Microscopes and microscopy methods

Abstract

The invention relates to a microscope comprising at least one light source for emitting light pulses for exciting a sample and an excitation light path with a microscope objective for guiding the light pulses onto or into the sample, wherein the excitation light path comprises a plurality of independent excitation channels designed for guiding the light pulses onto or into respectively different foci on or in the sample, a distribution unit for distributing the light pulses to the excitation channels, a scanner for changing the area on or in the sample illuminated by the light pulses, at least one detector for detecting emission light emitted as an optical response by the sample in response to the illumination by the light pulses, and a detection light 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 illuminated foci, the excitation light path is designed for sequentially illuminating the different foci one after the other through the excitation channels, each of the excitation channels comprising one optical fiber with a respective exit end, and, for guiding the light pulses onto the respectively different foci, the exit ends of the optical fibers are arranged with respectively different spacings relative to a light focusing assembly of the excitation light path. The invention also relates to a microscopy method.

Description

Technical Field

[0001] The present invention relates to a microscope as described in the preamble of claim 1 and a microsurgical method as described in the preamble of claim 24. Background Technology

[0002] A general-purpose microscope, particularly a multiphoton microscope, includes at least one light source for emitting light pulses to excite a sample; and an excitation optical path having microscope objectives for guiding the light pulses onto or into the sample. The excitation optical path includes multiple independent excitation channels designed to guide the light pulses to their respective different focal points on or within the sample; a distribution unit for distributing the light pulses to the excitation channels; and a scanner for varying the area on or within the sample illuminated by the light pulses. The general-purpose microscope also includes at least one detector for detecting emitted light emitted as an optical response by the sample in response to light pulse illumination; a detection optical path for guiding the emitted 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.

[0003] A general microscopy method, particularly a multiphoton microscopy method, includes the following steps: distributing light pulses to multiple excitation channels in an excitation optical path; guiding the light pulses to different focal points on or within a sample via different excitation channels and microscope objectives; using a scanner to change the area on or within the sample irradiated by the light pulses; and detecting the emitted light emitted by the sample as an optical response to the light pulse irradiation.

[0004] Common microscopes and common microscopy techniques are described, for example, in US 20170227748 A1

[12] .

[0005] Multiphoton imaging has become an important tool for monitoring neuronal activity. Optical tomography enables non-invasive optical access to deep structures within biological tissues, which is key to imaging neuronal populations in living and conscious animals. Combining multiphoton imaging with miniaturization techniques [1, 2] and hollow fiber [3] has produced small head-mounted microscopes that provide the ability to measure neuronal population activity in freely moving animals during behavioral tasks, where the neural networks under study are functional under their natural conditions [4-7].

[0006] The recently released open-source miniature two-photon microscope (Mini2P,

[13] ) provides the neuroscience community with such a technique. However, this technique is limited by the scanner’s throughput (the product of image size, spatial and temporal resolution). The Mini2P is equipped with an electrically tunable lens that allows switching between different groups of neurons within a certain depth range (about 250 μm). While this approach significantly increases the number of neurons observed and expands the range of structures observed, it distributes imaging time across multiple imaging planes, thus proportionally reducing the temporal resolution of each plane. Furthermore, the characteristics of the excitation employed (two-photon absorption) and the electrically tunable lens limit the maximum imaging depth to about 300 μm to 400 μm (half the depth of the mouse cortex) and the displacement range to about 250 μm, allowing only the study of the superficial portions of the cortex. This is also a significant limitation, as the cerebral cortex is functionally layered and neurons at different depths have distinctly different properties, making the ability to image multiple planes simultaneously at different depths highly desirable.

[0007] A simultaneous multiplanar imaging method has been validated on large microscopes [8,9], which involves splitting a strong laser pulse into a series of smaller pulses with increasing delays and increasing optical defocus (i.e., optical properties of the laser beam) in the tens of nanoseconds range. Defocusing coupled into the microscope causes the laser pulses to focus at different depths, thus enabling imaging at different depths, while the time delay allows for parallel detection of emission fluorescence from the sample at the detection electronics by demultiplexing the signal using the nanosecond-level delay introduced between pulses, achieving near-simultaneous multiplanar imaging without sacrificing temporal resolution. However, this design cannot be miniaturized because generating the delay for the laser pulses requires a long distance; for example, 10 ns corresponds to approximately 3 meters in air. It is also impossible to couple the already structured pulse sequence through optical fibers, as optical aberrations such as defocus are lost when coupled to single-mode fibers or similar materials (such as hollow-core fibers used in small multiphoton microscopes). Therefore, this method cannot be used to obtain simultaneous multiplanar imaging. Summary of the Invention

[0008] One of the problems of the present invention is to provide a microscope and microscopy method that are easy to miniaturize.

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

[0010] According to the present invention, the aforementioned universal microscope is further characterized in that: in order to separate the respective optical responses from the irradiated focal points in time, the excitation optical path is designed to sequentially irradiate different focal points one after another through the excitation channels, each of the excitation channels comprising an optical fiber having a respective exit end, and, in order to guide the light pulse to their respective different focal points, the exit ends of the optical fibers are arranged to have their respective different spacings relative to the optical focusing or collimating components of the excitation optical path.

[0011] According to the present invention, the above-described general microscopy method is further characterized in that the step of irradiating different focal points through the excitation channel is performed sequentially, one focal point after another, so as to separate the respective optical responses from the irradiated focal points in time, wherein each of the excitation channels includes an optical fiber having its own exit end, and, in order to guide the light pulse to its respective different focal point, the exit ends of the optical fibers are arranged to have their own different spacing relative to the optical focusing components of the excitation optical path.

[0012] The advantages of the present invention, as well as embodiments of the microscope and variations of the microscopy method according to the present invention, are described below with particular reference to the dependent claims and drawings.

[0013] The light source may include one or more lasers that provide excitation light with the desired spectral range and intensity. The excitation light may be electromagnetic radiation in the visible and adjacent ranges (i.e., the infrared and ultraviolet ranges). For example, typical pulse lengths for multiphoton microscopy can be as low as 100 femtoseconds, with repetition frequencies, for example, in the range of 80 to 100 MHz.

[0014] The term "excitation optical path" refers to all optical beam guiding and beam-modifying components, such as lenses, mirrors, prisms, gratings, filters, apertures, beam splitters, and modulators (e.g., spatial light modulators (SLMs)), through which the excitation light is guided from the light source to the sample under test. The excitation light is guided onto the sample via microscope objectives, particularly the same microscope objective that is also part of the detection optical path. For multiphoton microscopy, microscope objectives that have undergone chromatic aberration correction for both the excitation and detection wavelengths are preferred. The term "exit end" refers to the end where the excitation light leaves its respective optical fiber from the laser.

[0015] The back focal plane of a microscope objective and the plane optically conjugate thereto are also called the pupil plane. The term "intermediate image plane" refers to the plane in the excitation or detection optical path that is optically conjugate to the focal plane of the microscope objective.

[0016] The light emitted and / or deflected (e.g., scattered, typically radiated) by the sample under test in response to excitation light is referred to as emitted light or detection light, and reaches the detector via the detection optical path. The term "detection optical path" refers to all optical beam-guiding and beam-modifying components, such as lenses, mirrors, prisms, gratings, filters, apertures, beam splitters, and modulators (e.g., spatial light modulators (SLMs)), through which the emitted light is guided from the sample to the detector. This detection optical path may include the same microscope objective that is part of the excitation optical path.

[0017] The scanner can be, for example, a typical galvanometer scanner or a MEMS scanner (MEMS = Microelectromechanical Systems), or a combination of these components. The beam can also be scanned by appropriately manipulating the excitation light using a spatial light modulator (e.g., a phase-modulated spatial light modulator); that is, the scanner can include or be implemented by a spatial light modulator (SLM). The SLM is preferably arranged in the pupil plane of the excitation light path.

[0018] The detection light, such as fluorescence, is preferably detected using a high-bandwidth detector that generates a photocurrent lasting for several nanoseconds to minimize crosstalk between temporally adjacent channels. The detector can be, for example, a photomultiplier tube or a photodiode. The optical fiber can be single-mode fiber. Hollow-core fiber is preferred. The term "focal point" can, for example, refer to the diffraction-limited volume into which the excitation light is focused.

[0019] The first important idea of ​​this invention can be considered as follows: by spatially separating the output ends of each optical fiber, each channel achieves a distinct focal position within the sample. The second important idea of ​​this invention can be considered as follows: by sequentially illuminating different focal points one after another, the optical responses from the illuminated focal points are separated in time.

[0020] The first important advantage of this invention is that it allows for a variety of variations in the spatial arrangement of the fiber optic exit ends, thereby allowing for a variety of variations in the focal position within the sample. Another important advantage is that the arrangement of each fiber optic exit end can be easily miniaturized, thus the microscopy and microscopy methods according to the invention can be readily applied to living objects.

[0021] Generally, the microscope and microscopy methods of the present invention can be used with conventional laser scanning microscopes. However, the microscope and microscopy methods of the present invention are particularly suitable for multiphoton microscopes, such as two-photon microscopes and three-photon microscopes.

[0022] In a first important preferred embodiment, the emitting ends of the optical fibers are arranged at different intervals relative to the optical focusing assembly along the optical axis of the excitation optical path. Therefore, depth resolution of the sample can be achieved.

[0023] Alternatively or additionally, the emitting ends of the optical fibers may be laterally spaced apart from each other and / or laterally spaced relative to the optical axis of the excitation optical path. Embodiments where the emitting ends of the optical fibers are laterally spaced relative to the optical axis but have equal spacing relative to the optical focusing assembly correspond to multifocal microscopy. In this case, for example, it is preferable to arrange the emitting ends of the optical fibers and thus the illumination points on or within the sample along a line perpendicular to the direction in which the scanner moves the light spot on the sample. However, for HDR (High Dynamic Range) microscopy or DI (Dynamic Illumination) microscopy methods, for example, the emitting ends of the optical fibers and thus the illumination points on or within the sample may also be arranged along a line parallel to the direction in which the scanner moves the light spot on the sample. The optical fibers can be arranged at such a spacing that they do not contact each other within the region of their respective emitting ends.

[0024] In another preferred embodiment, the microscope further includes a collar in which the emitting end of the optical fiber is received. Such a collar defines the position of each emitting end of each optical fiber in the transverse direction relative to the optical axis and in the optical axis direction relative to the light focusing assembly. The microscope housing is preferably designed to receive the collar in the defined position and orientation. In another preferred embodiment, multiple different collars can be provided, each collar implementing a different illumination mode, for example, all illumination points are spaced apart from each other in the transverse direction along a line, or all illumination points are spaced apart from each other substantially only in the axial direction, and / or combinations thereof. Thus, a multi-mode microscope can be created where one method can be switched to another by changing the collar and, if necessary, also replacing the optical fiber itself and switching the control unit accordingly.

[0025] In a first embodiment of the microscope according to the invention, the distribution unit is designed to energy-distribute each light pulse to the excitation channel. In a corresponding embodiment of the microscopy method according to the invention, the light pulse is energy-divided and distributed to different excitation channels in the distribution step. That is, each light pulse is divided into multiple smaller pulses, and the number of smaller pulses is preferably equal to the number of excitation channels in the excitation optical path.

[0026] To energetically split light pulses and distribute them to different excitation channels, the distribution unit may include multiple beamsplitters for energetically splitting the light pulses and distributing them to different excitation channels. One, more, or all of these beamsplitters may be neutral beamsplitters. Choosing all beamsplitters as neutral beamsplitters is suitable for cases where the intention is to apply the same electromagnetic radiation spectrum at each focus. However, one, more, or all of these beamsplitters may also be dichroic beamsplitters. Choosing dichroic beamsplitters can be used, for example, in multifocal microscopy modes where the intention is to illuminate each focus with a different electromagnetic radiation spectrum, at least in some cases or all cases. There is also freedom regarding the pulse intensity. The light pulses of excitation light in each excitation channel may each have the same energy. However, they may also have increasing energies, for example, in HDR multifocal modes.

[0027] Furthermore, one, more, or all of these beamsplitters can be controllable beamsplitters, such as acousto-optic devices like AOTF or AOM. These devices will allow control over the energy and / or spectral composition of individual portions of the optical pulses in each excitation channel. If such acousto-optic devices are not fast enough to separate individual pulses at, for example, 80 MHz, it is preferable to use a slower laser pulse rate and / or separate pulses in groups of pulses (e.g., 5 to 10 pulses per group), thereby effectively reducing the required speed by a scaling factor.

[0028] In another embodiment, the microscope may further include a delay path in each of the excitation channels for applying different time delays to the energy-divided light pulses in their respective excitation channels. In a corresponding embodiment of the microscopy method, the energy-divided light pulses are subjected to different delays in each excitation channel.

[0029] The first important idea of ​​this embodiment can be considered as follows: to achieve time-separated illumination at different focal points by using different optical path lengths in each channel. These different optical path lengths in each channel can be achieved, for example, by using optical fibers of different lengths.

[0030] In another preferred embodiment of the microscope according to the invention, the dispensing unit is designed to guide individual light pulses from the light source to one of the excitation channels. In a corresponding preferred variant of the microscopy method according to the invention, in the dispensing step, each light pulse from the light source is guided to one of the excitation channels. In contrast to the previously described example (where each light pulse is divided into multiple partial light pulses), the basic idea of ​​this embodiment is to guide each light pulse from the light source as a whole, i.e., completely, to only one excitation channel.

[0031] A hybrid embodiment in which the pulse is energy-splitting and a portion of the pulse is subsequently guided to different excitation channels via an optical switch is also possible.

[0032] In this regard, the distribution unit may also include an optical switch for directing each optical pulse to one of the excitation channels. As the optical switch, an acousto-optic component, such as an AOTF, can be used. Using these components, the spectral composition of the optical pulses can also be shaped individually. If such an acousto-optic device is not fast enough to separate a single pulse at, for example, 80 MHz, it is preferable to use a slower laser pulse rate and / or separate pulses in groups (e.g., 5 to 10 pulses per group), thereby effectively reducing the required speed by a scaling factor. It is also preferable that the optical switch is a Pockels cell, which is faster than an acousto-optic component.

[0033] The actual measurement rate is not limited to the switching rate achievable by the optical switch used. Instead, with the repetition frequency of the laser source used being higher than the switching rate of the optical switch used, multiple consecutive sequences of light pulses from the light source are directed one after another into each of the excitation channels.

[0034] The method preferably further includes a step of demultiplexing the detection signal to evaluate the optical response from each irradiated point on or within the sample. This evaluation can be performed in a suitably programmed control unit.

[0035] In an advantageous embodiment of the invention, the microscope further includes a tube lens for generating a first intermediate image plane optically conjugate to the focal plane of the microscope objective. In principle, the emitting end of the optical fiber could be positioned in this first intermediate image plane immediately upstream of the tube lens in the excitation optical path. However, in that case, the scanner would have to be mounted in the rear focal plane of the microscope objective, which is impractical for various reasons. Therefore, it is preferable to additionally have an optical relay system comprising a first lens or lens group and a second lens or lens group for generating a second intermediate image plane optically conjugate to the first intermediate image plane. The emitting end of the optical fiber can then preferably be positioned in or near the second intermediate image plane.

[0036] In a further preferred embodiment, the scanner is arranged in or near the pupil plane, the pupil plane being optically conjugate to the back focal plane of the microscope objective and formed between the first lens or lens group and the second lens or lens group.

[0037] For example, for microscopic studies of live mice or other laboratory animals, a miniaturized microscope apparatus that can be easily applied to the animal being examined is preferred. In this case, the microscope may preferably also include a microscope head end, which can be easily applied to or attached to, for example, the head of the animal being examined. Advantageously, such a microscope head end may include the microscope objective, the scanner, and the exit end of the optical fiber, preferably arranged in a collar as described above. The microscope may also include a main beam splitter, such as a dichroic beam splitter, for separating the emitted light and light pulses from the excitation light path. The main beam splitter may preferably be housed and arranged in the microscope head end. The tube lens and the optical relay system may also preferably be housed and arranged in the microscope head end.

[0038] The microscope head may also include an optical interface for the transmission of emitted light. The at least one detector may be disposed at the optical interface.

[0039] The sample can be prepared using more than one fluorescent dye, for example, two different fluorescent dyes that emit fluorescence at different wavelengths. In this case, it is further preferred to have a detection module at the optical interface, which includes at least one color spectrometer and multiple detectors. Two color detection channels will be required when detecting two different wavelengths. The detection module can also be attached to the end of the microscope head.

[0040] Typically, this invention can be implemented using a microscope in which each of the excitation channels is designed to propagate light pulses containing the same wavelength spectrum. Using such a microscope, samples prepared with fluorescent dyes that can be excited by photons of the same wavelength can be studied.

[0041] In many cases, greater flexibility relative 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 to propagate a light pulse containing a different wavelength spectrum compared to the other excitation channels.

[0042] In another embodiment, the microscope further includes at least one dichroic mirror for combining light pulses exiting the first optical fiber with light pulses exiting the second optical fiber that have a different wavelength than the light pulses exiting the first optical fiber.

[0043] It is also advantageous that at least one of the excitation channels is designed to propagate at least a light pulse having a first wavelength spectrum and a light pulse having a second wavelength spectrum different from the first wavelength spectrum, or more specifically, at least one of the excitation channels is designed to propagate at least a light pulse having a first wavelength and a light pulse having a second wavelength different from the first wavelength.

[0044] The microscope in another embodiment of the present invention further includes at least one first dichroic beam splitter for separating light pulses with different wavelengths that leave the same optical fiber; and at least one second dichroic beam splitter located downstream of the first dichroic beam splitter for merging light pulses with different wavelengths, wherein the optical path length between the first dichroic beam splitter and the second dichroic beam splitter is different for light pulses with different wavelengths.

[0045] These variations may be advantageous for situations where samples need to be studied simultaneously using both two-photon and three-photon microscopy. Attached Figure Description

[0046] Other properties and advantages of the present invention will be described below with reference to the accompanying drawings.

[0047] Figure 1 The first embodiment of the microscope according to the present invention is shown;

[0048] Figure 2 : Showed in more detail Figure 1 The tip of a medium-sized microscope;

[0049] Figure 3 : Showed in more detail Figure 2 The fiber optic output end in the head of a medium-sized microscope;

[0050] Figure 4 : Shows the use Figure 2 Details of the focus produced by the microscope tip in the sample;

[0051] Figure 5 : A timeline is shown illustrating a first variation of the microscopy method according to the present invention;

[0052] Figure 6 The dispensing unit of a second embodiment of the microscope according to the present invention is shown;

[0053] Figure 7 : A timeline is shown illustrating a second embodiment of the microscopy method according to the present invention;

[0054] Figure 8 : This illustrates an alternative arrangement of the fiber optic exit end in a microscope according to the present invention; and

[0055] Figure 9 This illustrates yet another alternative arrangement of the fiber optic output end in a microscope according to the present invention.

[0056] In the accompanying drawings, the same components are usually represented by the same reference numerals. Detailed Implementation

[0057] Reference Figures 1 to 4 A first embodiment of the microscope 100 according to the present invention will be described. Then, reference will be made to... Figures 1 to 5 A first example of the microsurgical method according to the present invention is described.

[0058] Figure 1 The microscope 100 schematically shown in the diagram can be, in particular, a multiphoton microscope, and includes at least one light source 10 for emitting light pulses 14 to excite the sample 1, and a microscope objective 54 (see [reference]). Figure 2 , 4 The excitation optical path 10 is used to guide the light pulse 14 onto or into sample 1. The light source 10 can be a tunable laser, particularly one that provides near-infrared (NIR) excitation light. A commonly used laser for two-photon microscopy is a Ti:sapphire laser. This laser provides an average power greater than 2.3 W at 800 nm and its wavelength is tunable from 720 nm to 1060 nm. This allows users to target specific compounds for two-photon fluorescence imaging and photostimulation / photodeactivation. Laser pulses can be on the order of approximately 100 femtoseconds (fs). For example, for typical neuron imaging applications, a high repetition rate laser (80 MHz) is used for two-photon imaging (wavelength 920–980 nm), and a low repetition rate laser (< 4 MHz) is used for three-photon excitation at 1300 nm. However, the microscopy and microscopy methods of the present invention are not limited to these parameters. In order to keep the laser pulse at the desired pulse length and maintain high excitation efficiency, it is also preferable to have a pre-compensation or pre-chirped stage for the laser pulse to reduce dispersion, as detailed in references [4]-[7].

[0059] The excitation optical path includes multiple independent excitation channels ch#1, ch#2, and ch#N. These excitation channels are designed to guide the light pulse 14 to different focal points 51, 52, and 53 on or within sample 1 (see [link to sample 1]). Figure 4 Furthermore, the excitation optical path includes a distribution unit 20 for distributing the light pulse 14 to excitation channels ch#1, ch#2, and ch#N, and a scanner 46 for changing the area on or within sample 1 irradiated by the light pulse 14 (see [link to scanner]). Figure 2 ).

[0060] The microscope 100 also includes a detector 61 for detecting emitted light 16 emitted by the sample 1 as an optical response to illumination by the light pulse 14; and detection optical paths 54, 55, 56, and 63 for guiding the emitted light 16 onto the detector 61. Figure 1In the example, detector 61 is a first detector, and microscope 100 includes a second detector 62. The first detector 61 and the second detector 62 may be, for example, photomultiplier tubes (PMTs) or high-sensitivity semiconductor detectors. Preferably, detectors 61 and 62 have high bandwidth and are capable of generating photocurrents lasting several nanoseconds to minimize crosstalk between temporally adjacent excitation channel signals.

[0061] Control unit 90, such as PC, is used to control at least light source 10 and scanner 46, and to evaluate the light detected by detectors 61, 62.

[0062] According to the present invention, the excitation optical path is designed to sequentially illuminate different focal points 51, 52, and 53 one after another through excitation channels ch#1, ch#2, and ch#N. Therefore, the respective optical responses from the illuminated focal points 51, 52, and 53 can be separated in time. Each excitation channel ch#1, ch#2, and ch#N includes optical fibers 31, 32, and 3N with respective exit ends 71, 72, and 73 (see...). Figure 3 To guide the optical pulse 14 to its respective focal points 51, 52, and 53, the exit ends 71, 72, and 73 of optical fibers 31, 32, and 33 are arranged with different spacings d1, d2, and d3 relative to the optical focusing or collimating assembly 44 of the excitation optical path (see...). Figure 3 ) Fibers 31, 32, 33, and 3N can preferably be single-mode and / or hollow-core fibers.

[0063] exist Figure 1 In the example shown, the distribution unit 20 is designed to distribute the energy of each optical pulse 14 to the excitation channels ch#1, ch#2, and ch#N. This is in Figure 1 In the example, multiple neutral beam splitters 11 and 12 are used to energy-wise divide the optical pulse 14 and distribute it to different excitation channels ch#1, ch#2, and ch#N.

[0064] Neutral beam splitters 11 and 12 can preferably be designed such that the energy-splitting pulses (also referred to as partial pulses) guided into excitation channels ch#1, ch#2, and ch#N each have the same energy. For example, in the case where the distribution unit 20 comprises a total of three excitation channels and assuming no energy loss in the beam splitters 11 and 12, this can be achieved by directing 33% of the incident energy to the neutral beam splitter 11 in excitation channel 21 and by directing 50% of the incident energy to the neutral beam splitter 12 in excitation channel 22.

[0065] In addition, Figure 1In the example, each excitation channel includes its own specific delay paths 21, 22, 2N, used to apply different time delays to the energy-distributed optical pulses 14 in their respective excitation channels ch#1, ch#2, ch#N. Figure 1 In the example shown, each delay path adds another 10 ns relative to the previous excitation channel. This can be achieved, for example, separately using approximately 2 meters of optical fiber. The microscope 100 includes a microscope head end 40, into which optical fibers 31, 32, and 3N are introduced as an optical fiber bundle 30.

[0066] Figures 2 to 4 The microscope tip 40 is shown in more detail for a total of three excitation channels and therefore three corresponding optical fibers 31, 32, 33. Figure 3 Showing Figure 2 Details of the Chinese frame I. Figure 4 Showing Figure 2 Details of the Chinese frame II.

[0067] The bundle 30 of optical fibers 31, 32, and 33 is held in a ferrule 42, which is housed within the first tubular portion of the housing 41 of the microscope tip 40. (As...) Figure 3 As shown in detail, the output ends 71, 72, and 73 of optical fibers 31, 32, and 33 are arranged with different spacings d1, d2, and d3 relative to the optical focusing assembly 44 in the direction of the optical axis z' of the excitation optical path.

[0068] exist Figure 2 In the example shown, the focusing assembly 44 is a first lens group, which forms a relay optical system with a second lens group 48 arranged further downstream in the excitation optical path. An intermediate image plane is formed between the tube lens 50 and the second lens group 48, that is, an optical plane optically conjugate to the focal plane of the microscope objective lens 54 in sample 1. This intermediate image plane is imaged through the second lens group 48 and the first lens group 44 onto another intermediate image plane located near the exit ends 71, 72, 73 of the optical fibers 31, 32, 33 (see...). Figure 3 ).

[0069] Therefore, the different axial spacings d1, d2, d3 of the output ends 71, 72, 73 of optical fibers 31, 32, 33 relative to the first lens group 44 (see...) Figure 3 The ) are transformed into different axial depths D1, D2, and D3 corresponding to focal points 51, 52, and 53 in sample 1 (see Figure 4 Sample 1 could be, for example, the brain of a living animal.

[0070] The exit ends 71, 72, and 73 of optical fibers 31, 32, and 33 are spaced laterally relative to the optical axis z' of the excitation optical path. In the example shown, this lateral spacing is limited by the finite thickness of each optical fiber 31, 32, and 33, which are in close contact with each other within the ferrule 42 (see...). Figure 3 Due to the small lateral spacing of the output ends 71, 72, and 73 of optical fibers 31, 32, and 33, the corresponding focal points 51, 52, and 53 are also laterally spaced (i.e., in...). Figure 4 (In the middle, along the y-axis). But in reality, the lateral spacing is much smaller than the axial spacing. Figure 4 As shown. If a larger lateral spacing between the focal points in the sample is desired, the optical fibers can, of course, be arranged in the ferrule 42 such that they do not touch each other in the region of their respective exit ends.

[0071] Focuses 51, 52, and 53 can be moved laterally by scanner 46, i.e. Figure 4 In the x and y directions. Scanner 46 is a mirror that can pivot about two independent axes via a MEMS driver or a galvanometer driver. In the example shown, scanner 46 is arranged in the pupil plane of the excitation optical path, i.e., the plane optically conjugate to the back focal plane of microscope objective 54. The back focal plane of microscope objective 54 is imaged onto the plane where scanner 46 is located via main beam splitter 56, tube lens 50, and second lens group 48.

[0072] The detection light 16 emitted by sample 1, such as fluorescence generated from the fluorescent dye used to prepare sample 1 via a two-photon process, is collected by microscope objective 54 and directed to main beam splitter 55. Main beam splitter 55 is preferably a dichroic beam splitter that reflects the excitation light from scanner 46 but transmits the fluorescence from sample 1. Detection light 16 is then focused by lens 56 arranged at the optical interface 57 of the microscope housing toward color beam splitter 63, which separates the different colors of detection light 16.

[0073] The spectral portion of the detection light 16 that passes through the color spectrometer 63 is then detected by the first detector 61, while the spectral portion of the detection light 16 reflected at the color spectrometer 63 is detected by the second detector 62.

[0074] The microscopy method of the present invention can be particularly multiphoton microscopy, and includes methods that can be used with reference to Figures 1 to 4The microscope 100 described performs the following steps: distributing light pulses 14 to multiple excitation channels ch#1, ch#2, ch#N of the excitation optical path; guiding the light pulses 14 to different focal points 51, 52, 53 on or within the sample 1 via the different excitation channels ch#1, ch#2, ch#N and the microscope objective lens 54; changing the area on or within the sample 1 irradiated by the light pulses 14 using a scanner 46; and detecting the emitted light 16 emitted by the sample 1 as an optical response to the irradiation of the light pulses 14. According to the invention, the different focal points 51, 52, 53 are irradiated one after another through the excitation channels ch#1, ch#2, ch#N, so as to temporally separate the respective optical responses from the irradiated focal points 51, 52, 53.

[0075] Preferably, the microsurgical method may further include the step of demultiplexing the detection signal to evaluate the optical response from each irradiated point 51, 52, 53 on or within sample 1. This will refer to... Figure 5 To explain.

[0076] exist Figure 5 In Figure A, the intensity of the light pulses in each excitation channel ch#1, ch#2, and ch#N is schematically displayed in arbitrary units. Light pulses within the same excitation channel are represented by the same line type. The light pulses of the first excitation channel ch#1 are shown as solid lines, the light pulses of the second excitation channel ch#2 as dotted lines, and the light pulses propagating to the sample through the Nth excitation channel ch#N as dashed lines. Figure B shows the fluorescence detected from sample 1 in response to the light pulses shown in Figure A. Naturally, fluorescence only begins to occur after a time delay corresponding to the lifetime of the state excited in the fluorescent dye by the light pulse (e.g., via a two-photon process). Fluorescence originating from the respective light pulses of each excitation channel is shown in the corresponding boxes, each drawn with the same line type as the corresponding light pulse in Figure A.

[0077] The demultiplexing step of the method according to the invention includes accumulating the integral intensities of corresponding boxes within a plurality of such boxes. For example, in Figure 5 In the example, the signal in each box (shown as a solid line) belonging to the first excitation channel ch#1 can be integrated, and these integrals over multiple cycles can be summed to obtain the measurement for focus 51. The number of boxes for which the integrated signal is accumulated can be selected based on the scan rate. The same can be done for other focuses, i.e., for the other corresponding boxes in Figure B. In practice, the above method is limited to lasers with low repetition rates (< 4 MHz).

[0078] Now refer to Figure 6 and Figure 7Alternative embodiments of the dispensing unit 120 of the microscope according to the invention and corresponding variations of the method according to the invention are described, wherein a faster laser repetition rate can be used. In this example, the dispensing unit 120 is designed to guide 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 dispensing unit 120 includes optical switches 111, 112, 11N for guiding each light pulse 14 to one of the excitation channels ch#1, ch#2, ch#N, ch#N+1. That is, compared with reference to... Figure 1 In contrast to the described scenario (where each light pulse 14 from light source 10 is energy-splittered by beam splitters 11 and 12), in distribution unit 120, each light pulse 14 from light source 10 is ideally and completely guided to one of excitation channels ch#1, ch#2, ch#N, ch#N+1. Optical switches 111, 112, 11N can be Pockels cells, which can be controlled or driven by control unit 90.

[0079] A corresponding variation of the method of the present invention can preferably be carried out in such a manner that, in the allocation step, a sequence of multiple k consecutive light pulses 14 from the light source 10 is directed to each of the excitation channels ch#1, ch#2, ch#N, and ch#N+1. This will refer to... Figure 7 Describe it, where k=3.

[0080] exist Figure 7 In the diagrams, charts C, D, and E schematically depict... Figure 6 The control signals of the pucker cells 111, 112, and 11N change over time. Graph F schematically depicts the change in light pulse intensity over time in arbitrary units, and Graph G shows the detection light measured from the sample in response to light pulse illumination.

[0081] More specifically, during the time interval during which the Pockels box 111 receives a control signal from the control unit 90, the incident light pulses, i.e., the three light pulses shown in solid boxes in Figure F, will be guided by the Pockels box 111 to its output terminal 111b and into the optical fiber 31 of the first excitation channel ch#1.

[0082] Next, during the period when the control signal of Pockels cell 111 (Figure C) is low and the control signal of Pockels cell 112 (Figure D) is high, the three optical pulses shown in the dotted box in Figure F will first be guided through the output terminal 111a of Pockels cell 111 to Pockels cell 112, then to its output terminal 112b and into the fiber 32 of the second optical channel ch#2. Similarly, the optical pulses shown in the dashed box in Figure F will be guided through the output terminals 111a of Pockels cell 111 and 112a of Pockels cell 112, finally to the output terminal 11Nb of Pockels cell 11N and into the fiber 3N of the Nth excitation channel ch#N. The optical pulses propagating through the fiber 3N+1 of the (N+1)th excitation channel ch#N+1 do not... Figure 7 As shown in the image.

[0083] Since both the light transmitted through their respective Pockels and the light reflected at those Pockels are utilized, power loss can be avoided. Figure 6 The distribution unit 120 achieves temporal separation of the illumination of each focus 51, 52, and 53 by completely guiding the light pulse to only one excitation channel. Therefore, utilizing... Figure 6 The distribution unit 120 does not require a specific delay path. Instead, it is preferable that each fiber 31, 32, 3N, 3N+1 has the same length.

[0084] Figure 7 Demultiplexing in this case again involves integrating the detection signals in each box and accumulating the integrated signals from multiple boxes. The number of boxes for which the integrated signals are accumulated can also be selected based on the scan speed.

[0085] exist Figures 1 to 7 In this embodiment, each excitation channel ch#1, ch#2, ch#N, ch#N+1 is designed to propagate light pulses 14 containing the same wavelength spectrum, respectively. This wavelength spectrum can consist substantially of one wavelength. It can also include, for example, at least a first wavelength λ1 and a second wavelength λ2, which can be used to excite different fluorescent dyes. The fluorescence from these different fluorescent dyes can also differ at their respective wavelengths, and can be referred to above. Figure 2 The detection is performed using the first detector 61 and the second detector 62, respectively.

[0086] The axial offset range between optical fibers may be limited because fibers positioned beyond a certain distance can interfere with laser propagation from the distant fiber. In this regard, it is preferable to introduce a certain level of color multiplexing, i.e., combining laser pulses with different wavelengths from different fibers.

[0087] In such an embodiment, at least one excitation channel can be designed to propagate light pulses containing a different wavelength spectrum compared to other excitation channels.

[0088] Reference Figure 8 Examples describing this aspect. Figure 8 A first optical fiber 132 and a second optical fiber 232, belonging to the first and second excitation channels, are shown, respectively. The first excitation channel, containing the first optical fiber 132, is designed to propagate light pulse 110, while the second excitation channel, containing the second optical fiber 232, is designed to propagate light pulse 210 with a different wavelength than light pulse 110. Light pulse 110 can, for example, have a wavelength of 960 nm and can be used in two-photon microscopy. Light pulse 210 can have a wavelength of 1300 nm and can be used in three-photon microscopy. Three-photon microscopy (1300 nm) can be used, for example, to image deeper locations in the brain, while two-photon microscopy (960 nm) can be used, for example, to shallower layers, thus allowing simultaneous imaging of multiple layers at any location in the cortex without limiting temporal resolution. Light pulse 110 exiting the first optical fiber 132 and light pulse 210 exiting the second optical fiber 232 are merged or recombinated by a dichroic mirror 67, allowing them to further propagate through the same excitation path in the microscope and be focused at different depths. Figure 3 In this case, the beam splitter 67 will be positioned between the fiber optic output end and the lens 44. As previously mentioned, the output ends of the first fiber 132 and the second fiber 232 can be independently spaced apart from each other, either axially or laterally. That is, each output end of the first fiber 132 and the second fiber 232 can be in a specific spatial relationship relative to the lens 44 to achieve a desired axial or lateral offset. The specific time delay between optical pulse 110 and optical pulse 210 can be achieved by... Figure 8 The necessary delay stages are arranged upstream of the output ends of the optical fibers 132 and 232 shown to achieve this.

[0089] Temporal separation can also be achieved between light pulses with different wavelengths that propagate in the same excitation channel. (Refer to...) Figure 9 Examples describing this aspect. Figure 9 The output end of optical fiber 82 is shown. This optical fiber 82 is part of an excitation channel designed to propagate at least optical pulses 83 having a first wavelength λ1 (e.g., 920 nm) and optical pulses 84 having a second wavelength λ2 (e.g., 980 nm).

[0090] Light pulses 83 and 84, exiting fiber 82, first strike a first dichroic beamsplitter 87, which separates these light pulses 83 and 84. Light pulse 84 is transmitted through the first dichroic beamsplitter 87, while light pulse 83 is reflected towards mirrors 85 and 86. Light pulse 84, transmitted through the first dichroic beamsplitter 87, then strikes a second beamsplitter 88 and is transmitted again. Conversely, light pulse 83, reflected from the first dichroic beamsplitter 87, is then reflected by mirrors 85 and 86. Light pulse 83 then strikes the second beamsplitter 88 and is reflected again, causing the optical paths of light pulses 83 and 84 to re-merge downstream of the second beamsplitter 88. Since the optical path lengths between the first and second dichroic beamsplitters 87 and 88 are different for light pulses 83 and 84, the optical paths of light pulses 83 and 84 are re-merged. Figure 9 The settings allow these light pulses to be separated in time according to their wavelengths. For example, the path length of light pulse 83 can be adjusted by changing the positions of reflectors 85 and 86. Figure 9 The components also need to be arranged between the fiber optic output end and the lens 44 of the excitation optical path (see...). Figure 3 ).

[0091] This invention proposes a dispersive spatiotemporal multiplexing method for simultaneous multiplane imaging in multiphoton microscopy.

[0092] By arranging the respective emitting ends of the optical fibers in their own specific spatial relationships (e.g., at different distances) into the collimating lenses in the excitation optical path, the desired and necessary defocus aberrations are generated, for example causing these pulses to be focused at different depths in the sample by the microscope optics. Figure 3 , 4 ).

[0093] In one embodiment, a key aspect is the use of optical fibers that transmit pulses with unique time delays. Figure 1 This method is best suited for lasers with low laser repetition rates (< 4 MHz).

[0094] For faster repetition rates, a similar time-multiplexed laser pulse structure can be produced by using, for example, a fast Pockels cell to open the beam at different times. Figure 6 ).

[0095] By demultiplexing the fluorescence signal and separating it into multiple channels based on the specific temporal characteristics introduced by the light pulses in different excitation channels, simultaneous imaging of multiple illuminated locations in the sample can be achieved. Figure 5 , 7 ).

[0096] In all cases, the present invention can enable access to multiple planes distributed within a certain depth range, or to planes at the same depth but with lateral offset, thereby resulting in faster imaging of a single plane.

[0097] More broadly, dispersive spatiotemporal multiplexing can be used to combine multiple scanning paths or defocused beams in any scanning laser application to achieve a higher level of parallel imaging.

[0098] List of reference numerals

[0099] 1 sample

[0100] 10. Light source, laser

[0101] 11 First beam splitter

[0102] 12 Second beam splitter

[0103] 14 Excitation Light Pulse

[0104] 21 First Delay Path

[0105] 22 Second Delay Path

[0106] 2N Nth Delay Path

[0107] 30 fiber bundles

[0108] 31 First Fiber Optic

[0109] 32 Second optical fiber

[0110] 33 Third optical fiber

[0111] 3N Nth fiber

[0112] 40 Microscope tip

[0113] 41. Microscope casing

[0114] 42 ferrules for fiber bundle 30

[0115] 43. Microscope casing

[0116] 44 Focusing assembly, forming a first lens or lens group as a relay with the second lens or lens group 48, collimating lens

[0117] 46 Scanner, Pupil Surface

[0118] 48. The second lens or lens group forms a relay with the first lens or lens group 44.

[0119] 50mm tube scope

[0120] 51 First focal point, first focal volume

[0121] 52 Second focal point, second focal volume

[0122] 53. Third focal point, volume of the third focal point

[0123] 55. Main beam splitter, dichroic beam splitter

[0124] 54 Microscope Objectives

[0125] 56. Inspecting optical components, inspecting lenses.

[0126] 61 First Detector

[0127] 62 Second Detector

[0128] 63 Beam splitter, dichroic mirror

[0129] 67 Dichroic mirror

[0130] 71 End of fiber optic cable 31

[0131] 72. End of fiber optic cable 32

[0132] 73. End of fiber optic cable 33

[0133] 82 Fiber end, ferrule

[0134] 83 wavelength λ1 light

[0135] 84 wavelength λ2 light

[0136] 85 reflector

[0137] 86 Reflectors

[0138] 87 First Dichroic Mirror

[0139] 88 Second dichroic mirror

[0140] 90 Control Unit, PC

[0141] 100 Microscope according to the present invention

[0142] 111 First optical switch, Pockels box

[0143] 111a First output terminal of optical switch 111

[0144] 111b Second output terminal of optical switch 111

[0145] 112 Second optical switch, Pockels box

[0146] 112a First output terminal of optical switch 112

[0147] 112b Second output terminal of optical switch 112

[0148] 11N Nth optical switch, Pockels box

[0149] The first output terminal of the 11Na optical switch 11N

[0150] The second output terminal of the 11Nb optical switch 11N

[0151] Light with a wavelength of 110 λ1

[0152] 120 allocation units

[0153] 132 Fiber end cap, ferrule

[0154] Light with a wavelength of 210λ2

[0155] 232 Fiber optic tip, ferrule

[0156] ch#1 First Excitation Channel

[0157] ch#2 Second Excitation Channel

[0158] ch#N Nth activation channel

[0159] ch#N+1 The (N+1)th activation channel

[0160] d1 Distance between the end of fiber optic cable 31 and the surface of relay lens 44

[0161] d2 is the distance between the end of fiber optic cable 32 and the surface of relay lens 44.

[0162] d3 Distance between the end of fiber optic cable 33 and the surface of relay lens 44

[0163] D1 Distance between the exit surface of microscope objective 54 and the focal volume 51 corresponding to optical fiber 31

[0164] D2 Distance between the exit surface of the microscope objective 54 and the focal volume 52 corresponding to the optical fiber 32.

[0165] The distance between the exit surface of the D3 microscope objective 54 and the focal volume 53 corresponding to the optical fiber 33.

[0166] A. Laser pulse intensity

[0167] B. Detecting light intensity

[0168] C is the switching signal for the Pockmarked box 111 of the first optical fiber 31.

[0169] D is the switching signal for the Pockmarked box 112 of the second optical fiber 32.

[0170] E is the switching signal for the Pockels cell 11N of the Nth fiber 3N.

[0171] F 80 MHz laser pulse

[0172] G detects light intensity

[0173] The number of light pulses in the k-sequence. Figure 7 In the case of k=3

[0174] lateral direction in x sample

[0175] y-direction in the sample

[0176] z Optical axis direction of microscope objective 54

[0177] z' Optical axis direction in microscope housing 41

[0178] λ1 First wavelength

[0179] λ2 Second wavelength

[0180] References

[0181] 1. Piyawattanametha, W. et al., Fast scanning two-photon fluorescence imaging based on a two-dimensional scanning mirror using a microelectromechanical system. Opt Lett, 31, 2018-2020 (2006). https: / / doi.org:10.1364 / ol.31.002018

[0182] 2. Sawinski, J. et al., Visually induced activity in cortical cells during imaging in freely moving animals. Proceedings of the National Academy of Sciences 106, 19557-19562 (2009). https: / / doi.org:10.1073 / pnas.0903680106

[0183] 3. Russell, P. Photonic crystal fiber. Science 299, 358-362 (2003). https: / / doi.org:10.1126 / science.1079280

[0184] 4. Klioutchnikov, A. et al., Three-photon head-mounted microscopy for imaging deep cortex in freely moving rats. Nat Methods 17, 509-513 (2020). https: / / doi.org:10.1038 / s41592-020-0817-9

[0185] 5. Klioutchnikov, A. et al., Three-photon head-mounted microscope for imaging all layers of the visual cortex in freely moving mice. Nat Methods 20, 610-616 (2023). https: / / doi.org:10.1038 / s41592-022-01688-9

[0186] 6. Zong, W. et al., Large-scale two-photon calcium imaging in freely moving mice. Cell 185, 1240–1256e1230 (2022).

[0187] https: / / doi.org:10.1016 / j.cell.2022.02.017

[0188] 7. Zong, W. et al. Rapid, 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

[0189] 8. Beaulieu, DR., Davison, IG., Kilic, K., Bifano, TG., and Mertz, J., Simultaneous multiplanar imaging using reverberant two-photon microscopy. Nat Methods 17, 283-286 (2020). https: / / doi.org:10.1038 / s41592-019-0728-9

[0190] 9. Demas, J. et al., High-speed, cortical-scale volumetric recording of neural activity at cellular resolution using light-bead microscopy. Nat Methods 18, 1103-1111 (2021). https: / / doi.org:10.1038 / s41592-021-01239-8

[0191] 10. US11226474B2

[0192] 11. WO 2021 / 155330 A9

[0193] 12. US20170227748A1

[0194] 13. https: / / www.labmaker.org / collections / 2-photon-miniscope-mini2p

[0195] https: / / github.com / kavli-ntnu / MINI2P_toolbox / tree / main / Protocols。

Claims

1. Microscopes, especially multiphoton microscopes. Includes at least one light source (10) for emitting light pulses (14) to excite the sample (1); and an excitation optical path having a microscope objective (54) for guiding the light pulses (14) onto or into the sample (1). The excitation optical path includes • Multiple independent excitation channels (ch#1, ch#2, ch#N, ch#N+1) are designed to guide the light pulse (14) onto the sample (1) or to their respective focal points (51, 52, 53) within the sample (1). • Distribution unit (20; 120) for distributing optical pulses (14) to the excitation channels (ch#1, ch#2, ch#N, ch#N+1). • Scanner (46) for changing the area on or within the sample (1) illuminated by the light pulse (14), It also includes at least one detector (61) for detecting emitted light (16) emitted by the sample (1) as an optical response to illumination by a light pulse (14); and detection optical paths (54, 55, 56, 63) for guiding the emitted light (16) onto the detector (61), and Includes 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). Its features are, In order to temporally separate the respective optical responses from the illuminated focal points (51, 52, 53), the excitation optical path is designed to sequentially illuminate the different focal points (51, 52, 53) one after another through the excitation channels (ch#1, ch#2, ch#N, ch#N+1). Each of the excitation channels (ch#1, ch#2, ch#N, ch#N+1) includes an optical fiber (31, 32, 33, 3N) having its own exit end (71, 72, 73), and in order to guide the optical pulse (14) to its respective different focal points (51, 52, 53), the exit ends (71, 72, 73) of the optical fiber (31, 32, 33) are arranged with their own different spacings (d1, d2, d3) relative to the optical focusing component (44) of the excitation optical path.

2. The microscope according to claim 1, Its features are, The emitting ends (71, 72, 73) of the optical fibers (31, 32, 33) are arranged at different intervals (d1, d2, d3) relative to the optical focusing assembly (44) in the direction of the optical axis (z') of the excitation optical path.

3. The microscope according to claim 1 or 2, Its features are, The emitting ends (71, 72, 73) of the optical fibers (31, 32, 33) are laterally spaced from each other relative to the optical axis (z') of the excitation optical path.

4. The microscope according to any one of claims 1 to 3, Its features are, The optical fibers (31, 32, 33) do not contact each other in the region of their respective emitting ends (71, 72, 73).

5. The microscope according to any one of claims 1 to 4, Its features are, The distribution unit (20) is designed to distribute the energy of each optical pulse (14) onto the excitation channels (ch#1, ch#2, ch#N).

6. The microscope according to claim 5, Its features are, The distribution unit (20) includes multiple neutral beam splitters (11, 12) for energy-level splitting of optical pulses (14) and distributing the optical pulses (14) to different excitation channels (ch#1, ch#2, ch#N).

7. The microscope according to claim 5 or 6, Also includes The delay paths (21, 22, 2N) in each of the excitation channels (ch#1, ch#2, ch#N) are used to apply different time delays to the energy-distributed light pulses (14) in their respective excitation channels (ch#1, ch#2, ch#N).

8. The microscope according to any one of claims 1 to 7, Its features are, The distribution unit (120) is designed to guide each light pulse from the light source to one of the excitation channels (ch#1, ch#2, ch#N, ch#N+1).

9. The microscope according to claim 8, Its features are, The distribution unit (120) further includes optical switches (111, 112, 11N) for directing each optical pulse (14) to one of the excitation channels (ch#1, ch#2, ch#N, ch#N+1).

10. The microscope according to claim 9, Its features are, The optical switches (111, 112, 11N) are Pockels boxes.

11. The microscope according to any one of claims 1 to 10, Also includes The tube lens (50) is used to generate a first intermediate image plane that is optically conjugate to the focal plane of the microscope objective (54), and An optical relay system comprising a first lens or lens group (44) and a second lens or lens group (48) for generating a second intermediate image plane optically conjugate to the first intermediate image plane, wherein the emitting ends (71, 72, 73) of the optical fibers (31, 32, 33) are arranged in or near the second intermediate image plane.

12. The microscope according to claim 11, Its features are, The scanner (46) is arranged in or near the pupil plane, which is optically conjugate to the back focal plane of the microscope objective (54) and is formed between the first lens or lens group (44) and the second lens or lens group (48).

13. The microscope according to any one of claims 1 to 12, Also includes Microscope head end (40), which at least accommodates the microscope objective (54), the scanner (46) and the output ends (71, 72, 73) of the optical fibers (31, 32, 33).

14. The microscope according to claim 13, Its features are, The microscope tip (40) includes an optical interface (57) for the transmission of emitted light (16).

15. The microscope according to any one of claims 1 to 14, Also includes A ferrule (42) is provided in which the exit ends (71, 72, 73) of the optical fibers (31, 32, 33) are housed.

16. The microscope according to claim 14 or 15, Its features are, The at least one detector (61) is arranged at the optical interface (57).

17. The microscope according to any one of claims 14 to 16, Also includes The detection module at the optical interface (57) includes at least one color spectrometer (63) and multiple detectors (61, 62).

18. The microscope according to any one of claims 1 to 17, Its features are, Each of the excitation channels (ch#1, ch#2, ch#N, ch#N+1) is designed to propagate light pulses containing the same wavelength spectrum (14), respectively.

19. The microscope according to any one of claims 1 to 18, Its features are, The light source (10) provides excitation light having at least a first wavelength (λ1) and a second wavelength (λ2).

20. The microscope according to any one of claims 1 to 19, Its features are, At least one of the excitation channels is designed to propagate a light pulse containing a different wavelength spectrum compared to the other excitation channels.

21. The microscope according to claim 20, Also includes At least one dichroic mirror (67) is used to combine an optical pulse (110) leaving the first optical fiber (132) with an optical pulse (210) leaving the second optical fiber (232) and having a different wavelength than the optical pulse (110) leaving the first optical fiber (132).

22. The microscope according to any one of claims 1 to 21, Its features are, At least one of the excitation channels is designed to propagate at least a light pulse (83) having a first wavelength (λ1) and a light pulse (84) having a second wavelength (λ2) different from the first wavelength (λ1).

23. The microscope according to claim 22, Also includes At least one first dichroic beam splitter (87) is used to separate light pulses (83, 84) that leave the same optical fiber (82) and have different wavelengths. And at least one second dichroic beam splitter (88), which is located downstream of the first dichroic beam splitter (87), for combining light pulses (83, 84) with different wavelengths. in, The optical path length between the first dichroic beam splitter (87) and the second dichroic beam splitter (88) is different for light pulses (83, 84) with different wavelengths.

24. Microsurgical techniques, especially multiphoton microscopy. The method includes the following steps: The light pulse (14) is distributed to multiple excitation channels (ch#1, ch#2, ch#N, ch#N+1) of the excitation optical path. The light pulses (14) are guided to the sample (1) or to their respective focal points (51, 52, 53) in the sample (1) via different excitation channels (ch#1, ch#2, ch#N, ch#N+1) and microscope objectives (54). Using a scanner (46), the area on or within the sample (1) illuminated by the light pulse (14) is changed. The emitted light (16) emitted by the sample (1) in response to the illumination of the light pulse (14) is detected as an optical response. The method is further characterized in that, The steps of illuminating different focal points (51, 52, 53) through excitation channels (ch#1, ch#2, ch#N, ch#N+1) are performed sequentially, one focal point (51, 52, 53) after another, in order to temporally separate the respective optical responses from the illuminated focal points (51, 52, 53). Each of the excitation channels (ch#1, ch#2, ch#N, ch#N+1) includes an optical fiber (31, 32, 33) with its own output end (71, 72, 73). Furthermore, in order to guide the light pulses (14) to their respective focal points (51, 52, 53), the emitting ends (71, 72, 73) of the optical fibers (31, 32, 33) are arranged to have their respective spacings (d1, d2, d3) relative to the light focusing components (44) of the excitation optical path.

25. The method according to claim 24, Its features are, In the allocation step, the optical pulse (14) is energy-divided and allocated to different excitation channels (ch#1, ch#2, ch#N).

26. The method according to claim 25, Its features are, The energy-divided light pulses (14) are subjected to different delays in each of the excitation channels (ch#1, ch#2, ch#N).

27. The method according to any one of claims 24 to 26, Its features are, In the distribution step, each light pulse (14) from the light source (10) is directed to one of the excitation channels (ch#1, ch#2, ch#N, ch#N+1).

28. The method according to claim 27, Its features are, In the allocation step, a sequence of multiple (k) light pulses (14) from the light source (10) that are adjacent to each other is guided one after another to each of the excitation channels (ch#1, ch#2, ch#N, ch#N+1).

29. The method according to any one of claims 24 to 28, Also includes The step of demultiplexing the detection signal to evaluate the optical response from each irradiated point (51, 52, 53) on or in sample (1).

Images