WO2011023593A1

WO2011023593A1 - Method of and apparatus for imaging a cellular sample

Info

Publication number: WO2011023593A1
Application number: PCT/EP2010/061961
Authority: WO
Inventors: Mathieu Ducros; Serge Charpak; Valentina Emiliani; Laurent Moreaux
Original assignee: INSERM (Institut National de la Santé et de la Recherche Médicale); Centre National De La Recherche Scientifique - Cnrs -
Priority date: 2009-08-24
Filing date: 2010-08-17
Publication date: 2011-03-03

Abstract

A method of imaging a cellular sample by multi-photon imaging, the method comprising: providing a multi-photonic excitation beam; splitting the multi-photonic excitation beam into a plurality of N sub-beams; modulating the amplitude of the plurality of N sub-beams to create N respective temporal modulation pattern; focusing the plurality of N modulated sub-beams to N respective spatially separated focal points P₁... P_N within the cellular sample; simultaneously detecting fluorescent signals emitted from the N focal points P₁... P_N; and demodulating the detected fluorescent signals so that fluorescence data relating to the respective focal points P₁...P_N may be obtained. An apparatus for imaging a cellular sample is also described.

Description

METHOD OF AND APPARATUS FOR IMAGING A CELLULAR SAMPLE

Field of the Invention

The present invention relates in general to a method and apparatus for imaging a cellular sample. Particularly, but not exclusively, the invention relates to a method of imaging a cellular sample by means of photonic excitation, for example by two-photon microscopy.

Background of the Invention

Photonic excitation is often used in the investigation of small target objects, such as biological tissues and chemical molecules which are too small to be seen by the unaided eye. In photonic excitation incident photons interact with the target object to emit a corresponding photon which is detected and used to generate an image of the target object.

The target object may be a cellular sample made up oi single or multiple biological cells, clusters populations or networks of such cells. The cells may be in a living or non-living state and be from a plant, animal, bacterial source, or the like. The investigation of such cellular samples may have applications such as screening of drug activity, diagnosis of disease state, research into cellular functions and transport properties including inter-cellular signalling in cell populations.

Fluorescence microscopy is a type of microscopy in which a fluorescent material is used to mark a specimen or objects of interest in the specimen. The fluorescent material is then illuminated with a wavelength of light providing an energy level sufficient to excite the fluorescent material to emit emission light. The image of the specimen is detected by collecting the emission light. Multi-photon imaging techniques such as two-photon microscopy are becoming increasingly used in neurophysiology studies. In two-photon microscopy an intrinsic fluorophore or a fluorescent dye attached to the target object is excited by a pair of incident photons causing the emission of a single fluorescence photon which can then be used to generate an image of the target object. Two-photon laser scanning microscopy was first described by Denk et al (Denk et al. (1990). "Two-photon laser scanning fluorescence microscopy." Science 248(4951): 73-6) and was rapidly applied in biology and mainly the field of neuroscience (See reviews such as Konig (2000). "Multiphoton microscopy in life sciences." J

Microsc 200(Pt 2): 83-104 , Garaschuk et al. (2006). "Optical monitoring of brain function in vivo: from neurons to networks." Pflugers Arch 453(3): 385-96 and Wilt et al. (2009). "Advances in light microscopy for neuroscience." Annu Rev Neurosci 32: 435-506. Multi-photon microscopy has several advantages over other fluorescence microscopy techniques. Near infra red wavelengths allow deeper imaging in biological tissues than UV or blue light that are strongly attenuated. Furthermore the fluorescence excitation is restricted to very small volumes near the microscope objective focal spot making multi-photon microscopy intrinsically optically resolved. Thus, wide field fluorescence collection is possible.

A challenging task in neurophysiology consists in recording activity simultaneously in a number of cells labelled with ion or voltage sensitive reporters. The simultaneous recording of electrical activity in a population of neurones in vivo is an essential step in the study of cerebral function. Two photon microscopy coupled with the use of fluorescent molecules provides micrometre spatial resolution enabling individual cells to be observed and identified, is non-invasive and enables neuronal activity to be visualised in vivo up to a depth of 500μm.

A drawback of conventional two-photon microscopes is the use of scanning mechanisms employing optical-mechanical devices such as mirrors mounted on galvanometers leading to a limited acquisition speed which does not allow electrical signals on a number of cells to be recorded simultaneously, and a limited imaging depth. Various attempts have been made to overcome these drawbacks. For example, Lillis, et al. (2008). "Two-photon imaging of spatially extended neuronal network dynamics with high temporal resolution." J Neurosci Methods 172(2): 178-84, describes a two-photon fluorescence mirror-based scanning imaging system, for monitoring the dynamics of spatially extended neuronal networks. Another approach consist in using acousto-optic deflectors to steer the laser beam very fast (less than 10 μs) between cells or cellular regions (See for example Otsu et al. (2008). Optical monitoring of neuronal activity at high frame rate with a digital random-access multiphoton (RAMP) microscope." J Neurosci Methods 173(2): 259-70 and Grewe et al. "High-speed in vivo calcium imaging reveals neuronal network activity with near-millisecond precision." Nat Methods 7(5): 399-405. Other techniques, such as for example that described in Nikolenko et al. (2008). "SLM Microscopy: Scanless Two-Photon Imaging and Photostimulation with Spatial Light Modulators." Front Neural Circuits 2: 5.

involve the use of array detectors such as CCD cameras. However the resulting increase in acquisition speed achieved by such techniques is detrimental to the image quality in terms of signal noise ratio, point spread function (PSF) or imaging depth.

Summary of the Invention

To better address one or more of the foregoing concerns, a first aspect of the invention provides a method of imaging a cellular sample in multi-photon imaging, the method comprising: providing a multi-photonic excitation beam; splitting the multi- photonic excitation beam into a plurality of N sub-beams;

applying a different amplitude modulation pattern on each sub beam (for example by modulating the amplitude of the plurality of N sub-beams to N respective frequencies fi...f_N) or with N different binary temporal patterns; focusing the plurality of N modulated sub-beams to N respective spatially separated focal points P₁... P_N within the cellular sample; simultaneously detecting fluorescent signals emitted from the N focal points P_I ...P_N; and demultiplexing the detected fluorescent signals so that fluorescence data relating to the respective focal points PI ...PN may be obtained. A second aspect of the invention provides an apparatus for imaging a cellular sample in multi-photon imaging, the apparatus comprising: a light source for providing a multi-photonic excitation beam; a beam divider for splitting the photon excitation beam into a plurality of N sub-beams; a modulator to apply a different amplitude modulation pattern to the plurality of N sub-beams (for example with N respective frequencies fi...fN or with N different binary temporal patterns); an objective for focusing the plurality of N modulated sub-beams to N respective spatially separated focal points Pi... P_N within the cellular sample; a photodetector for simultaneously detecting fluorescent signals emitted from the N focal points Pi ... P_N; and a demultiplexer for demultiplexing the detected

fluorescent signals; and a processor for processing the demodulated fluorescent signals to obtain fluorescence data relating to the respective focal points P_{1 ^} - PN-

The position of the recorded fluorescence signal is thus coded by a specific modulation pattern. This in turn reduces the need to move the laser beam by optomechanical techniques between the points of interest within the cellular sample since the fluorescence signals from a number of points within the sample can be measured simultaneously. Consequently since the laser beam does not need to be scanned along the cellular sample to image a number of points of interest the imaging acquisition speed may be increased and the time resolution improved.

In embodiments of the invention:

the step of simultaneously detecting fluorescent signals may include collecting a combined fluorescence signal composed of fluorescent signals emitted from the N spatially separated focal points PI ... PN; and wherein

fluorescence data relating to the respective focal points Pi ... P_N is obtained by extracting the signal components of the combined fluorescence signals .

The temporal patterns applied to the N sub-beams can be any set of signals that permit de-multiplexing of signals that are summed onto the

photodetector. For example, the temporal patterns could be sinusoidal

modulations at frequencies U to fN that may be separated in the frequency domain. They could also be binary patterns such as complementary Golay sequences: the particular correlation properties of the Golay complementary sequences allow demultiplexing in the temporal domain. (See Golay (1949). "Multi-Slit Spectrometry." Journal of the Optical Society of America 39(6): 437-444)

The N temporal patterns may be applied to each of the N focal points using the technique called focal modulation microscopy (Chen, N., C. H. Wong, et al. (2008). "Focal modulation microscopy." Opt Express 16(23): 18764-9), i.e.

applying a rapid spatial phase modulation to one half of each sub beam. In the implementation where sub beams are modulated at frequencies U to f_fM, the amplitude of the power spectrum may be measured at each frequency fi to fN N fluorescent signals corresponding to the N focal points P₁...P_N may be displayed

Parts of the methods according to embodiments of the invention may be computer implemented. Parts of the methods of embodiments may be implemented in software on a programmable apparatus. Methods may be implemented in a combination of software and hardware.

Since parts of embodiments of the present invention can be implemented in software, the parts of embodiments of the present invention can be embodied as computer readable code for provision to a programmable apparatus on any suitable carrier medium. A tangible carrier medium may comprise a storage medium such as a floppy disk, a CD-ROM, a hard disk drive, a magnetic tape device or a solid state memory device and the like. A transient carrier medium may include a signal such as an electrical signal, an electronic signal, an optical signal, an acoustic signal, a magnetic signal or an electromagnetic signal, e.g. a microwave or RF signal.

Brief Description of the Drawings Embodiments of the invention will now be described, by way of example only, and with reference to the following drawings in which:-

Figure 1 is a schematic diagram of an apparatus for imaging a cellular sample according to a general embodiment of the invention;

Figure 2 is a schematic diagram of an apparatus for imaging a cellular sample according to a particular embodiment of the invention; and Figure 3 schematically illustrates a method and graphically illustrates results obtained according to the particular embodiment of the invention.

Figure 4 schematically illustrates an apparatus for imaging cellular sample according to a particular embodiment of the invention

Figure 5 graphically illustrates results obtained using the apparatus described in Figure 4

Detailed description

An apparatus in a general form for imaging a cellular sample according to at least one embodiment of the invention will be described with reference to Figure 1. A two-photon microscopy apparatus comprises a pulsed laser excitation source L providing a laser beam 11 , and a laser beam divider BD for dividing the laser beam 11 , into a plurality of N sub beams 11 -1... 11 -N. In the illustrated example the laser beam is divided into 3 sub-beams 11 -1 , 11 -2 and 11 -3. A modulating device M is provided for modulating each sub-beam 11-1 , 11 -2 and 11-3 with a different modulation pattern ml , m2, m3 (e.g. to a respective frequency f1 , f2 and f3). The apparatus further includes a first objective lens Obj-1 for focusing each sub-beam 11 -1 , 11-2 and 11 -3 to a respective distinct position Pi , P₂ and P₃ within the cellular sample S. A second objective lens Obj-2, which may be identical to Obj-1 in epifluorescence mode, is provided for collecting and focussing fluorescence signals emitted from the cellular sample onto a photo-detector D such that the photodetector simultaneously detects a sum of the signals s(P,) emitted by each point P,. Photodetector D provides an electrical signal in response to the collected fluorescent signal. A de-modulator device driven by the modulation patterns ml , m2, m3 from modulator M is used to de-modulate and separate the signal S(P₁) =s(rτij) and detect the envelope of each fluorescence originating in positions Pi, P₂, and P3 of the cellular sample, representing the physiological fluorescence variation.

Modulation patterns ml , m2 and m3 differ to one another. In this way the position of the recorded fluorescence signal is coded by its temporal modulation pattern. For example, if sinusoidal modulations at frequencies f 1 , f2 and f3 are employed the position of the recorded fluorescence is coded in frequency. In general, frequencies of all modulation patterns are in the order of 10-100 times greater than the highest frequency of the fluorescence signals collected by the photodetector reporting physiological activity of the cellular sample.

Figure 2 illustrates in more detail a two-photon microscopy apparatus according to a particular embodiment of the invention. In this embodiment the apparatus comprises a femtosecond pulsed laser FL as a two-photon fluorescence excitation source. The femtosecond pulsed laser provides a pulsed excitation beam 11. The excitation laser beam 11 is divided into 5 sub-beams 11 -1 , 11-2, 11 -3, 11 -4 and 11-5 by a diffractive optical element DOE. The 5 sub-beams 11 -1 , 11 -2, 11-3, 11-4 and 11-5 are relayed by two lenses L1 (for example a Thorlabs, LA1509B, focal length f=100mm) and L2 (for example Thorlabs LA1608- B, f=75mm) onto an arrangement of XY galvanometric mirrors GS (for example, General scanning GS120). A 5-channel chopper C rotated at a speed of approximately 5700rpm by a DC motor M, and positioned between lens L1 and lens L2 modulates each sub-beam 11-1 , 11-2, 11 -3, 11-4 and 11-5 at frequencies f 1=3.08, f2= 3.27, f3=3.47 , f4=3.66, f5=3.85kHz, respectively. The galvanometric mirrors arrangement GS deflects the sub-beams relayed by lens L1 and l_2 onto lenses L3 (f=80mm) and L4 (f=250mm) which magnify the combined beam diameter to fill the back aperture of an objective OB. The sub-beams 11-1 , 11 -2, 11 -3, 11-4 and 11 -5 are focused by objective OB to 5 respective positions within the cellular sample S. A row of five excitation spots Pi (i=1-5) are thus created inside the sample (S). The fluorescent signals emitted by the excitations spots are collected by the objective OB and projected onto a photomultiplier tube PMT by a dichroic element DIC and a lens L5. The fluorescent signals collected by the photomultiplier tube are converted into a corresponding electrical current. This photocurrent is then amplified and discriminated by a photon counter Amp+PhC. The signal is then analysed by a computer in order to measure the amplitude of the power spectrum at frequencies f1 to f5 and the fluorescence signals at all 5 spots are displayed. By closing diaphragm D positioned between the chopper C and lens L2, and leaving the chopper C in a position that transmits light for the central sub-beam, a conventional 2-photon image may also be obtained.

By virtue of the process of frequency multiplexing and demultiplexing the spatial resolution is conserved despite the scattering in the biological material of sample S. This is not the case for systems using a camera simultaneously recording fluorescence from a number of points because scattering leads to a widening of the point spread function (PSF).

While the first embodiment of the invention relates to a two-photon microscopy apparatus, it will be appreciated that the invention relates to multi- photon imaging apparatus. In the described embodiment the laser excitation source may be a femtosecond laser for example a Coherent Mira laser. However it will be appreciated that any known laser or light source for providing an excitation source for multi-photon excitation may be used such as a Ti:Sapphire femtosecond laser. Multi-photon microscopy requires very high focal intensities around 1GW/cm² in the near infra red spectral region to efficiently excite biocompatible fluorescent molecules. For example Ti.Sapphire femtosecond laser are commonly employed in two-photon microscopy because this type of laser provides an average power of >1W with ~100fs pulses and a repetition rate of ~80MHz in the near infra red region from 700 to 1000nm. The number of points that can be observed simultaneously will depend on the power of the laser used.

The photo detector in this embodiment may be provided by a photomultiplier tube (PMT) for example a Hamamatsu H7422p. It will be appreciated that any suitable photo detection device for detecting a fluorescence signal may be used. The objective OB in this embodiment may for example be a Leica 63x NA0.9 or any objective device suitable for focussing laser sub-beams into different points of a cellular sample. The dichroic may be for example a Chroma dcxr695.

It will also be appreciated that the types of lens used and the corresponding focal lengths have been given as suitable examples only. The application of the apparatus and method according to embodiment of the invention described in Figure 2 may be demonstrated by imaging a sample containing two fluorescent beads. Figure 3 presents such a method and the results obtained.

In this illustrative example, we imaged a test sample S consists of 2 fluorescent beads B1 and B2 (6μm in diameter) spaced apart by a distance of 25μm as illustrated in part A of Figure 3 using the apparatus described in Figure 2. The sample S is moved at a speed of 25μm/s so that each bead intercepts successively the five two-photon excitation focii (a,b,c,d and e). The five focii are also spaced by 25μm as shown in B presenting an image of two grey beads (small white arrows) that are aligned with the 5 focii. In Fig 3B the first bead is about to reach the first sub-beam focus point a. Part C of Figure 3 presents a graphical illustration of signal intensity versus time. Fluorescence signals are recorded simultaneously at point a, b, c, d and e as the two beads cross through the five focii. As each bead crosses a focus point a, b, c, d or e it creates a peak signal at the frequency corresponding to the particular location. When the two beads coincide with two foci (at time 2, 3, 4 and 5s) they create a signal with 2 frequency components that can be separated.

The same experiment is repeated with a scattering media (for example, 0.5μm diameter latex particles in water, concentration 0.02% weight, thickness 970μm, 2.7 OD at 800nm, or 6.7 OD at 515nm) placed between the objective OB and the fluorescent beads B1 and B2, as illustrated in part D, E and F of Figure 3. It will be noted that even through a scattering layer the signals from different locations can be de-multiplexed in the frequency domain.

In alternative embodiment of the invention, the diffractive optical element

(DOE) can be replaced by a spatial light modulator (SLM), such as a liquid crystal SLM that generates multiple sub beams targeted to specific sample locations.

(see Papagiakoumou et al. (2008). "Patterned two-photon illumination by spatiotemporal shaping of ultrashort pulses." Opt Express 16(26): 22039-47) This helps to efficiently redirect the light power to a few targeted spots or to any user defined patterns within the sample Since light is not directed to regions in the sample of low interest, for example regions devoid of cells, the technique is more light efficient.

In alternative embodiments of the invention, the chopper may be replaced by a digital micro-mirror device (DMD), a matrix of -1000x700 micro-electromechanical system (MEMS) micro-mirrors (each ~10x10μm²), or any like device suitable for modulating the sub-beams at frequencies in the order of 10-10OkHz. In DMDs each micro mirror is typically 10x10μm² and can be flipped between two stable angular positions. Currently the fastest switching rate of the entire micro- mirror matrix is around 2OkHz. Faster DMD could be engineered in the future. Therefore a DMD can generate fast binary (ON-OFF) modulations of each sub- beam at frequencies in the order of 10-3OkHz.

Instead of modulating the sub-beams at different frequencies, temporal binary patterns, such as complementary Golay sequences, can also be applied to each sub-beam. Golay sequences are orthogonal binary functions, originally developed for the optical problem of dynamic multi slit spectroscopy (Golay

(1949). "Multi-Slit Spectrometry." Journal of the Optical Society of America 39(6):

437-444). In the context of our invention Golay temporal sequences permit separation of the signals originating from different locations. For example if points

Pi, P₂ and P₃ are excited with modulating sequences M-i, M₂, M₃ where

M₁(I) = [OJ ₁O₁I ₁O₁I ₁O₁I]

M₂(t) = [0,0,1 ,1 ,0,0,1 ,1]

M₃(t) = [0,1 ,1 ,0,0,1 ,1 ,0]

"0" and "1" corresponding to ON and OFF illumination state. In the case of spatial modulation with a DMD, the illumination state is determined by the angular position of the DMD micro-mirrors for the sub beams corresponding to points Pi, P₂ and P₃. Each ON or OFF state can last as little as 50μs with the current commercial DMD technology. During the short time interval of a sequence M, the photon emission at each point has a constant amplitude equal to Si, S₂ and S₃. At the photodetector we record the sum of all signals

S(t) = SiM₁ (t) + S₂M₂W + S₃M₃(Q For each M, sequence there is a complementary "orthogonal" sequence M₁ ^* such that

∑M,(t)xM,^*(t) = 0 if i≠j and∑M,(t)xM,^*(t) = N/4 where "Σ" is the sum over all elements of the sequence and "x" is the element-to- element vector multiplication, and N is the number of elements in a sequence.

M₁^t) = [-1 ,0,-1 ,0,0,1 ,0,1]

M₂ ^*(t) = [-1 ,-1 ,0,0,0,0,1 ,1]

M₃ ^* (t) = [-1 ,0,0,-1 ,0,1 ,1 ,0]

For example ∑Mi(t)xM₂ ^*(t) = (0x-1 )+(1x-1 )+(0x0)+(1x0)+(0x0)+(1x0)+(0x1)+(1x1 ) = 0 and

= 2 As a consequence

ΣS(t)xM₂ ^*(t) = 2s₂

ΣS(t)xM₃ ^*(t) = 2s₃

We see that by multiplying the photodetector signal by the M^* sequences we can compute the amplitude of the signal at each point Pi, P₂ and P₃. This could be done online by electronic circuits operating on the photodetector output voltage, or in a post processing step using a fast algorithm on digitally recorded data. To separate the signal from 6 points maximum a sequence with 8 binary states is required. For 14 points a sequence of 16 states is necessary. Techniques to extract longer Golay sequences have also been devised (Golay, M. J. E. (1961 ). "Complementary Series." IRE Transactions on InformationTheory: 82-87)

Figure 4 illustrates in more detail a two-photon microscopy apparatus according to a particular embodiment of the invention that uses an SLM to target specific sites inside the sample and a DMD to create binary sequences on each sub beams. In this embodiment the apparatus comprises a femtosecond pulsed laser femtosecond laser FL1 (Tsunami Spectra Physics) for the acquisition of a conventional laser scanning microscopy image. FL1 excitation wavelength is set to 925 nm. The femtosecond laser beam is sent to a galvanometric scanner (GS, General Scanning GS 120), magnified by the telescope composed of lenses L2 (f=100mm) and L4 (f=300mm), transmitted through both dichroic mirrors DIC1 (900DCXRU Chroma) and DIC2 (695DCXRU Chroma), and focalized into the sample (S) by a microscope objective (OB 63x NA 0.9 Leica). Fluorescence emitted at the focal spot is collected in epifluorescence mode by the same objective, reflected by a dichroic mirror DIC2 and imaged onto a photomultiplier tube (PMT, R6357 Hamamatsu) by lens L5. Raster scanning of GS allows acquisition of a conventional 2D two-photon image. The user manually places the excitation points with a mouse click onto the image using a computer interface (e.g. P1 , P2 and P3 in inset I.) These points are located in cells or sites where the user wants to measure the fast neuronal activity simultaneously.

A secondary femtosecond laser FL2 (Tsunami Spectra Physics) laser provides a pulsed excitation beam at 800nm sent to a spatial light modulator SLM (Hamamatsu LCOS X10468-02). A computer sends an holographic pattern to the SLM to generate the user specified illumination pattern (i.e. points P1 , P2 and P3) at the microscope objective focal plane (see Lutz et al. (2008). "Holographic photolysis of caged neurotransmitters." Nat Methods 5(9): 821 -7 and Papagiakoumou et al. (2008). "Patterned two-photon illumination by spatiotemporal shaping of ultrashort pulses." Opt Express 16(26): 22039-47) for more detail on this technique). Lens L1 (f=500mm) images the SLM Fourier plane onto the DMD mirror matrix creating a set of points PV PZ and P3' that are conjugate points of P1 , P2 and P3. The DMD pixels corresponding PV, P2' and P3' are modulated according to temporal binary sequence M1 , M2 and M3 as illustrated in the inset II. The sequences are repeated in a loop for the duration of the user-specified acquisition. All other pixels in the DMD are turned OFF. The DMD surface is imaged into the sample by a telescope composed of lens L4 and the microscope objective OB. Modulated fluorescence signals emitted at points P1 , P2 and P3 are collected in epifluorescence mode and imaged onto the PMT where they add up. The sum of all signals is processed as explained above (multiplication by the demodulating sequences M1 ^*, M2^* or M3^*) to extract the signals originating from P1 , P2 and P3 (S(P1 ,t), S(P2,t) and S(P3,t)) as illustrated by inset III. Note that when the conventional 2D image is acquired, the beam from laser FL2 is blocked by a shutter (not shown in Figure 4)), and when acquisition of signals at points P1 , P2 and P3 take place, the beam from laser FL1 is blocked by another shutter.

The application of the apparatus and method according to embodiment of the invention described in Figure 4 may be demonstrated by recording fast activity in a neuron labeled with a fluorescent calcium indicator in an acute mouse brain slice. Figure 5 presents such a method and the results obtained.

A mitral cell in the mouse olfactory bulb was patched with a borosilicate patch pipette (indiacted by white arrow in Figure 5A) filled with a solution containing Oregon Green Bapta 2. This calcium sensitive dye diffused inside the cell in a few minutes. The cell depth is around 60μm. We acquired a 2D image (Figure 5A, scale bar 20μm) using laser FL1 and galvanometric scanner GS. Then we placed three target points onto the cell in the apical dendrite (P1), at the base of the apical dendrite (P2) and in the soma (P3). The computer calculated and sent the phase hologram to the SLM in order to diffract the laser beam from FL2 into the three points P1 , P2 and P3. The DMD pixels corresponding to P1 , P2 and P3 where modulated according to the binary Golay sequences described above and illustrated in Figure 4, inset II. The Golay sequence lasted 2ms, which is the recording time resolution. With the current commercially available DMD and using the shortest Golay sequence, a time resolution down to 0.5ms is achievable for up to 6 points. It goes up to 1 ms for 14 points. We applied a 2OmV voltage step (ΔV) for 10ms to trigger 1-3 action potentials that created a very fast calcium influx inside the cell. The fluorescence signals where demodulated using the method described above. A trace for a single stimulation is sow in Figure 5B, while an average of 10 traces is displayed in Figure 5C. Traces are displayed as ΔF/F, i.e. the relative fluorescence variation with respect to the control signal measured over the first 150ms. This result demonstrates that our method allows to measure fluorescence variations elicited by electrical stimulation simultaneously in a number of points of a labelled sample at the millisecond temporal resolution. The method according to the embodiments of the invention can find applications in areas such as neurophysiology and in particular in imaging activity in cellular networks. It will be appreciated, however, that the invention may be applied in any biological or pharmacological domain which would benefit from high speed image acquisition, a resolution of the order of micrometres and an image depth to the order of 500 microns in scattering media. Such areas include pharmacology, development biology, cardiovascular imaging, dermatology, ophthalmology etc. The targeted temporal resolution in the context of an application in neurophysiology corresponds to the dynamics of the electrical-physiological signals studies is in the order to 1 ms. In the present invention the temporal resolution δt is not limited to 1 ms. The temporal resolution will depend on factors such as the element used to modulate the sub-beams, the frequency response of the photo-detectors and the power of the laser. It should be noted that the temporal resolution δt is inversely proportional to the frequency resolution δf .

Accordingly, in order to reduce the temporal resolution δt, the frequency

separation of the N sub-beams should thus be increased. For example, in order to obtain a temporal resolution of 0.1 ms the frequency of the N sub beams should be separated by at least a frequency of 2OkHz (2^*δt). To obtain a temporal resolution of 1 ms the frequencies should be separated by 2kHz.

The number of points N in the sample that may be simultaneously imaged is limited by the power of the laser and the photo-toxicity. With existing

commercial lasers with a maximum power in the order of 5W the limit is around 500 points (1 OmW per sub-beam). However with the development of more powerful femtosecond lasers imaging systems capable of imaging 100-1000 points may be provided. The approach adopted by the embodiments of the present invention enables the fluorescence signature of a multi-cellular sample to be obtained from a number of points simultaneously, thereby allowing wide field imaging to be obtained at increased imaging acquisition speed. A spatial resolution of the order of 1 μm and a temporal resolution of the order of 1 ms can be obtained. The imaging depth is greater than 350μm in brain tissue.

Although the present invention has been described hereinabove with reference to specific embodiments, the present invention is not limited to the specific embodiments, and modifications will be apparent to a skilled person in the art which lie within the scope of the present invention.

In the claims, the word "comprising" does not exclude other elements or steps, and the indefinite article "a" or "an" does not exclude a plurality. The mere fact that different features are recited in mutually different dependent claims does not indicate that a combination of these features cannot be advantageously used. Any reference signs in the claims should not be construed as limiting the scope of the invention.

Claims

1. A method of imaging a cellular sample by multi-photon imaging, the method comprising:

providing a multi-photonic excitation beam;

splitting the multi-photonic excitation beam into a plurality of N sub- beams;

modulating the amplitude of the plurality of N sub-beams to create N respective temporal modulation patterns;

focusing the plurality of N modulated sub-beams to N respective spatially separated focal points Pi... P_N within the cellular sample;

simultaneously detecting fluorescent signals emitted from the N focal points PI ...PN^", and

demodulating the detected fluorescent signals so that fluorescence data relating to the respective focal points P_I ...P_N may be obtained.

2. A method according to claim 1 , wherein the step of simultaneously detecting fluorescent signals comprises collecting a combined fluorescence signal composed of fluorescent signals emitted from the N focal points Pi...P_N; and wherein fluorescence data relating to the respective focal points P-_I ...P_N is obtained by extracting the signal components of the combined modulated fluorescence signals .

3. A method according to claim 1 or 2 wherein modulating the amplitude of the plurality of N sub-beams creates N respective temporal modulation patterns with N different binary temporal patterns.

4. A method according to claim 3 wherein the N different binary temporal patterns are Golay sequences.

5. A method according to claim 1 or 2, wherein modulating the amplitude of the plurality of N sub-beams creates N respective temporal modulation patterns with N different frequencies fi ...fN

6. A method according to any one of the preceding claims, further comprising measuring the amplitude of the power spectrum at each frequency Mo f_N or demultiplexing the signals from the mixed temporal modulations sequences.

7. A method according to any one of the preceding claims wherein the frequency of each of the N frequencies is over 100 times greater than the highest frequency of the fluorescence signal

8. A method according to any one of the preceding claims wherein the frequencies fi to f_N of the N sub beams are separated by at least a frequency of 2kHz

9. A method according to any one of the preceding claims wherein splitting the multi-photonic excitation beams includes directing each of the plurality of sub- beams to a predetermined location within the sample.

10. An apparatus for imaging a cellular sample, the apparatus comprising:

a light source for providing a multi-photon excitation beam a beam divider for splitting the multi-photon excitation beam into a plurality of N sub-beams;

a modulator for modulating the amplitude of the plurality of N sub- beams with N respective temporal modulation patterns;

an objective for focusing the plurality of N modulated sub-beams to N respective spatially separated focal points P₁... P_N within the cellular sample;

a photodetector for simultaneously detecting fluorescent signals emitted from the N focal points P_{1 ^} P_N; and

a demodulator for demodulating the detected fluorescent signals; and

a processor for processing the demodulated fluorescent signals to obtain fluorescence data relating to the respective focal points P_{1 1} - PN.

1 1. An apparatus according to claim 10, wherein the photodetector is arranged to collect a combined fluorescence signal composed of fluorescent signals emitted from the N focal points P_{1 ^} P_N; and wherein the processor is configured to extract the signal components of the combined fluorescence signal modulated according to each temporal pattern.

12. An apparatus according to claim 10 or 11 , wherein the modulator comprises a digital mirror device for creating N respective temporal modulation patterns with

N different binary temporal patterns.

13. An apparatus according to claim 12, wherein the digital mirror device is operable to create N different Golay sequences.

14. An apparatus according to claim 10 or 11 , wherein the modulator is operable to modulate the amplitude of the plurality of N sub-beams to create N respective temporal modulation patterns with frequencies fi...f_N.

15. An apparatus according to any one of claims 10 to 14 wherein the beam divider comprises a spatial light modulator for directing each sub-beam to a predetermined location within the sample.

16. An apparatus according to any one of claims 10 to 15, wherein the processor is configured to measure the amplitude of the power spectrum at each frequency f i to f_N

17. An apparatus according to any one of claims 10 to 16 further comprising a display for displaying N fluorescent signals corresponding to the N focal points

18. An apparatus according to any one of claims 10 to 17 wherein the modulator is configured to modulate the frequency of each of the N frequencies to be over 100 times greater than the highest frequency of the fluorescence signal

19. An apparatus according to any one of claims 10 to 18 wherein the modulator is configured to modulate the frequency frequencies U to f_N of the N sub beams are separated by at least a frequency of 2kHz .