device, e.g. microprocessor, for instance it may be a micro-controller. In particular, it may include a programmable scan controller, for instance a programmable digital logic device such as a Programmable Array Logic (PAL), a Programmable Logic Array, a Programmable Gate Array, especially a Field Programmable Gate Array (FPGA). The use of an FPGA allows subsequent programming of the detection device or a scanning device thereof, e.g. by downloading the required settings of the FPGA. The control system furthermore typically may comprise an input/output means 46 for inputting information about the scan path to be followed. The control system furthermore may be adapted for receiving information from the tracking and/or focusing means which may be used as input for adapting the control signals to be provided. The control system also may be adapted for providing information to the detection unit or a control or evaluation unit thereof, to provide information on which portion of the substrate is scanned at that moment.

In particular, the control system may be adapted for controlling a high speed bi-directional scanning system within an optical detection system 100 for scanning a substrate 8 with an excitation irradiation beam. The control system 300 may comprise software or hardware means for controlling scanning of the substrate 8 using a first (Z') and possibly a second (Z") and third (Z'") motion, and a sequence in which the scanning using the motion(s) is carried out, i.e. the sequences of scanning enabling to complete the scanning of at least part of the substrate 8. The control system is especially suitable for controlling the scanning motions as described in more detail in the first aspect of the present invention. The present invention also includes that components of optical scanning system 200 are machine settable, e.g. control system 300 sets the parameters for scanning, e.g. using three motions Z', Z", Z'", e.g. in accordance with an optimized algorithm e.g. for tuning the velocities of the different motions to ensure that the whole substrate 8 is scanned. In accordance with embodiments of the present invention a bi-directional beam scanner in accordance with the present invention may be programmed. Accordingly, the present invention includes a computer program product which provides the functionality of any of the methods according to the present invention when executed on a computing device. Further, the present invention includes a data carrier such as a CD-ROM or a diskette which stores the computer product in a machine readable form and which executes at least one of the methods of the invention when the program stored on the data carrier is executed on a computing device. Nowadays, such software is often offered on the Internet or a company Intranet for downloading, hence the present invention includes transmitting the detection- related or scanning-related computer product according to the present invention over a local or wide area network.

According to a third aspect, the present invention provides a method for detecting luminescence sites on a substrate 8. The detection method thereby is adapted for scanning a substrate 8 with at least one excitation irradiation beam in an efficient way. The method comprises generating at least one excitation irradiation beam for exciting luminescence sites on the substrate 8, deflecting the at least one excitation irradiation beam with an optical deflector on a single focusing element on the irradiation path between the optical deflector and the substrate and focusing the excitation irradiation beam on the substrate 8. Deflecting thereby typically comprises varying an angle of incidence of the at least one excitation irradiation beam on the focusing element 7 with respect to an optical axis of the single focusing element 7, thus providing a first scanning motion of the at least one excitation irradiation beam over the substrate 8. It is to be noticed that only a single focusing element is used for focusing the deflected excitation irradiation beam on the substrate. In other words, no further focusing elements are present on the irradiation path between the deflector and the substrate. The at least one excitation irradiation beam may be a plurality of excitation irradiation beams, such as two, three or more excitation irradiation beams allowing to excite different labels. The plurality of excitation irradiation beams may be generated simultaneously or the excitation irradiation beam having the most appropriate excitation behavior, e.g. the most appropriate excitation wavelength or wavelength range, may be selected for use. The method furthermore typically may comprise, collecting a luminescence irradiation beam generated from the luminescence sites on the substrate 8. The collecting thereby may comprise detecting the luminescence via the same focusing element and using the same deflector, thus allowing to obtain a beam that is substantially parallel to the excitation irradiation beam. The method according to the present invention further may comprise providing at least one further scanning motion by moving the focusing element 7 and the deflector 5 with respect to the substrate. This movement may be such that the focusing element 7 and the deflector 5 may be moved, e.g. on a slider, with respect to other components, e.g. optical components, of the detection system such as e.g. with respect to a beam splitter for splitting reflected excitation irradiation and luminescence irradiation, with respect to the irradiation source, with respect to the detector unit, etc. The at least one further scanning motion may be a second scanning motion in a substantially different direction than the scanning motion obtained by deflecting the excitation irradiation beam. The at least one further scanning motion furthermore may additionally be a third scanning motion, e.g. a discontinuous motion, in substantially the same direction as the direction of the scanning motion obtained by deflecting the excitation irradiation beam. Typically the second scanning motion may be substantially slower than the first scanning motion, i.e. the first optical scanning motion may be at least twice as fast, preferably at least ten times at fast as the second scanning motion. The second scanning motion may be provided by moving a sliding means comprising the focusing element 7 and the deflector 5 with respect to the remaining optics of the detection system and with respect to the substrate. The latter allows detection using a split optics embodiment, having the advantage that the whole optical system of the detection system is not to be moved with respect to the substrate while interconnecting the substrate to the external fixed world, e.g. for cleaning or heating or providing sample, still is feasible.

The applications of the present invention are in the field of molecular diagnostics: clinical diagnostics, point-of-care diagnostics, advanced bio molecular diagnostic research, biosensors, gene and protein expression arrays, environmental sensors, food quality sensors, etc, but are not limited thereto. The present invention allows a large number of useful bioassays to be run in a cost-effective package. Multiple chromogenic labels may also be used in micro-array technology, flow cytometry, detection based on fluorescence resonance energy transfer (FRET) which occurs due to the interaction between the electronic excited states of two chromogenic dye molecules, molecular beacons based detection technology such as e.g. real-time nucleic acid detection and real-time PCR quantification, surface enhanced detection techniques such as surface-enhanced Raman spectroscopy (SERS), surface-enhanced fluorescence (SEF) or surface-enhanced resonance Raman spectroscopy (SERRS), microfluidic detection, etc. In preferred embodiments, the detection system of the present invention is an epi-fluorescence biosensor meaning that the light is incident on the surface from above, but it could also be a transmission biosensor, meaning that the light is incident from below and transmitted through the microarray.

As indicated above, embodiments of the present invention provide or use an optical scanning system for detection methods which involve beam scanning. In today's high through-put molecular diagnostics there is a need to detect fluorescent labels with high speed and the present invention provides a cost effective solution thereto. The focal point shifts generated by the beam deflection (fast scanning in the Z' direction) combined with the scanning motion generated by the slider (slow scanning in the Z" direction) allows fast detection of individual labels is the goal. An advantage of particular embodiments of the present invention is that a surface can be scanned and fluorescent labels present on this surface can be detected with high speed. It is an advantage of particular embodiments of the present invention that time- effective detection is obtained, e.g. due to the provision of at least two scanning motions in an orchestrated way. Other arrangements for accomplishing the objectives of high speed bidirectional beam scanning embodying the invention will be obvious for those skilled in the art. It is to be understood that although preferred embodiments, specific constructions and configurations, as well as materials, have been discussed herein for devices according to the present invention, various changes or modifications in form and detail may be made without departing from the scope and spirit of this invention.

Claims

CLAIMS:

1. A detection system (100) for detecting luminescence sites on a substrate (8), the detection system (100) comprising: an irradiation unit for generating at least one excitation irradiation beam for exciting luminescence sites on the substrate (8), - a first optical scanning means (200) comprising an optical deflector (5) for deflecting said at least one excitation irradiation beam, and a single focusing element (7) on the irradiation path between the optical deflector (5) and the substrate (8), the single focusing element (7) having an optical axis and being adapted for receiving the deflected at least one excitation irradiation beam and for focusing said deflected at least one excitation irradiation beam on the substrate (8), said optical deflector adapted for varying an angle of incidence of said at least one excitation irradiation beam on said single focusing element (7) with respect to an optical axis of said single focusing element (7), said varying providing a first scanning motion (Z') of the at least one excitation irradiation beam over said substrate (8).

2. A detection system (100) according to the previous claim, wherein the optical deflector (5) is moveable with respect to said single focusing element (7).

3. A detection system (100) according to claim 1, wherein said detection system (100) furthermore comprises a second scanning means (202) adapted for providing a relative motion between said single focusing element (7) and said substrate (8) thus providing a second scanning motion (Z") of said at least one excitation irradiation beam over said substrate (8) simultaneously with said first scanning motion (Z').

4. A detection system (100) according to claim 3, wherein said second scanning means (202) comprises a slider (22) whereon the focusing element (7) and the optical deflector (5) are mounted, said slider (22) being moveable with respect to said substrate (8) and other components of said detection system (100) for generating said second scanning motion (Z").

5. A detection system (100) according to claim 3, wherein said first optical scanning motion is substantially faster than said second scanning motion.

6. A detection system (100) according to the previous claim, wherein said slider

(22) is part of a sliding means (20) which is moveable with respect to a substrate and other components of the detection system according to a third scanning motion (Z'") in a direction that is substantially parallel to said first scanning motion.

7. A detection system (100) according to the previous claim, wherein the third scanning motion (Z'") is a discontinuous scanning motion.

8. A detection system (100) according to the previous claim, wherein the angle of incidence CC is smaller than 1°.

9. A detection system (100) according to claim 1 wherein the distance between the substrate (8) and the focusing element (7) can be changed in order to focus the at least one excitation irradiation beam.

10. A detection system (100) according to claim 1, wherein said detection system

(100) furthermore comprises a detection unit (108) having at least a detector element (106) and said detection system (100) is adapted for guiding luminescence irradiation beams via said focusing element (7) and said optical deflector (5) to said at least a detector element (106).

11. A detection system (100) according to claim 1, wherein the optical deflector

(5) is a reflective device being any of a plane mirror, a spherical mirror, or an aspherical mirror.

12. A detection system (100) according to claim 1, wherein the optical deflector

(5) is a reflective device being a rotating mirror.

13. A detection system (100) according to claim 1, wherein the optical deflector

(5) is a reflective device translated with a variable linear velocity v in an alternating forward and backward direction.

14. A control system (300) for controlling a detection system for detecting luminescence sites on a substrate (8), the control system (300) comprising an optical deflector driver (51) for controlling movement of an optical deflector (5) adapted for deflecting at least one excitation irradiation beam thus varying an angle of incidence of said at least one excitation irradiation beam on a single focusing element (7) on an irradiation path between the optical deflector (5) and the substrate (8), said varying providing a first scanning motion (Z') of the at least one excitation irradiation beam over said substrate (8).

15. A method for detecting luminescence sites on a substrate (8), the method comprising generating at least one excitation irradiation beam for exciting luminescence sites on the substrate (8), deflecting the at least one excitation irradiation beam with a deflector on a single focusing element on the irradiation path between a deflector and the substrate, - focusing said at least one excitation irradiation beam on said substrate, said deflecting comprising varying an angle of incidence of said at least one excitation irradiation beam on the focusing element (7) with respect to an optical axis of the focusing element (7), said varying providing a first scanning motion (Z') of the at least one excitation irradiation beam over said substrate (8).