Microscope and microscopy method

Abstract

The invention relates to a microscope comprising at least one light source for emitting light pulses for exciting a sample and an excitation beam path with a microscope objective for directing the light pulses on or into the sample, wherein the excitation beam path comprises a plurality of separate excitation channels being designed for guiding light pulses into in each case different focal spots on or in the sample, a distribution unit for distributing the light pulses to the excitation channels, a scanner for varying a region on or in the sample being irradiated by the light pulses, comprising further at least one detector for the detection of emission light emitted by the sample as an optical response to irradiation by the light pulses and a detection beam path for guiding the emission light onto the detector, and comprising a control unit for controlling at least the light source and the scanner and for evaluating the light detected by the detector. According to the invention, the microscope is characterized in that for temporally separating the respective optical responses from the irradiated focal spots, the excitation beam path is designed for irradiating the different focal spots through the excitation channels sequentially one after another, each of the excitation channels comprises an optical fiber having in each case an exit end, and, for guiding light pulses into the respectively different focal spots the exit ends of the optical fibers are arranged with an in each case different spacing with respect to a light focussing component of the excitation beam path. The invention relates furthermore to a microscopy method.

Description

[0001] Microscope and microscopy method

[0002] The invention relates to a microscope according to the preamble of claim 1 and to a microscopy method according to the preamble of claim 24.

[0003] A generic microscope, in particular a multi-photon-microscope, comprises at least one light source for emitting light pulses for exciting a sample and an excitation beam path with a microscope objective for directing the light pulses on or into the sample. The excitation beam path comprises a plurality of separate excitation channels being designed for guiding light pulses into in each case different focal spots on or in the sample, a distribution unit for distributing the light pulses to the excitation channels, a scanner for varying a region on or in the sample being irradiated by the light pulses. The generic microscope comprises further at least one detector for the detection of emission light emitted by the sample as an optical response to irradiation by the light pulses and a detection beam path for guiding the emission light onto the detector, and a control unit for controlling at least the light source and the scanner and for evaluating the light detected by the detector.

[0004] A generic microscopy method, in particular a multi-photon-microscopy method, comprises the steps of: distributing light pulses to a plurality of excitation channels of an excitation beam path, guiding light pulses via the different excitation channels and a microscope objective into respectively different focal spots on or in the sample, varying with a scanner a region on or in the sample being irradiated by the light pulses, detecting emission light emitted by the sample as an optical response to irradiation by the light pulses.

[0005] Generic microscopes and generic microscopy methods are described, e.g., in US 20170227748 A1

[0012] , Multiphoton imaging has become an essential tool to monitor neuronal activity. Optical sectioning, granting optical access to deep structures within biological tissue in a non- invasive way, is the key for imaging neuronal populations in-vivo and in awake animals. Combining multiphoton imaging with miniaturization techniques [1 , 2] and hollow-core optical fibers [3], resulting in miniature head-mounted microscopes, provides the ability to measure activity of neuronal populations in freely-moving animals during behavioral tasks, when the studied neuronal network is functional in its natural condition [4-7],

[0006] Recently published open-source miniature two-photon microscopes provide such technology to the neuroscience community, (Mini2P,

[0013] ). This technology is however limited by the throughput of the scanner (product of the size of the image, spatial and temporal resolution). Mini2P comes with an electrically tunable lens that allows to switch between neuronal populations over a range of depths (~250 pm). While this method significantly increases the number of observed neurons and expands the range of observed structures, it subdivides the imaging time on the number of imaging planes, proportionally lowering the temporal resolution for each plane. In addition, the employed excitation (two photon absorption) and the characteristics of the electric lens limit the maximal imaging depth to about 300 pm - 400 pm (half the depth of cortex in mice) and the displacement range to about ~ 250 pm allowing only the superficial part of cortex to be investigated. This also is a significant limitation as the cerebral cortex is functionally structured in layers and neurons at different depths have dramatically different properties making the capability of imaging multiple planes at once and at different depths highly desirable.

[0007] A simultaneous multiplane imaging approach has been demonstrated for large microscopes [8,9], consisting of separating one intensive laser pulse into series of smaller pulses with increasing delays in the range of tens of nanoseconds range and increasing optical defocus, i.e. , the optical properties of a laser beam. The defocus coupled into the microscope results in focusing of the laser pulse into different depths and thus imaging at different depths, while the time delay allows to detect the emitted fluorescence from the sample in parallel at the detection electronics by de-multiplexing the signal using nanosecond level delays, introduced between pulses, producing virtually simultaneous imaging of multiple planes without sacrificing temporal resolution. But such a design cannot be miniaturized, since producing delay for laser pulses requires long distances, e.g., 10 ns correspond to ~ 3 m in air. It is also not possible to couple already structured pulse train through a fiber, as the optical aberrations such as defocus are lost through the coupling into single more fibers or alike (hollow core fibers, used in miniature multiphoton microscopes). It is therefore not possible to use this approach to gain simultaneous multiplane imaging.

[0008] It can be considered to be a problem of the invention to provide a microscope and a microscopy method which can be readily miniaturized.

[0009] This problem is solved by the microscope having the features of claim 1 and the microscopy method having the features of claim 24.

[0010] According to the invention, the above generic microscope is further characterized in that for temporally separating the respective optical responses from the irradiated focal spots, the excitation beam path is designed for irradiating the different focal spots through the excitation channels sequentially one after another, each of the excitation channels comprises an optical fiber having in each case an exit end, and, for guiding light pulses into the respectively different focal spots the exit ends of the optical fibers are arranged with an in each case different spacing with respect to a light focussing or collimating component of the excitation beam path.

[0011] According to the invention, the above generic microscopy method is further characterized in that the step of irradiating the different focal spots through the excitation channels is carried out sequentially one focal spot after another for temporally separating the respective optical responses from the irradiated focal spots, wherein each of the excitation channels comprises an optical fiber having in each case an exit end, and, for guiding light pulses into the respectively different focal spots the exit ends of the optical fibers are arranged with an in each case different spacing with respect to a light focussing component of the excitation beam path.

[0012] Advantages of the invention and embodiments of the microscope according to the invention and variants of the microscopy method are described below in particular with respect to the dependent claims and the figures. The light source can comprise one or more lasers which provide excitation light in the needed spectral range and with the needed intensity. The excitation light can be electromagnetic radiation in the visible and adjacent ranges, i.e., in the infrared and the ultraviolet range. Typical pulse lengths, e.g., for multi-photon-microscopy, can be, e.g., as low as 100 femtoseconds at a repetition rate of e.g., in the range of 80 to 100 MHz.

[0013] The term excitation beam path denotes all optical beam-guiding and beam-modifying components, for example lenses, mirrors, prisms, gratings, filters, stops, beam splitters, modulators, e.g., spatial light modulators (SLM), by means of which and via which the excitation light from the light source is guided to the sample to be examined. The excitation light is guided onto the sample by the microscope objective, in particular the same microscope objective which is also part of the detection beam path. For multi- photon-microscopy microscope objectives are preferably used which are chromatically corrected for the used wavelength of the excitation light as well as the wavelengths of the detection light. The term exit end denotes the end of an optical fiber where the excitation light from the laser leaves the respective optical fiber.

[0014] The back focal plane of the microscope objective and planes optically conjugate thereto are also referred to as pupil planes. The term intermediate image plane denotes planes in the excitation beam path or the detection beam path which are optically conjugate to a focal plane of the microscope objective.

[0015] Light that is emitted and / or deflected, for example scattered, generally radiated, by the sample to be examined as a consequence of the irradiation by the excitation light can be referred to as emission light or detection light and reaches the detector via the detection beam path. The term detection beam path denotes all beam-guiding and beammodifying optical components, for example lenses, mirrors, prisms, gratings, filters, stops, beam splitters, modulators, e.g., spatial light modulators (SLM), by means of which and via which the emission light is guided from the sample to be examined to the detector. This detection beam path can comprise the same microscope objective that is part of the excitation beam path.

[0016] The scanner can, e.g., be a typical galvanometric scanner or a MEMS-scanner (MEMS = Micro Electro Mechanical System) or a combination of such components. It is also possible to scan the beam by suitable manipulation of the excitation light with a spatial light modulator, e.g., a phase modulating spatial light modulator, i.e. , the scanner can comprise a spatial light modulator (SLM) or can be realized by an SLM. The SLM can preferably be arranged in a pupil plane of the excitation beam path.

[0017] Detection light, e.g., fluorescence light, is preferably detected with high-bandwidth detectors, producing photo-electric current with few nanosecond durations to minimize cross-talk between temporally adjacent channels. The detector can, e.g., be a photomultiplier or photodiode. The optical fibers can be single-mode-optical fibers. Preferably, hollow core fibers are used. The term focal spot can , e.g., be a diffraction-limited volume, into which the excitation light is focused.

[0018] As a first important idea of the present invention can be considered the concept of achieving for each of the channels a respectively different location for the respective focal spot in the sample by spatially separating the exit ends of the respective optical fibers. As a second important idea of the present invention can be considered the concept of irradiating different focal spots sequentially one after another and thus of temporally separating the optical responses from the irradiated focal spots.

[0019] A first important advantage of the present invention is that it allows many variations of the spatial arrangement of the exit ends of the respective optical fibers and, thus, many variations of the locations of the focal spots in the sample. A further important advantage is that the setup with the exit ends of the respective optical fibers can easily be miniaturized and, thus, the microscope and the microscopy method according to the invention can readily be applied to living objects.

[0020] Generally, it is possible to use the microscope and the microscopy method of the invention for usual laser scanning microscopy. The microscope and the microscopy method of the invention are particularly well suited, though, for the multi-photon-mi- croscopy, e.g., two-photon-microscopy and three-photon-microscopy.

[0021] In a first important preferred embodiment, the exit ends of the optical fibers are arranged with respect to the light focussing component with an in each case different spacing in a direction of an optical axis of the excitation beam path. Thus, a depth resolution of the sample can be achieved.

[0022] Alternatively or additionally, the exit ends of the optical fibers can be laterally spaced from each other and / or laterally spaced with respect to a direction of an optical axis of the excitation beam path. An embodiment where the exit ends of the optical fibers are laterally spaced with respect to the optical axis but have, in each case, the same spacing with respect to the light focussing component would correspond to a multi-spot- microscope. It would then be preferable, e.g., to arrange the exit ends of the optical fibers and thus the illumination spots on or in the sample along a line which is perpendicular to the direction in which a scanner moves the spots over the sample. It would also be possible, though, e.g., for HDR-microscopy (High-Dynamic-Range-micros- copy) or Dl-microscopy (Dynamic Illumination microscopy) methods, to arrange the exit ends of the optical fibers and thus the illumination spots on or in the sample along a line which is parallel to the direction in which a scanner moves the spots over the sample. The optical fibers can be arranged with such spacings that they do not touch each other in the region of their respective exit ends.

[0023] In a further preferred embodiment, the microscope further comprises a ferrule in which the exit ends of the optical fibers are received. Such a ferrule can define the position of each of the exit ends of each of the fibers with respect to the optical axis in the lateral direction and with respect to the light focussing component in the direction of the optical axis. The housing of the microscope can preferably be designed for the reception of the ferrule at a defined location and in a defined orientation. In a further preferred embodiment, it is possible to have a plurality of different ferrules, each of which realizes a different illumination mode, e.g., all illumination spots spaced from each other in a line in a lateral direction or all illumination spots spaced from each other essentially only in an axial direction and / or combinations thereof. Thus, a multi-modal microscope can be created where it is possible to switch from one method to another by changing the ferrule and, if necessary, also the fibers themselves and switching the control unit correspondingly.

[0024] In a first embodiment of the microscope according to the invention, the distribution unit is designed for energetically distributing each of the light pulses over the excitation channels. In the corresponding embodiment of the microscopy method according to the invention, the light pulses are energetically divided in the distributing step and distributed over the different excitation channels. I.e., each and every one of the light pulses is divided into a plurality of smaller pulses and the number of the smaller pulses is preferably equal to the number of excitation channels in the excitation beam path.

[0025] For the purpose of energetically dividing and distributing the light pulses over the different excitation channels, the distribution unit can comprise a plurality of beam splitters for energetically dividing and distributing the light pulses over the different excitation channels. One, a plurality, or all of these beam splitters may be neutral beam splitters. The option of all beam splitters being neutral beam splitters would be chosen where it is intended to subjugate in each and every one of the focal spots with the same spectrum of electromagnetic radiation. But it is also possible that one, a plurality, or all of these beam splitters are dichroic beam splitters. The option of the beam splitters being dichroic beam splitters could be chosen, e.g., for a multi-spot microscopy mode where it is intended to subjugate the separate focal spots with an at least in some cases or in all cases different spectrum of electromagnetic radiation. As regards the intensity of the pulses there are also degrees of freedom. The pulses the light pulses of the excitation light in the separate excitation channels can have, in each case, the same energy. But it would also be possible to have increasing energies, e.g., for a HDR-multi-spot mode.

[0026] It is furthermore possible that one, a plurality or all of the beam splitters are controllable beam splitters, e.g., acousto-optical devices such as AOTFs orAOMs. These devices would allow controlling the energy and / the spectral composition of the individual partial light pulses in the separate excitation channels. If such acousto-optical devices are not fast enough to separate single pulses at, e.g., 80 MHz it might be preferable to use either a slower laser pulse rate and / or to separate the pulses by groups of pulses, e.g., by groups of 5 to 10 pulses, thus effectively reducing the required speed by a proportional factor.

[0027] In a further embodiment, the microscope can further comprise a delay path in each of the excitation channels for temporally delaying the energetically distributed light pulses in each case differently in the respective excitation channels. In the corresponding embodiment of the microscopy method the energetically divided light pulses are subjugated to a different delay in each of the excitation channels.

[0028] A first important idea of this embodiment can be considered the concept of bringing about the temporally separated irradiation of different focal spots by using different optical path lengths in the respective channels. Said different optical path lengths in the respective channels can be, e.g., brought about by using optical fibers having, in each case, different lengths.

[0029] In a further preferred embodiment of the microscope according to the invention, the distribution unit is designed for guiding of individual light pulses from the light source to one of the excitation channels. In the corresponding preferred variant of the microscopy method according to the invention, in the distributing step, each of the light pulses from the light source is directed to one of the excitation channels. As opposed to the previously described examples, where each of the light pulses is divided into partial light pulses, the essential idea in this embodiment is to direct light pulses from the light source in each case as a whole, i.e., in their entirety, to only one of the excitation channels.

[0030] Hybrid embodiments where the pulses are energetically divided and the partial pulses are then directed to different excitation channels by means of optical switches are also possible.

[0031] In this respect, the distribution unit can further comprise optical switches for directing each of the light pulses into one of the excitation channels. As optical switches, acousto-optical components, e.g., AOTFs, can be used. With such components, it would also be possible to individually shape the spectral composition of the light pulses. If such acousto-optical devices are not fast enough to separate single pulses at, e.g., 80 MHz it might be preferable to use either a slower laser pulse rate and / or to separate the pulses by groups of pulses, e.g., by groups of 5 to 10 pulses, thus effectively reducing the required speed by a proportional factor. It may also be preferred that the optical switches are Pockels-cells which are faster as acousto-optical components. The actual measurement rates are not limited to achievable switching rates of use optical switches. Rather, in the case where the repetition rates of the use laser sources are higher than the switching rates of used optical switches, sequences of a plurality of light pulses from the light source immediately following one another are directed one after another into each of the excitation channels.

[0032] The method preferably comprises further the step of de-multiplexing a detection signal for evaluating the optical response from each of the irradiated spots on or in the sample. Such evaluations can be done in suitably programmed control unit.

[0033] In advantageous embodiments of the invention, the microscope comprises further a tube lens for generating a first intermediate image plane being optically conjugate to a focal plane of the microscope objective. In principle, it would be possible to arrange the exit ends of the optical fibers in this first intermediate image plane immediately upstream of a tube lens in the excitation beam path. Then, however, the scanner would have to be mounted in the back focal plane of the microscope objective which is not possible for a couple of reasons. It is therefore preferred to additionally have an optical relay comprising a first lens or lens group and a second lens or lens group for generating a second intermediate image plane being optically conjugate to the first intermediate image plane. The exit ends of the optical fibers can then preferably be arranged in or in the vicinity of said second intermediate image plane.

[0034] In further preferred embodiments, the scanner is arranged in or in the vicinity of a pupil plane that is optically conjugate to a back focal plane of the microscope objective and that is formed between the first lens or lens group and the second lens or lens group.

[0035] E.g., for the purpose of microscope investigations of living mice or other laboratory animals, it is preferred to have miniaturized microscope equipment which can be readily applied to the animals to be investigated. In such cases, the microscope can preferably further comprise a microscope head which can readily be applied or attached to, e.g., the head of an animal to be investigated. Advantageously, such a microscope head can comprise the microscope objective, the scanner, and the exit ends of the optical fibers, preferably being arranged in a ferrule as describe above. The micro- scope can further comprise a main beam splitter, e.g., a dichroic beam splitter, for separating emission light and light pulses from the excitation beam path. The main beam splitter can preferably be received and arranged in the microscope head. The tube lens and the optical relay can preferably also be received and arranged in the microscope head.

[0036] The microscope head can further comprise an optical interface for the passage of emission light. The at least one detector can be arranged at said optical interface.

[0037] A sample can be prepared with more than one fluorescent dye, e.g., two different fluorescent dyes which emit fluorescence light at different wavelengths. In such cases it is furthermore preferred to have a detection module at the optical interface, the detection module comprising at least one color splitter and a plurality of detectors. In the case of two different wavelengths to be detected, one would need two chromatic detection channels. This detection module can also be attached to the microscope head.

[0038] Generally, the invention can be realized with the microscope, where each of the excitation channels is designed for propagating in each case light pulses comprising the same spectrum of wavelengths. With such a microscope samples being prepared with fluorescent dyes that can be excited with photos of the same wavelengths can be investigated.

[0039] In many cases, a higher flexibility with respect to different fluorescent dyes is desired. For these cases, the light source can preferably provide excitation light having at least a first wavelength and a second wavelength. At least one of the excitation channels can be designed for propagating light pulses comprising another spectrum of wavelengths as compared to the other excitation channels.

[0040] In a further embodiment, the microscope further comprises at least one dichroic mirror for merging light pulses exiting a first optical fiber with light pulses exiting a second optical fiber and having a different wavelength as the light pulses exiting the first optical fiber.

[0041] It can also be convenient that at least one of the excitation channels is designed for propagating at least light pulses having a first spectrum of wavelengths and light pulses having a second spectrum of wavelengths which is different from the first spectrum of wavelengths, or, more specifically, that at least one of the excitation channels is designed for propagating at least light pulses having a first wavelength and light pulses having a second wavelength which is different from the first wavelength.

[0042] Still another embodiment of the microscope further comprises at least one first dichroic beam splitter for separating light pulses exiting the same optical fiber and having different wavelengths and at least one second dichroic beam splitter downstream of the first dichroic beam splitter for merging the light pulses having different wavelengths, wherein the optical path lengths between the first dichroic beam splitter and the second dichroic beam splitter are in each case different for the light pulses having different wavelengths.

[0043] These variants may be advantageous for cases where a sample is to be investigated simultaneously by both two-photon-microscopy and three-photon-microscopy.

[0044] Further properties and advantages of the invention will be described in the following with respect to the attached figures.

[0045] Figure 1 : shows a first embodiment of a microscope according to the invention;

[0046] Figure 2: shows the microscope head of the microscope of figure 1 in closer detail;

[0047] Figure 3: shows exit ends of optical fibers in the microscope head of figure 2 in closer detail;

[0048] Figure 4: shows details of the focal spots created in a sample with the microscope head of figure 2;

[0049] Figure 5: shows time charts for illustrating a first variant of the microscopy method according to the invention;

[0050] Figure 6: shows a distribution unit of a second embodiment of a microscope according to the invention; Figure 7: shows time charts for illustrating a second embodiment of the microscopy method according to the invention;

[0051] Figure 8: shows an alternative arrangement of the exit ends of optical fibers in a microscope according to the invention; and

[0052] Figure 9: shows a further alternative arrangement of the exit end of an optical fiber in a microscope according to the invention.

[0053] In the figures, identical components are generally denoted by the same reference numbers.

[0054] A first embodiment of a microscope 100 according to the invention will be described with respect to figures 1 to 4. A first example of a microscopy method according to the invention will then be described with reference to figures 1 to 5.

[0055] The microscope 100 which is schematically shown in figure 1 can in particular be a multi-photon-microscope and comprises at least one light source 10 for emitting light pulses 14 for exciting a sample 1 and an excitation beam path with a microscope objective 54 (see figures 2, 4) for directing the light pulses 14 on or into the sample 1. The light source 10 can be a tunable laser and in particular a laser providing excitation light in the near infrared range (NIR). One commonly used laser for two-photon microscopy is the Ti:sapphire laser. This laser offers an average power greater than 2.3 W at 800 nm and a wavelength that is tunable from 720 nm to 1060 nm. This allows the user to target specific compounds for two-photon fluorescence imaging and photostimula- tion / uncaging. The lasers pulses can be of the order of 100 femtoseconds (fs). E.g., for a typical neuronal imaging application high repetition rate laser (80 MHz) are used for two-photon imaging (wavelength 920-980 nm) and low repetition rates (< 4 MHz) at 1300 nm are used for three-photon excitation. But the microscope and the microscopy method of the invention are not limited to these parameters. To keep the laser pulses at a desired pulse length and maintain a high excitation efficiency it is furthermore preferable to have a precompensation or prechirp stage for the laser pulses to reduce dispersion, as detailed in references [4-[7], The excitation beam path comprises a plurality of separate excitation channels ch#1 , ch#2, ch#N that are designed for guiding light pulses 14 into in each case different focal spots 51 , 52, 53 (see figure 4) on or in the sample 1 . Furthermore, the excitation beam path comprises a distribution unit 20 for distributing the light pulses 14 to the excitation channels ch#1 , ch#2, ch#N and a scanner 46 (see figure 2) for varying a region on or in the sample 1 being irradiated by the light pulses 14.

[0056] The microscope 100 comprises further a detector 61 for the detection of emission light 16 emitted by the sample 1 as an optical response to irradiation by the light pulses 14 and a detection beam path 54, 55, 56, 63 for guiding the emission light 16 onto the detector 61 . In the example of figure 1 , the detector 61 is a first detector and the microscope 100 comprises a second detector 62. The first detector 61 and the second 62 can, e.g., be photomultiplier tubes (PMTs) or high sensitivity semiconductor detectors. Preferably, the detectors 61 and 62 have a high-bandwidth and are able to produce photo-electric currents with few nanosecond durations to minimize cross-talk between the signals of temporally adjacent excitation channels.

[0057] A control unit 90, e.g., a PC, serves the purpose of controlling at least the light source 10 and the scanner 46 and of evaluating the light detected by the detectors 61 , 62.

[0058] According to the invention, the excitation beam path is designed for irradiating the different focal spots 51 , 52, 53 through the excitation channels ch#1 , ch#2, ch#N sequentially one after another. Thus, the respective optical responses from the irradiated focal spots 51 , 52, 53 can be temporally separated. Each of the excitation channels ch#1 , ch#2, ch#N comprises an optical fiber 31 , 32, 3N having in each case an exit end 71 , 72, 73 (see figure 3). For guiding the light pulses 14 into the respectively different focal spots 51 , 52, 53 the exit ends 71 , 72, 73 of the optical fibers 31 , 32, 33 are arranged with an in each case different spacing d1 , d2, d3 (see figure 3) with respect to a light focussing or collimating component 44 of the excitation beam path. The optical fibers 31 , 32, 33, 3N can preferably be single-mode and / or hollow core fibers.

[0059] In the example shown in figure 1 , the distribution unit 20 is designed for energetically distributing each of the light pulses 14 over the excitation channels ch#1 , ch#2, ch#N. This is achieved in the example of figure 1 by a plurality of neutral beam splitters 11 , 12 for energetically dividing and distributing the light pulses 14 over the different excitation channels ch#1 , ch#2, ch#N.

[0060] The neutral beam splitters 11 , 12 can preferably be designed in such a way that the energetically divided pulses which can also be termed partial pulses and that are directed into the excitation channels ch#1 , ch#2, ch#N have, in each case, the same energy. E.g., in a case, where the distribution unit 20 comprises a total of three excitation channels and with the assumption that no energy is lost in the beam splitters 11 , 12, this could be realized by a neutral beam splitter 11 directing 33% of the incoming energy into the excitation channel 21 and furthermore by a neutral beam splitter 12 directing 50% of the incoming energy into the excitation channel 22.

[0061] Furthermore, in the example of figure 1 , each of the excitation channels comprises an in each case specific delay path 21 , 22, 2N for temporally delaying the energetically distributed light pulses 14 in each case differently in the respective excitation channels ch#1 , ch#2, ch#N. In the example shown in figure 1 each of the delay paths adds another 10 ns to the previous excitation channel. This could in each case, e.g., be achieved by ca. 2m of an optical fiber. The microscope 100 comprises a microscope head 40 into which the optical fibers 31 , 32, 3N are introduced as a fiber bundle 30.

[0062] Figures 2 to 4 show the microscope head 40 in more detail for the example of a total of three excitation channels and, thus three corresponding optical fibers 31 , 32, 33. Figure 3 shows details of the box I in figure 2. Figure 3 shows details of the box II in figure 2.

[0063] The bundle 30 of the optical fibers 31 , 32, 33 are held in a ferrule 42 which is received in a first tubelike portion of a housing 41 of the microscope head 40. As depicted in detail in figure 3, the exit ends 71 , 72, 73 of the optical fibers 31 , 32, 33 are arranged with respect to the light focussing component 44 with an in each case different spacing d1 , d2, d3 in a direction of an optical axis z' of the excitation beam path.

[0064] In the example shown in figure 2, the focusing component 44 is a first lens group which forms a relay optics with a second lens group 48 being arranged further downstream in the excitation beam path. Between a tube lens 50 and the second lens group 48 an intermediate image plane is formed, i.e. , an optical plane which is optically conjugate to a focal plane of the microscope objective 54 in the sample 1 . This intermediate image plane is imaged through the second lens group 48 and the first lens group 44 into a further intermediate image plane which is near the exit ends 71 , 72, 73 of the optical fibers 31 , 32, 33 (see figure 3).

[0065] Thus, the different axial spacings d1 , d2, d3 of the exit ends 71 , 72, 73 of the optical fibers 31 , 32, 33 with respect to the first lens group 44 (see figure 3) are translated into different axial depths D1 , D2, D3 of the corresponding focal spots 51 , 53, 53 in the sample 1 (see figure 4). The sample 1 can, e.g., be the brain of a living animal.

[0066] The exit ends 71 , 72, 73 of the optical fibers 31 , 32, 33 are spaced to some extent laterally with respect to a direction of an optical axis z' of the excitation beam path. In the shown example, this lateral spacing is confined to the spacing which is brought about by the finite thickness of the respective optical fibers 31 , 32, 33 which are in close contact with each other in the ferrule 42 (see figure 3). Caused by the small lateral spacings of the exit ends 71 , 72, 73 of the optical fibers 31 , 32, 33 the corresponding focal spots 51 , 53, 53 are, in each case, also laterally spaced (i.e., in figure 4, in the direction of the y-axis). In reality, though, the lateral spacing is much smaller as compared to the axial spacing than shown in figure 4. If a larger lateral spacing of the focal spots in the sample were desired, it would, of course, also be possible to have the optical fibers arranged in the ferrule 42 in such a way that they do not touch each other in the region of their respective exit ends.

[0067] The focal spots 51 , 52, and 53 can be moved laterally, i.e., in the x- and y-direction in figure 4, with the scanner 46. The scanner 46 is a mirror which can be pivoted in two independent axes with a MEMS-drive or a galvanometric drive. In the shown example, the scanner 46 is arranged in a pupil plane of the excitation beam path, i.e., a plane that is optically conjugate to a back focal plane of the microscope objective 54. The back focal plane of the microscope objective 54 is imaged into the plane of the scanner 46 via the main beam splitter 56, the tube lens 50, and the second lens group 48. Detection light 16 emitted by the sample 1 , e.g., fluorescence light generated in two- photon-processes from fluorescent dyes with which the sample 1 has been prepared, is collected by the microscope objective 54 and directed to the main beam splitter 55. The main beam splitter 55 is preferably a dichroic beam splitter reflecting the excitation light coming from the scanner 46 but transmitting fluorescence light from the sample 1 . The detection light 16 is then focused by lens 56 being arranged at an optical interface 57 of the microscope housing in the direction of a color splitter 63 serving the purpose of separating different colors of the detection light 16.

[0068] The spectral portion of the detection 16 that is transmitted through the color splitter 63 is then detected by the first detector 61 and the spectral portion of the detection light 16 that is reflected at the color splitter 63 is detected by the second detector 62.

[0069] The microscopy method of the present invention can in particular be a multi-photon- microscopy method and comprises the following steps which can be carried out with the microscope 100 as described with reference to figures 1 to 4: distributing the light pulses 14 to the plurality of excitation channels ch#1 , ch#2, ch#N of the excitation beam path, guiding the light pulses 14 via the different excitation channels ch#1 , ch#2, ch#N and the microscope objective 54 into the respectively different focal spots 51 , 52, 53 on or into the sample 1 , varying with the scanner 46 a region on or in the sample 1 being irradiated by the light pulses 14, and detecting emission light 16 emitted by the sample 1 as an optical response to irradiation by the light pulses 14. According to the invention, the different focal spots 51 , 52, 53 are being irradiated through the excitation channels ch#1 , ch#2, ch#N one after another for temporally separating the respective optical responses from the irradiated focal spots 51 , 52, 53.

[0070] Preferably, the microscopy method can further comprise the step of de-multiplexing a detection signal for evaluating the optical response from each of the irradiated spots 51 , 52, 53 on or in the sample 1 . This will be explained with reference to figure 5.

[0071] In figure 5, the chart A schematically shows in arbitrary units the intensity of the respective light pulses in the excitation channels ch#1 , ch#2, ch#N. Light pulses of the same excitation channel are represented, in each case, with the same line type. Light pulses of the first excitation channel ch#1 are shown as solid lines, light pulses of the second excitation channel ch#2 are shown as dotted lines, and light pulses being propagated to the sample through the Nth excitation channel ch#N are shown as dashed lines. Chart B shows the detected fluorescence from the sample 1 in response to the irradiation with the light pulses as shown in chart A. Naturally, the fluorescence light sets in only with a time delay corresponding to the lifetime of the states excited by the light pulses by, e.g., two-photon-processes, in the fluorescent dyes. The fluorescence stemming from respective light pulses of the individual excitation channels are shown in respective boxes which are drawn, in each case, in the same line type has the corresponding light pulses in chart A.

[0072] The de-multiplexing step of the method according to the invention consists in adding the integrated intensities of corresponding boxes for a number of these boxes. E.g., in the example of figure 5, the signal in each of the boxes shown in solid lines and belonging to the first excitation channel ch#1 can be integrated and these integrals can be summed up for a number of periods to yield a measurement value for the focal spot 51 . The number of boxes for which the integrated signals are summed up can be chosen dependent on the scanning velocity. The same can be done for the other focal spots, i.e. , for the other corresponding boxes in chart B. In practice the approach described above is limited to comparatively low laser repetition rates lasers (< 4 MHz).

[0073] An alternative embodiment of a distribution unit 120 of a microscope according to the invention and a corresponding variant of a method according to the invention where faster laser repetition rates are possible will now be described with reference to figures 6 and 7. In this example, the distribution unit 120 is designed for guiding of individual light pulses from the light source to one of the excitation channels ch#1 , ch#2, ch#N, ch#N+1. For this purpose, the distribution unit 120 comprises optical switches 111 , 112, 11 N for directing each of the light pulses 14 into one of the excitation channels ch#1 , ch#2, ch#N, ch#N+1. I.e., contrary to the situation described with respect to figure 1 where each and every one of the light pulses 14 coming from the light source 10 is energetically divided by the beam splitters 11 and 12, in the distribution unit 120 each of the light pulses 14 from the light source 10 is directed, ideally in its entirety, to one of the excitation channels ch#1 , ch#2, ch#N, ch#N+1. The optical switches 111 , 112, 11 N can be Pockels-cells which can be controlled or driven by the control unit 90.

[0074] The corresponding variant of the method according to the invention can preferably carried out in such a way that in the distributing step, sequences of a plurality k of light pulses 14 from the light source 10 immediately following one another are directed into each of the excitation channels of the excitation channels ch#1 , ch#2, ch#N, ch#N+1. This will be described with reference to figure 7 where k = 3.

[0075] In figure 7, charts C, D, and E, schematically depict the control signals over time for the Pockels-cells 111 , 112, 11 N of figure 6, respectively. Chart F depicts schematically the intensity of the light pulses in arbitrary units over time and chart G shows the measured detection light from the sample in response to the irradiation with the light pulses.

[0076] More specifically, in the time interval where Pockels-cell 111 receives the control signal from the control unit 90, incoming light pulses, i.e. , the three light pulses in the boxes shown in solid lines in chart F will be guided by the Pockels-cell 111 to its output 111 b and into the optical fiber 31 of the first excitation channel ch#1 .

[0077] Moving on to the period in which the control signal (chart C) for the Pockels-cell 111 is low and the control signal (chart D) for the Pockels-cell 112 is high, the three light pulses in the boxes shown in dotted lines in chart F will be guided first through output 111 a of Pockels-cell 111 to Pockels-cell 112 and then to its output 112b and into the optical fiber 32 of the second optical channel ch#2. In the same fashion, the light pulses shown in the boxes having dashed lines in chart F will be guided through output 111 a of Pockels-cell 111 and output 112a of Pockels-cell 112 finally to output 11 Nb of Pockels-cell 11 N and into the optical fiber 3N of the Nth excitation channel ch#N. Light pulses being propagated through the optical fiber 3N+1 of the N+1 th excitation channel ch#N+1 are not shown in figure 7.

[0078] Since both light that is transmitted through the respective Pockels-cells and light that is reflected at the Pockels-cells is in each case used power losses can be avoided. With the distribution unit 120 of figure 6, the temporal separation of the irradiation of individual focal spots 51 , 52, 53 is achieved by the guiding of the light pulses in their entirety to only one of the excitation channels. Thus, with the distribution unit 120 of figure 6, no specific delay paths are necessary. Rather, it may be preferable that each of the optical fibers 31 , 32, 3N, 3N+1 have the same lengths.

[0079] The de-multiplexing in the situation of figure 7 again consists in integrating the detection signals in the individual boxes and summing up the integrated signals for a number of boxes. The number of boxes for which the integrated signals are summed up can be chosen again dependent on the scanning velocity.

[0080] In the embodiments of figures 1 to 7, each of the excitation channels ch#1 , ch#2, ch#N, ch#N+1 is designed for propagating in each case light pulses 14 comprising the same spectrum of wavelengths. This spectrum of wavelengths can consist essentially of one wavelength. It can also comprise, e.g., at least a first wavelength A1 and a second wavelength A2 with which different fluorescent dyes can be excited. Fluorescence light coming from these different fluorescent dyes can also differ in its respective wavelength and can be detected, as described above with respect to figure 2, separately with the first detector 61 and the second detector 62.

[0081] The range of axial offset between the optical fibers may be limited as the fibers positioned beyond a certain distance will disturb the laser propagation from fibers positioned remotely. In this regard it may be preferred to introduce a chromatic level of multiplexing, i.e. , a combination of laser pulses with different wavelengths coming from different optical fibers.

[0082] In such an embodiment, at least one of the excitation channels can be designed for propagating light pulses comprising another spectrum of wavelengths as compared to the other excitation channels.

[0083] An example therefor will be described with respect to figure 8. Figure 8 shows a first optical fiber 132 and a second optical fiber 232 which respectively belong to a first excitation channel and a second excitation channel. The first excitation channel comprising the first optical fiber 132 is designed for propagating light pulses 110 and the second excitation channel comprising the second fiber 232 is designed for propagating light pulses 210 having a different wavelength as the light pulses 110. The light pulses 110 can, e.g., have a wavelength of 960 nm and can be used for two-photon- microscopy. The light pulses 210 can have a wavelength of 1300 nm and can be used for three-photon-microscopy. Three-photon-microscopy (1300 nm) can be used, e.g., for imaging at deeper locations in the brain and two-photon-microscopy (960 nm) can be used, e.g., for more shallow layers, and thus simultaneous imaging of multiple layers is possible without limiting temporal resolution, anywhere in the cortex. Light pulses 110 exiting the first optical fiber 132 are merged or recombined with light pulses 210 exiting the second optical fiber 232 by a dichroic mirror 67 allowing them to propagate further through the same excitation beam path in the microscope and being focused at various depths. In the situation of figure 3, this beam splitter 67 would be arranged between the exit ends of the optical fibers and the lens 44. As before, the exit ends of the first optical fiber 132 and the second optical fiber 232 can be spaced independently from each other, both axially and laterally. I.e. , each of the exit ends of the first optical fiber 132 and the second optical fiber 232 can be in a specific spatial relation with respect to the lens 44 for achieving a desired axial or lateral offset. A specific time delay between the light pulses 110 and the light pulses 210 can be achieved by arranging the necessary delay stages upstream of the exit ends of the fibers 132 and 232 shown in figure 8.

[0084] It is also possible to achieve a temporal separation for light pulses having different wavelengths and being propagated in the same excitation channel. An example for this will be described with reference to figure 9. Figure 9 shows the exit end of an optical fiber 82 which is part of an excitation channel that is designed for propagating at least light pulses 83 having a first wavelength A1 , e.g., 920 nm, and light pulses 84 having a second wavelength A2 , e.g., 980 nm.

[0085] The light pulses 83 and 84 exiting the optical fiber 82 first impinge on a first dichroic beam splitter 87 which serves the purpose of separating these light pulses 83, 84. Light pulses 84 are transmitted by the first dichroic beam splitter 87 whereas light pulses 83 are reflected in the direction of mirrors 85 and 86. The light pulses 84 transmitted through the first dichroic beam splitter 87 then impinge on a second beam splitter 88 and are again transmitted. The light pulses 83 on the other hand being reflected from the first dichroic beam splitter 87 are then reflected by the mirrors 85 and 86. Then The light pulses 83 impinge upon the second beam splitter 88 and are again reflected such that downstream of the second beam splitter 88 the beam path of the light pulses 83 and 84 reunited. Since the optical path lengths between the first dichroic beam splitter 87 and the second dichroic beam splitter 88 are different for the light pulses 83 and the light pulses 84, a temporal separation of these light pulses in accordance with their wavelengths can be achieved by the setup of figure 9. By varying, e.g., the position of the mirrors 85 and 86, the path length for the light pulses 83 can be adapted. The components of figure 9 would also need to be arranged between the exit end of an optical fiber and the lens 44 of the excitation beam path (see figure 3).

[0086] The present invention proposes chromatic spatio-temporal multiplexing for simultaneous multiplane imaging in multiphoton miniature microscopy.

[0087] A desired and required defocus aberration is produced by placing the respective exit ends of optical fibers with an in each case specific spatial relation, e.g., at different distances, to a collimation lens in the excitation beam path resulting, e.g., in focusing these pulses through the microscope optics at different depths in the sample (figures 3, 4).

[0088] In one embodiment, one key aspect is the use of optical fibers that deliver, in each case, pulses with a unique temporal delay (figure 1 ). This approach is best suited for comparatively low laser repetition rates lasers (< 4 MHz).

[0089] For faster repetition rates it is possible to use, e.g., fast Pockels-cells opening the beams at different times, and thus generating a similar temporal multiplexed laser pulse structure (figure 6).

[0090] A simultaneous imaging of the plurality of illuminated locations in the sample is achieved by de-multiplexing of the fluorescence signal into multiple channels according to the in each case specific temporal signature introduced by the light pulses in the different excitation channels (figures 5, 7).

[0091] In all cases the invention can enable access to several planes distributed over a certain depth range or at the same depth, but with lateral offset, resulting in a faster imaging of a single plane. More generally, Chromatic Spatio-Temporal Multiplexing can be used to combine multiple scanning paths or defocused beams for higher level of parallel imaging in any scanning laser application.

[0092] List of reference numbers

[0093] I sample

[0094] 10 light source, laser

[0095] II first beam splitter

[0096] 12 second beam splitter

[0097] 14 excitation light pulses

[0098] 21 first delay path

[0099] 22 second delay path

[0100] 2N Nth delay path

[0101] 30 bundle of optical fibers

[0102] 31 first optical fiber

[0103] 32 second optical fiber

[0104] 33 third optical fiber

[0105] 3N Nth optical fiber

[0106] 40 microscope head

[0107] 41 housing of microscope

[0108] 42 ferrule for bundle 30 of optical fibers

[0109] 43 housing of microscope

[0110] 44 focussing component, first lens or lens group forming a relay with second lens or lens group 48, collimating lens

[0111] 46 scanner, pupil plane second lens or lens group, forming a relay with first lens or lens group 44 tube lens first focal spot, first focal volume second focal spot, second focal volume third focal spot, third focal volume main beam splitter, dichroic beam splitter microscope objective detection optics, detection lens first detector second detector beam splitter, dichroic mirror dichroic mirror end of optical fiber 31 end of optical fiber 32 end of optical fiber 33 end of optical fiber, ferrule light with wavelength A1 light with wavelength A2 mirror mirror first dichroic mirror second dichroic mirror control unit, PC microscope according to the invention first optical switch, Pockels-cell 111a first output of optical switch 111

[0112] 111 b second output optical switch 111

[0113] 112 second optical switch, Pockels-cell

[0114] 112a first output of optical switch 112

[0115] 112b second output optical switch 112

[0116] 11 N Nth optical switch, Pockels-cell

[0117] 11 Na first output of optical switch 11 N

[0118] 11 Nb second output optical switch 11 N

[0119] 110 light with wavelength A1

[0120] 120 distribution unit

[0121] 132 end of optical fiber, ferrule

[0122] 210 light with wavelength A2

[0123] 232 end of optical fiber, ferrule ch#1 first excitation channel ch#2 second excitation channel ch#N N-th excitation channel ch#N+1 N+1 th excitation channel d1 distance between end of optical fiber 31 and surface of relay lens 44 d2 distance between end of optical fiber 32 and surface of relay lens 44 d3 distance between end of optical fiber 33 and surface of relay lens 44

[0124] D1 distance between exit surface of microscope objective 54 and focal volume 51 corresponding to optical fiber 31

[0125] D2 distance between exit surface of microscope objective 54 and focal volume 52 corresponding to optical fiber 32

[0126] D3 distance between exit surface of microscope objective 54 and focal volume 53 corresponding to optical fiber 33 A intensity of laser pulses

[0127] B intensity of detected light

[0128] C switching signal of Pockels-cell 111 for first optical fiber 31

[0129] D switching signal of Pockels-cell 112 for second optical fiber 32

[0130] E switching signal of Pockels-cell 11 N for N-th optical fiber 3N

[0131] F 80 MHz laser pulses

[0132] G intensity of detected light k number of light pulses in sequence, k = 3 in Fig. 7 x lateral direction in the sample y lateral direction in the sample z direction of the optical axis of microscope objective 54 z' direction of the optical axis in the microscope housing 41

[0133] A1 first wavelength

[0134] A2 second wavelength

[0135] References

[0136] 1 Piyawattanametha, W. et al. Fast-scanning two-photon fluorescence imaging based on a microelectromechanical systems two- dimensional scanning mirror. Opt Lett 31 , 2018-2020 (2006). https: / / doi.org: 10.1364 / ol.31 .002018

[0137] 2 Sawinski, J. et al. Visually evoked activity in cortical cells imaged in freely moving animals. Proceedings of the National Academy of Sciences of the United States of America 106, 19557-19562 (2009). https: / / doi.org: 10.1073 / pnas.0903680106

[0138] 3 Russell, P. Photonic crystal fibers. Science 299, 358-362 (2003). https: / / doi.org: 10.1126 / science.1079280 4 Klioutchnikov, A. et al. Three-photon head-mounted microscope for imaging deep cortical layers in freely moving rats. Nat Methods 17, 509-513 (2020). https: / / doi.org: 10.1038 / s41592-020-0817-9

[0139] 5 Klioutchnikov, A. et al. A three-photon head-mounted microscope for imaging all layers of visual cortex in freely moving mice. Nat Methods 20, 610-616 (2023). https: / / doi.org: 10.1038 / s41592-022-01688-9

[0140] 6 Zong, W. et al. Large-scale two-photon calcium imaging in freely moving mice. Cell 185, 1240-1256 e1230 (2022). https: / / doi.org: 10.1016 / j.cell.2022.02.017

[0141] 7 Zong, W. et al. Fast high-resolution miniature two-photon microscopy for brain imaging in freely behaving mice. Nat Methods 14, 713-719 (2017). https: / / doi.org: 10.1038 / nmeth.4305

[0142] 8 Beaulieu, D. R., Davison, I. G., Kilic, K., Bifano, T. G. & Mertz, J. Simultaneous multiplane imaging with reverberation two-photon microscopy. Nat Methods 17, 283-286 (2020). https: / / doi.org: 10.1038 / s41592-019-0728-9

[0143] 9 Demas, J. et al. High-speed, cortex-wide volumetric recording of neuroactivity at cellular resolution using light beads microscopy. Nat Methods 18, 1103-1111 (2021 ). https: / / doi.org: 10.1038 / s41592-021-01239-8

[0144] 10 US11226474B2

[0145] 11 WO 2021 / 155330 A9

[0146] 12 US20170227748A1

[0147] 13 https: / / www.labmaker.org / collections / 2-photon-miniscope-mini2p https: / / github.com / kavli-ntnu / MINI2P_toolbox / tree / main / Protocols

Claims

Claims1. Microscope, in particular multi-photon-microscope, comprising at least one light source (10) for emitting light pulses (14) for exciting a sample (1 ) and an excitation beam path with a microscope objective (54) for directing the light pulses (14) on or into the sample (1 ), wherein the excitation beam path comprises• a plurality of separate excitation channels (ch#1 , ch#2, ch#N, ch#N+1 ) being designed for guiding light pulses (14) into in each case different focal spots (51 , 52, 53) on or in the sample (1 ),• a distribution unit (20; 120) for distributing the light pulses (14) to the excitation channels (ch#1 , ch#2, ch#N, ch#N+1 ),• a scanner (46) for varying a region on or in the sample (1 ) being irradiated by the light pulses (14), comprising further at least one detector (61 ) for the detection of emission light (16) emitted by the sample (1 ) as an optical response to irradiation by the light pulses (14) and a detection beam path (54, 55, 56, 63) for guiding the emission light (16) onto the detector (61 ), and comprising a control unit (90) for controlling at least the light source (10) and the scanner (46) and for evaluating the light detected by the detector (61 ), characterized in that for temporally separating the respective optical responses from the irradiated focal spots (51 , 52, 53), the excitation beam path is designed for irradiating the different focal spots (51 , 52, 53) through the excitation channels (ch#1 , ch#2, ch#N, ch#N+1 ) sequentially one after another, each of the excitation channels (ch#1 , ch#2, ch#N, ch#N+1 ) comprises an optical fiber (31 , 32, 33, 3N) having in each case an exit end (71 , 72, 73), and, for guiding light pulses (14) into the respectively different focal spots (51 , 52, 53) the exit ends (71 , 72, 73) of the optical fibers (31 , 32, 33) are arranged with an in each case different spacing (d1 , d2, d3) with respect to a light focussing component (44) of the excitation beam path.

2. Microscope according to claim 1 , further characterized in that the exit ends (71 , 72, 73) of the optical fibers (31 , 32, 33) are arranged with respect to the light focussing component (44) with an in each case different spacing (d1 , d2, d3) in a direction of an optical axis (z1) of the excitation beam path.

3. Microscope according to claim 1 or 2, further characterized in that the exit ends (71 , 72, 73) of the optical fibers (31 , 32, 33) are laterally spaced from each other with respect to a direction of an optical axis (z1) of the excitation beam path.

4. Microscope according to one of the claims 1 to 3, further characterized in that that the optical fibers (31 , 32, 33) do not touch each other in the region of their respective exit ends (71 , 72, 73).

5. Microscope according to one of the claims 1 to 4, further characterized in that the distribution unit (20) is designed for energetically distributing each of the light pulses (14) over the excitation channels (ch#1 , ch#2, ch#N).

6. Microscope according to claim 5, further characterized in that the distribution unit (20) comprises a plurality of neutral beam splitters (11 , 12) for energetically dividing and distributing the light pulses (14) over the different excitation channels (ch#1 , ch#2, ch#N).

7. Microscope according to claim 5 or 6, further comprising a delay path (21 , 22, 2N) in each of the excitation channels (ch#1 , ch#2, ch#N) for temporally delaying the energetically distributed light pulses (14) in each case differently in the respective excitation channels (ch#1 , ch#2, ch#N).

8. Microscope according to one of the claims 1 to 7, further characterized in that the distribution unit (120) is designed for guiding of individual light pulses from the light source to one of the excitation channels (ch#1 , ch#2, ch#N, ch#N+1 ).

9. Microscope according to claim 8, further characterized in that the distribution unit (120) further comprises optical switches (111 , 112, 11 N) for directing each of the light pulses (14) into one of the excitation channels (ch#1 , ch#2, ch#N, ch#N+1 ).

10. Microscope according to claim 9, further characterized in that the optical switches (111 , 112, 11 N) are Pockels-cells.11 . Microscope according to one of the claims 1 to 10, further comprising a tube lens (50) for generating a first intermediate image plane being optically conjugate to a focal plane of the microscope objective (54) and an optical relay comprising a first lens or lens group (44) and a second lens or lens group (48) for generating a second intermediate image plane being optically conjugate to the first intermediate image plane, the exit ends (71 , 72, 73) of the optical fibers (31 , 32, 33) being arranged in or in the vicinity of said second intermediate image plane.

12. Microscope according to claim 11 , further characterized in that the scanner (46) is arranged in or in the vicinity of a pupil plane that is optically conjugate to a back focal plane of the microscope objective (54) and that is formed between the first lens or lens group (44) and the second lens or lens group (48).

13. Microscope according to one of the claims 1 to 12, further comprising a microscope head (40), in which at least the microscope objective (54), the scanner (46), and the exit ends (71 , 72, 73) of the optical fibers (31 , 32, 33) are received.

14. Microscope according to claim 13, further characterized in that the microscope head (40) comprises an optical interface (57) for the passage of emission light (16).

15. Microscope according to one of the claims 1 to 14, further comprising a ferrule (42) in which the exit ends (71 , 72, 73) of the optical fibers (31 , 32, 33) are received.

16. Microscope according to one of the claims 14 or 15, further characterized in that the at least one detector (61 ) is arranged at the optical interface (57).

17. Microscope according to one of the claims 14 to 16, further comprising a detection module at the optical interface (57), the detection module comprising at least one color splitter (63) and a plurality of detectors (61 , 62).

18. Microscope according to one of the claims 1 to 17, further characterized in that each of the excitation channels (ch#1 , ch#2, ch#N, ch#N+1 ) is designed for propagating in each case light pulses (14) comprising the same spectrum of wavelengths.

19. Microscope according to one of the claims 1 to 18, further characterized in that the light source (10) provides excitation light having at least a first wavelength (A1 ) and a second wavelength (A2).

20. Microscope according to one of the claims 1 to 19, further characterized in that at least one of the excitation channels is designed for propagating light pulses comprising another spectrum of wavelengths as compared to the other excitation channels.21 . Microscope according to claim 20, further comprising at least one dichroic mirror (67) for merging light pulses (110) exiting a first optical fiber (132) with light pulses (210) exiting a second optical fiber (232) and having a different wavelength as the light pulses (110) exiting the first optical fiber (132).

22. Microscope according to one of the claims 1 to 21 , further characterized in that at least one of the excitation channels is designed for propagating at least light pulses (83) having a first wavelength (A1 ) and light pulses (84) having a second wavelength (A2) which is different from the first of wavelength (A1 ).

23. Microscope according to claim 22, further comprising at least one first dichroic beam splitter (87) for separating light pulses (83, 84) exiting the same optical fiber (82) and having different wavelengths, and at least one second dichroic beam splitter (88) downstream of the first dichroic beam splitter (87) for merging the light pulses (83, 84) having different wavelengths, wherein the optical path lengths between the first dichroic beam splitter (87) and the second dichroic beam splitter (88) are in each case different for light pulses (83, 84) having different wavelengths.

24. Microscopy method, in particular multi-photon-microscopy method, the method comprising the steps of: distributing light pulses (14) to a plurality of excitation channels (ch#1 , ch#2, ch#N, ch#N+1 ) of an excitation beam path, guiding light pulses (14) via the different excitation channels (ch#1 , ch#2, ch#N, ch#N+1 ) and a microscope objective (54) into respectively different focal spots (51 , 52, 53) on or in the sample (1 ), varying with a scanner (46) a region on or in the sample (1 ) being irradiated by the light pulses (14), detecting emission light (16) emitted by the sample (1 ) as an optical response to irradiation by the light pulses (14), the method being further characterized in the step of irradiating the different focal spots (51 , 52, 53) through the excitation channels (ch#1 , ch#2, ch#N, ch#N+1) is carried out sequentially one focal spot (51 , 52, 53) after another for temporally separating the respective optical responses from the irradiated focal spots (51 , 52, 53), wherein each of the excitation channels (ch#1 , ch#2, ch#N, ch#N+1 ) comprises an optical fiber (31 , 32, 33) having in each case an exit end (71 , 72, 73), and, for guiding light pulses (14) into the respectively different focal spots (51 , 52, 53) the exit ends (71 , 72, 73) of the optical fibers (31 , 32, 33) are arrangedwith an in each case different spacing (d1 , d2, d3) with respect to a light focussing component (44) of the excitation beam path.

25. Method according to claim 24, further characterized in that in the distributing step, the light pulses (14) are energetically divided and distributed over the different excitation channels (ch#1 , ch#2, ch#N).

26. Method according to claim 25, further characterized in that the energetically divided light pulses (14) are subjugated to a different delay in each of the excitation channels (ch#1 , ch#2, ch#N).

27. Method according to one of the claims 24 to 26, further characterized in that in the distributing step, each of the light pulses (14) from the light source (10) is directed to one of the excitation channels of the excitation channels (ch#1 , ch#2, ch#N, ch#N+1 ).

28. Method according to claim 27, further characterized in that in the distributing step, sequences of a plurality (k) of light pulses (14) from the light source (10) immediately following one another are directed one after another into each of the excitation channels of the excitation channels (ch#1 , ch#2, ch#N, ch#N+1 ).

29. Method according to one of the claims 24 to 28, further comprising the step of de-multiplexing a detection signal for evaluating the optical response from each of the irradiated spots (51 , 52, 53) on or in the sample (1 ).

Images