Optical equipment, imaging system, and imaging method used in a compact multiphoton microscope

Abstract

The present disclosure provides an optical instrument, imaging system, and imaging method for use in a compact multiphoton microscope, and relates to an optical arrangement area. The optical instrument has a fluorescence excitation light path and a fluorescence collection light path, and includes a condenser lens, the condenser lens including a first lens group and a second lens group spaced apart from each other, the first lens group being located in the fluorescence collection light path, the second lens group being located in the fluorescence excitation light path and the fluorescence collection light path, and the second lens group being closely attached to a microscope objective lens. The solution disclosed herein improves the collection efficiency of the compact multiphoton microscope for scattered fluorescence and improves the imaging signal-to-noise ratio when observing a sample with scattering properties.

Description

[Technical Field]

[0001] The present disclosure relates to the field of optical arrangements, and more particularly to optical instruments, imaging systems, and imaging methods used in miniature multiphoton microscopes. [Background technology]

[0002] Compact multiphoton microscopes are characterized by their light weight, small volume, and flexible use. They utilize the multiphoton absorption and fluorescence emission properties of fluorescent agents to image fluorescently labeled or autofluorescent biological tissues in real time. Currently, the fluorescence collection optical path of compact multiphoton microscopes is relatively simple, consisting of a microscope objective lens and a focusing lens spaced apart from the microscope objective lens. This optical device can meet the needs of collecting ballistic fluorescence photons. However, when the target sample has scattering properties, such as biological tissue, fluorescence photons are scattered in the tissue and deviate from their original trajectory. This optical device configuration cannot ensure that all scattered fluorescence photons enter the collection optical path, limiting the signal-to-noise ratio and imaging depth of fluorescence imaging. Summary of the Invention [Problem to be solved by the invention]

[0003] The present disclosure is made to solve the above-mentioned problems. Embodiments of the present disclosure provide an optical instrument, an imaging system, and an imaging method for use in a miniature multiphoton microscope. [Means for solving the problem]

[0004] According to a first aspect, one embodiment of the present disclosure provides an optical instrument having a fluorescence excitation light path and a fluorescence collection light path, the optical instrument including a condenser lens, the condenser lens including a first lens group and a second lens group spaced apart from each other, the first lens group being located in the fluorescence collection light path, the second lens group being located in both the fluorescence excitation light path and the fluorescence collection light path, and the second lens group being closely attached to a microscope objective lens.

[0005] Based on the first aspect, in some embodiments of the first aspect, the optical instrument further includes a third lens group located in the fluorescence excitation light path, wherein the arrangement of the third lens group and the second lens group in the fluorescence excitation light path is a tube lens.

[0006] Based on the first aspect, in some embodiments of the first aspect, the optical instrument further includes a spectroscopic optical element located in the fluorescence excitation optical path and the fluorescence collection optical path, the spectroscopic optical element having a function of transmitting or reflecting the light beam depending on the wavelength of the light beam.

[0007] According to the first aspect, in some embodiments of the first aspect, the dispersing optical element includes a dichroic mirror.

[0008] Based on the first aspect, in some embodiments of the first aspect, the first lens group and the second lens group are separated by a dichroic mirror, and the second lens group and the third lens group are separated by a dichroic mirror.

[0009] Based on the first aspect, in some embodiments of the first aspect, the optical instrument further includes a femtosecond laser device located in the fluorescence excitation light path, and the femtosecond laser device is used to simultaneously absorb at least two photons in a sample to be detected mounted in the optical instrument.

[0010] Based on the first aspect, in some embodiments of the first aspect, the optical instrument further includes a collimating lens, a galvanometer mirror, and a scanning lens located in the fluorescence excitation light path, wherein the collimating lens is used to collimate the received initial laser light beam and emit the collimated light beam, the galvanometer mirror is used to reflect and scan the collimated light beam, and the scanning lens is used to focus the collimated light beam reflected by the galvanometer mirror onto a focal plane of the scanning lens to obtain a focused light beam, and emit the focused light beam to the third lens group.

[0011] Based on the first aspect, in some embodiments of the first aspect, the collimating lens is composed of one negative lens and one cemented doublet lens.

[0012] Based on the first aspect, in some embodiments of the first aspect, the microscope objective comprises an infinity objective, and the objective is located in the fluorescence excitation light path and the fluorescence collection light path.

[0013] Based on the first aspect, in some embodiments of the first aspect, the length of the infinity objective is 4.34 mm or less, and the physical aperture of the optical lens of the infinity objective is 3 mm or less.

[0014] Based on the first aspect, in some embodiments of the first aspect, the second lens group arrangement is a tube lens in the fluorescence excitation light path.

[0015] Based on the first aspect, in some embodiments of the first aspect, the optical instrument further includes a fourth lens group, and in the fluorescence excitation light path, the fourth lens group and the second lens group are arranged as a tube lens, and the fourth lens group and the second lens group are closely spaced groups.

[0016] Based on the first aspect, in some embodiments of the first aspect, the second lens group is used to focus the fluorescent beam emitted by the microscope objective to emit the first focused beam.

[0017] According to the first aspect, in some embodiments of the first aspect, the first lens group is used to emit a second focused beam based on the first focused beam, and the diameter of the second focused beam is smaller than the diameter of the first focused beam.

[0018] According to a second aspect, one embodiment of the present disclosure provides an imaging system, including an optical instrument according to the first aspect configured to collect optical signals of a sample to be detected, and a signal processing module configured to convert the optical signals collected by the optical instrument into an image.

[0019] According to a third aspect, one embodiment of the present disclosure provides an imaging method, the imaging method comprising: determining an imaging target area of ​​a target sample; and detecting an optical signal of the imaging target area by the optical instrument according to the first aspect, so as to generate an image corresponding to the imaging target area based on the optical signal. [Effects of the Invention]

[0020] An optical instrument according to an embodiment of the present disclosure includes a focusing lens, the focusing lens including a first lens group and a second lens group spaced apart from each other. The first lens group is located in the fluorescence collection optical path, and the second lens group is located in both the fluorescence excitation optical path and the fluorescence collection optical path. The second lens group is closely attached to the microscope objective lens and can immediately focus the scattered fluorescence beam emitted by the objective lens and prevent the scattered light beam from exiting the fluorescence collection optical path. The first lens group can further focus the scattered light beam so that the focused scattered light beam enters the fluorescence collection optical fiber bundle and is captured by a detector. [Brief explanation of the drawings]

[0021] The above and other objects, features, and advantages of the present disclosure will be more clearly understood with reference to the drawings. The drawings are used to further deepen understanding of the embodiments of the present disclosure and are used to explain the present disclosure together with the embodiments of the present disclosure as part of the specification, but are not intended to limit the present disclosure. In the drawings, the same reference numerals generally represent the same parts or steps. [Figure 1] 1 is a partial structural schematic diagram of a related optical device according to one exemplary embodiment of the present disclosure. [Figure 2] FIG. 10 is a partial structural schematic diagram of a related optical device according to another exemplary embodiment of the present disclosure. [Figure 3] 1 is a structural schematic diagram of an optical device according to one exemplary embodiment of the present disclosure. [Figure 4] 1 is a structural schematic diagram of an excitation light path of an optical device according to one exemplary embodiment of the present disclosure. [Figure 5]1 is a structural schematic diagram of a collection light path of an optical instrument according to one exemplary embodiment of the present disclosure. [Figure 6] FIG. 1 is a structural schematic diagram of a condenser lens according to an exemplary embodiment of the present disclosure. [Figure 7] 1 is a line graph of fluorescence collection efficiency for three optical instruments according to one exemplary embodiment of the present disclosure. [Figure 8] FIG. 1 is a fluorescence propagation trajectory analysis diagram of a collection light path of an optical instrument according to one exemplary embodiment of the present disclosure. [Figure 9] FIG. 1 is a structural schematic diagram of an imaging system according to an exemplary embodiment of the present disclosure. [Figure 10] 1 is a flowchart of an imaging method according to one exemplary embodiment of the present disclosure. DETAILED DESCRIPTION OF THE INVENTION

[0022] Hereinafter, the technical solutions in the embodiments of the present disclosure will be described clearly and completely with reference to the drawings in the embodiments of the present disclosure. Needless to say, the described embodiments are only some embodiments of the present disclosure, and are not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present disclosure without creative work fall within the scope of protection of the present disclosure.

[0023] <Microscope> It is an optical device made up of a combination of optical lenses and used to magnify tiny objects to a state where they can be observed by the naked eye or electronic devices.

[0024] <Fluorescence microscope> The object is illuminated with a light source to emit fluorescent light, and the shape and position of the object are observed.

[0025] <Single-photon fluorescence microscope> A light source illuminates the specimen, and fluorescent molecules in the specimen absorb one photon and then emit one fluorescence photon by radiative transition, which is detected by the microscope.

[0026] <Multiphoton fluorescence microscope> At high photon densities, fluorescent molecules in the analyte can simultaneously absorb two or more long wavelength photons, and after a short excited state lifetime, the analyte emits a short wavelength fluorescence photon that is detected by the microscope, where the short wavelength fluorescence photon is short compared to the long wavelength photon.

[0027] <Finite objective lens> Both the object and its image are a finite distance away from the objective lens.

[0028] <Infinity objective lens> After the object passes through the objective lens, the image is located at infinity, that is, the light beam emerges parallel from the objective lens, and cannot be imaged directly, but must be imaged through a tube lens.

[0029] <Tube lens> In a microscope imaging system, when using an infinity objective, the light coming out of the objective is parallel, and a group of lenses must be used to focus the parallel light onto the image plane, and this group of lenses is called a tube lens.

[0030] <Scanning lens> In a scanning optical system, a collimated light beam is vibrated by a galvanometer mirror, and the emitted light beam passes through a group of lenses to form an image on an image plane. This group of lenses is called a scanning lens.

[0031] <Dichroic mirror> An optical element that transmits light of a certain wavelength almost completely and reflects light of some other wavelengths almost completely.

[0032] <Fluorescence excitation light path>

[0033] Laser light emitted from a laser device is transmitted to the sample tissue via an optical element, causing fluorescent molecules in the sample tissue to absorb the laser light and emit fluorescence; this light transmission path is called a fluorescence excitation optical path.

[0034] <Fluorescence collection optical path> The fluorescent light emitted by fluorescent molecules in the sample tissue when they absorb the laser light passes through the objective lens and is then collected by a condenser lens onto a detector. This optical transmission path is called the fluorescence collection optical path.

[0035] For ease of understanding, the following will be described first with reference to examples of related optical equipment.

[0036] FIG. 1 is a partial structural schematic diagram of a related optical instrument according to one exemplary embodiment of the present disclosure. The partial structural schematic diagram is a schematic diagram of a fluorescence collection light path of the related optical instrument. As shown in FIG. 1, the optical instrument includes a finite miniature objective lens 120, a dichroic mirror 130, and a monolithic focusing lens 140, and such an optical instrument is used to observe neurofunctional imaging of living brain tissue 110. For example, femtosecond laser light (e.g., the wavelength of the femtosecond laser light is 920 nm) emitted from a hollow photonic crystal fiber is collimated by a collimating lens, reflected by a galvanometer mirror to enter a scanning lens, and focused by the scanning lens. After that, it is reflected by a dichroic mirror 130 to enter the finite miniature objective lens 120 and focussed on the sample tissue. The fluorescent protein in the sample tissue is then excited by the two-photon effect to emit green fluorescence (for example, the green fluorescence wavelength is 520 nm±20 nm), which is focused by the finite miniature objective lens 120, passes through the dichroic mirror 130, and is focused into a flexible optical fiber bundle by the monolithic focusing lens 140. The green fluorescence is then introduced into a highly sensitive photoelectric detector via the flexible optical fiber bundle, where it is detected.

[0037] Because both the laser light and the fluorescence are transmitted through flexible optical fibers, a small probe corresponding to the optical device in Figure 1 can be attached to the animal's head, allowing for on-body observation of the animal's brain neurofunction signals without affecting the animal's free movement. However, because animal brain tissue is a chaotic scattering tissue, the penetration depth of the 920 nm laser light used is limited. While it can excite fluorescence with a sufficient signal-to-noise ratio at a depth of 400 μm in animal brain tissue, the signal-to-noise ratio of the fluorescence signal in deeper brain regions is poor.

[0038] FIG. 2 is a partial structural schematic diagram of a related optical instrument according to another exemplary embodiment of the present disclosure. The partial structural schematic diagram is a schematic diagram of a fluorescence collection light path of the related optical instrument. As shown in FIG. 2, the optical instrument includes an infinity miniature objective lens 220, a dichroic mirror 230, and a monolithic focusing lens 240, and such an optical instrument may be used to observe neurofunctional imaging of living brain tissue 210. Similarly, femtosecond laser light (e.g., laser light wavelength is 1300 nm) emitted from a hollow photonic crystal fiber is collimated by a collimator lens, reflected by a galvanometer mirror to enter a scanning lens, focused by the scanning lens, collimated by a tube lens, reflected by a dichroic mirror 230, and then enters the infinity miniature objective lens 220 to be focused on the sample tissue. The fluorescent protein in the sample tissue is then excited by the three-photon effect to emit green fluorescence (for example, the green fluorescence wavelength is 520 nm±20 nm), which is then emitted as a light beam through the infinity miniature objective lens 220, transmitted through the dichroic mirror 230, and focused into a plastic optical fiber by the monolithic condenser lens 240. The green fluorescence is then introduced into a highly sensitive photoelectric detector via the plastic optical fiber, where it is detected. Compared with the excitation wavelength of 920 nm, the laser light wavelength of 1300 nm has a deeper penetration depth in scattering brain tissue.

[0039] The fluorescence collection configurations of the related optical instruments shown in Figures 1 and 2 are both relatively simple, consisting of a single infinite or finite objective lens combined with a monolithic focusing lens. While these simple fluorescence collection configurations are sufficient for collecting ballistic photons, the fluorescence is scattered and deviated from its original trajectory in scattering tissue, making them unable to meet the needs of collecting scattered fluorescence. Taking green fluorescence as an example, because green fluorescence is strongly scattered in brain tissue, most of the fluorescence emitted from brain tissue 1 mm deep deviates from the ballistic trajectory and scatters randomly after reaching the brain surface.

[0040] In response to the above problems, the present disclosure provides an optical device to improve the collection efficiency of scattered fluorescence, thereby increasing brain tissue imaging depth and imaging signal-to-noise ratio.

[0041] Figure 3 is a structural schematic diagram of an optical instrument according to an exemplary embodiment of the present disclosure, Figure 4 is a structural schematic diagram of an excitation light path of the optical instrument according to an exemplary embodiment of the present disclosure, and Figure 5 is a structural schematic diagram of a collection light path of the optical instrument according to an exemplary embodiment of the present disclosure.

[0042] The structure of the optical device according to the embodiment shown in FIG. 3 will be described in detail below with reference to FIGS.

[0043] 3 , the optical device according to the embodiment of the present disclosure includes a condenser lens 310 (the condenser lens 310 includes a first lens group 311 and a second lens group 312 separated from each other), a tube lens 320 (the tube lens 320 includes a second lens group 312 and a third lens group 321), a dichroic mirror 330, a collimator lens 340, a galvanometer mirror 350, a scanning lens 360, an infinity objective lens 370, and a flexible optical fiber bundle 390. Here, the excitation optical path 400 includes a femtosecond laser device and a hollow optical fiber (not shown) that transmits the femtosecond laser light, the collimator lens 340, the galvanometer mirror 350, the scanning lens 360, the tube lens 320, the dichroic mirror 330, and the infinity objective lens 370. The collection path 500 includes an infinity objective lens 370 , a dichroic mirror 330 , a collection lens 310 , and a flexible fiber optic bundle 390 .

[0044] In this manner, the dichroic mirror 330, the second lens group 312, and the infinity objective lens 370 are simultaneously located in the excitation light path 400 and the collection light path 500. The second lens group 312 and the infinity objective lens 370 are closely spaced. In the excitation light path 400, the second lens group 312 and the third lens group 321 are arranged as a tube lens 320, and the second lens group 312 and the third lens group 321 are separated by the dichroic mirror 330. In the collection light path 500, the first lens group 311 and the second lens group 312 are arranged as a condenser lens 310, and the first lens group 311 and the second lens group 312 are separated by the dichroic mirror 330. Note that "closely spaced" means that the relative distance between the second lens group and the infinity objective lens is smaller than a predetermined distance threshold. Here, the relative distance is equal to the ratio of the absolute distance s between the second lens group and the infinity objective to the objective diameter D, and the predetermined distance threshold is typically one-third.

[0045] In another embodiment, the tube lens 320 may include only the second lens group in close contact with the objective lens.

[0046] Furthermore, the operation process of the excitation light path of the optical device according to the embodiment of the present disclosure will be described in detail with reference to FIG.

[0047] 4 , the femtosecond laser device emits a femtosecond laser light beam, which is transmitted through a hollow optical fiber and collimated by a collimating lens 340 at the transmission exit of the hollow optical fiber to obtain a collimated light beam. The collimated light beam is reflected and scanned by a galvanometer mirror 350 and enters a scanning lens 360. The collimated light beam is imaged on a focal plane corresponding to the scanning lens 360. After passing through the focal plane, the light beam diverges and enters the third lens group 321 of the tube lens 320. After being reflected by a dichroic mirror 330, the light beam enters the second lens group 312 of the tube lens 320. After being collimated by the tube lens 320, the light beam enters the infinity objective lens 370. The light beam passes through the infinity objective lens 370 and is focused on an immersion sample 380.

[0048] Further, with reference to FIG. 5, the operation process of the collection light path of the optical instrument according to the embodiment of the present disclosure will be described in detail.

[0049] 5 , the immersion sample 380 is excited by a femtosecond laser beam emitted by a femtosecond laser device to generate a fluorescent beam. The fluorescent beam enters the infinity objective lens 370 and then enters the second lens group 312 of the focusing lens 310. The second lens group 312 focuses the scattered fluorescent beam to obtain a first focused beam related to the fluorescent beam. The first focused beam passes through the dichroic mirror 330, which filters the transmitted beam to obtain a transmitted beam within the fluorescence band. The transmitted beam passes through the first lens group 311 of the focusing lens 310, which focuses the transmitted beam to obtain a second focused beam with a smaller diameter than the first focused beam. After passing through the first lens group 311, the second focused beam can smoothly enter the flexible optical fiber bundle 390.

[0050] The fluorescent light beam emitted from the deep tissue of the sample is scattered by the sample tissue, and by the time it reaches the infinite objective lens 370, the fluorescent light beam has already deviated from its original propagation trajectory. The deeper the tissue of the sample, the greater the deviation of the scattered light beam in the fluorescent light beam. In the embodiment of the present disclosure, by closely attaching the second lens group 312 of one focusing lens 310 to the exit of the infinite objective lens 370, the random scattered light beam can be effectively focused within a minimum time, and after passing through the dichroic mirror, can be further focused by the first lens group 311 of the focusing lens 310 to obtain a second focused beam that meets the optical fiber focusing size of the flexible optical fiber bundle 390. Furthermore, because the second lens group 312 is closely attached to the infinity objective lens 370, the second lens group is necessarily present simultaneously in the excitation optical path 400 and the collection optical path 500. Based on this, the embodiment of the present disclosure introduces a tube lens 360 having two spaced-apart lens groups in the excitation optical path 400, where the second lens group 312 is shared by the collection lens 310 and the tube lens 360, and a dichroic mirror 330 simultaneously separates the first lens group 311 and the second lens group 312, and the third lens group 321 and the second lens group 312. The above-described structure of the optical device compactly combines the excitation optical path 400 and the collection optical path 500 in the optical device, achieving the functions of multiphoton fluorescence excitation and fluorescence collection, and maximizing the collection efficiency of scattered fluorescence.

[0051] 6 is a structural schematic diagram of a focusing lens according to an exemplary embodiment of the present disclosure. As shown in FIG. 6, in an exemplary embodiment of the present disclosure, the optical device includes a focusing lens 310 having a first lens group 311 and a second lens group 312, where the first lens group 311 and the second lens group 312 are spaced apart. The focusing lens 310 may be attached to a related optical device structure, where the second lens group 312 is in close contact with an objective lens in the related optical device structure and is used to focus the light beam emitted by the objective lens, and the first lens group 311 is used in addition to the second lens group 312 to further focus the light beam. Furthermore, the first lens group 311 of the focusing lens 310 according to the embodiment of the present disclosure is located in the fluorescence collection light path in the related optical device structure, and the second lens group 312 is simultaneously located in both the fluorescence excitation light path and the fluorescence collection light path in the related optical device structure.

[0052] The focusing lens in the embodiments of the present disclosure can instantly focus the light beam emitted by the objective lens in the associated optical equipment structure, improve the collection efficiency for the scattered light beam, and further increase the imaging depth and imaging signal-to-noise ratio of the sample tissue.

[0053] In an exemplary embodiment of the present disclosure, the optical device includes a third lens group 321, which forms a new optical arrangement with the condenser lens 310 in the above embodiment. Here, the third lens group 321 and the second lens group 312 form a tube lens 320, which is located in the excitation light path 400.

[0054] The tube lens 320 may include only one lens group or two or more lens groups. The lens groups in the tube lens 320 may be spaced apart or closely spaced. The embodiments of the present disclosure merely provide preferred tube lens arrangements and do not impose specific limitations on the tube lens structure. Those skilled in the art can specifically select the structure of the tube lens according to the actual application, as long as the function of the tube lens in the embodiments of the present disclosure can be achieved.

[0055] The technical solution according to the embodiment of the present disclosure can not only improve the collection efficiency of the scattered light beam emitted by the objective lens in the optical device structure, but also make the layout of the optical device according to the embodiment of the present disclosure more compact by having the tube lens 320 and the condenser lens 310 share the second lens group 312.

[0056] In one exemplary embodiment of the present disclosure, the optical equipment includes a focusing lens 310, a tube lens 320, and a dichroic mirror 330, where the dichroic mirror is used to separate the first lens group 311 from the second lens group 312, and the third lens group 321 from the second lens group 312.

[0057] The dichroic mirror may be replaced with any other spectroscopic optical element that has the function of transmitting or reflecting a light beam depending on the wavelength of the light beam.

[0058] The optical instrument according to the present disclosure allows for tight coupling of the fluorescence excitation and collection optical paths in an associated optical microscope.

[0059] In one exemplary embodiment of the present disclosure, the infinity objective lens 370 is a simplified infinity miniature objective lens. Here, "simplified" means that the objective lens has a small number of lenses, a short length, and relatively flat lens surfaces. The length of the simplified infinity miniature objective lens in the embodiment of the present disclosure is 4.34 mm, and the physical diameter of the optical lens is 3 mm.

[0060] It should be noted that the embodiments of the present disclosure merely provide an explanation of exemplary parameters and related structures of one infinity objective lens 370, and are not specific limitations on the infinity objective lens 370. Those skilled in the art may replace the infinity objective lens 370 with other types of objective lenses according to actual circumstances.

[0061] The simplified infinity lenslet can shorten the transmission distance of the scattered fluorescence therein, and can further reduce the loss of scattered fluorescence in the simplified infinity lenslet.

[0062] In one exemplary embodiment of the present disclosure, the collimating lens 340 is composed of one negative lens and one cemented doublet lens. Note that the arrangement of the collimating lens is not limited to one particular arrangement, and any arrangement of the collimating lens may be used as long as it can achieve the collimating function, satisfy the structural size requirements, and obtain the desired collimating configuration.

[0063] Experiments on scattered fluorescence collection efficiency were conducted based on the optical configuration shown in Figures 1, 2, and 3. Specifically, the propagation trajectory of scattered fluorescence was simulated and tracked using the optical design software Zemax, and an ideal emission point was transplanted into the scattering sample tissue to be detected to simulate the fluorescence excitation point, and one million light beams were emitted. The scattering attributes for the scattering sample tissue were set to a scattering length of 75 μm and a scattering anisotropy coefficient of 0.92. Monte Carlo tracking was performed using the software to obtain the fluorescence collection efficiency for the optical configuration shown in Figures 1, 2, and 3.

[0064] FIG. 7 is a line graph of the fluorescence collection efficiency of three optical instruments according to one exemplary embodiment of the present disclosure. Here, m2PM is an abbreviation for the optical instrument according to the embodiment shown in FIG. 1, and m3PM is an abbreviation for the optical instrument according to the embodiment shown in FIG. 2. The m2PM has a maximum numerical aperture of 0.5 and a working distance of 1 mm, while the m3PM has a maximum numerical aperture of 0.9 and a working distance of 1.75 mm. The arrangement shown in FIG. 3 of the present disclosure has a maximum numerical aperture of 0.65 and a working distance of 1.75 mm. The optical instrument shown in FIG. 3 of the present disclosure has the same size as the final fluorescence collection surfaces of the m2PM and m3PM, both of which are 1.7 mm.

[0065] The mean free path in Figure 7 refers to the average length over which one scattering event occurs. As shown in Figure 7, when the sample tissue depth is 1 mm, the fluorescence collection efficiency of the disclosed arrangement is 3.2%, the fluorescence collection efficiency of m3PM is 2.14%, and the fluorescence collection efficiency of m2PM is 1.03%. When the sample tissue depth is 1.5 mm, the fluorescence collection efficiency of the disclosed arrangement is 1.47%, and the fluorescence collection efficiency of m3PM is 0.75%. That is, after the inflection point of the scattering-enhanced region, the fluorescence collection efficiency of the disclosed arrangement is always superior to that of m2PM and m3PM.

[0066] Table 1 shows a comparison of the fluorescence collection efficiency data for three optical devices according to one exemplary embodiment of the present disclosure. As shown in Table 1, simulation results show that, with the same objective lens numerical aperture (NA) of 0.65, the fluorescence collection efficiency of the arrangement of the present disclosure is 2.58 times and 3.59 times that of the m3PM at depths of 1 mm and 1.5 mm, respectively. With the same objective lens NA of 0.5, the fluorescence collection efficiency of the arrangement of the present disclosure is 2.18 times that of the m2PM at a depth of 1 mm. An "*" indicates that the objective lens NA was changed to the specified value using an aperture.

[0067] JPEG2025537854000002.jpg69170

[0068] Therefore, according to the simulation calculations, the optical arrangement of the present disclosure is clearly superior in terms of improving the scattered fluorescence collection efficiency compared to the arrangements of m2PM and m3PM.

[0069] To explain the principle of the scattered fluorescence collection efficiency of an optical instrument according to an embodiment of the present disclosure, the present disclosure further simulates the propagation trajectory of scattered fluorescence and provides FIG. 8. FIG. 8 shows an analysis diagram of the fluorescence propagation trajectory of the collection light path of an optical instrument according to one exemplary embodiment of the present disclosure. As shown in FIG. 8, the optical instrument shown in FIG. 8 includes only the objective lens 370, the optical instrument shown in FIG. 8 includes the objective lens 370 and the second lens group 312, and the optical instrument shown in FIG. 8 includes the objective lens 370, the second lens group 312, and the first lens group 311. (b) shows the size and irradiance distribution of the scattered fluorescence received by the detector due to the effect of the optical instrument shown in FIG. 8, (d) shows the size and irradiance distribution of the scattered fluorescence received by the detector due to the effect of the optical instrument shown in FIG. 8, and (f) shows the size and irradiance distribution of the scattered fluorescence received by the detector due to the effect of the optical instrument shown in FIG. 8.

[0070] The specific experimental procedure was to implant a point light source at a depth of 1 mm in the heterogeneously scattering brain tissue. The scattering properties of the scattering sample tissue were set to a scattering length of 75 μm and a scattering anisotropy coefficient of 0.92. A detector (with an area of ​​18 × 18 mm) was placed at the position of the fluorescence collection optical fiber bundle. 2 (set to 0.05°C), allowing the detector to receive all scattered fluorescence.

[0071] While the diagrams shown in a, c, and e only show the ray propagation without considering brain tissue scattering, the corresponding diagrams shown in b, d, and e show the size and irradiance distribution of the scattered fluorescence received by the detector when brain tissue scattering is considered. As shown, due to the effect of the optical devices shown in a, c, and e, the full width at half maximum of the scattered fluorescence size at the detector shown in b, d, and e was reduced from 5.2 mm to 3 mm, and finally to 1.1 mm. Therefore, the combination of the objective lens, the second lens group 312, and the first lens group 311 can maximize the collection efficiency of the scattered fluorescence entering the fluorescence collection fiber bundle.

[0072] The optical apparatus of the present disclosure has been described above in detail with reference to Figures 1 to 8. Hereinafter, embodiments of the imaging system and imaging method of the present disclosure will be described with reference to Figures 9 and 10. Note that the descriptions of the embodiments of the imaging system and imaging method correspond to the descriptions of the embodiments of the optical apparatus, so that parts that are not described in detail may be referred to in the previous method embodiments.

[0073] 9 is a structural schematic diagram of an imaging system according to an exemplary embodiment of the present disclosure. As shown in FIG. 9, the imaging system according to an embodiment of the present disclosure includes an optical device 910 and a signal processing module 920.

[0074] The optical instrument 910 is used to collect the optical signal of the sample to be detected, and the optical instrument may be the optical instrument according to any one of the embodiments of the present disclosure.

[0075] The signal processing module 920 is used to convert the optical signals collected by the optical instrument into an image.

[0076] 10 is a flowchart of an imaging method according to an exemplary embodiment of the present disclosure. As shown in FIG. 10, the imaging method includes steps S1010 and S1020.

[0077] In step S1010, an imaging target area of ​​the detection target sample is determined.

[0078] In step S1020, an optical signal of the imaging target area is detected by an optical device according to any one of the embodiments of the present disclosure, so as to generate an image corresponding to the imaging target area based on the optical signal.

[0079] The imaging method shown in FIG. 10 may determine an image corresponding to an imaging target region of a brain tissue sample, and based on the image corresponding to the imaging target region of the brain tissue sample, brain function of the imaging target region of the brain tissue sample may be studied.

[0080] The basic principles of the present disclosure have been described above with reference to specific embodiments. However, the advantages, merits, effects, etc. mentioned in the present disclosure are merely examples and are not limiting. These advantages, merits, effects, etc. are not necessarily provided by each embodiment of the present disclosure. Furthermore, the specific details disclosed above are merely for illustration and understanding and are not limiting. The above details do not necessarily limit the present disclosure to be realized by adopting the above specific details.

[0081] Block diagrams of elements, devices, apparatus, and systems according to the present disclosure are merely illustrative examples and are not intended to require or imply that they be connected, arranged, or configured in the manner shown in the block diagrams. Those skilled in the art may connect, arrange, or configure these elements, devices, apparatus, and systems as desired. For example, terms such as "include," "comprise," and "have" are open-ended terms and may be used interchangeably to mean "including but not limited to." As used herein, "or" and "and" refer to "and / or" and may be used interchangeably unless the context clearly indicates otherwise. As used herein, "for example," means "including but not limited to," and may be used interchangeably.

[0082] In the devices, apparatuses, and methods of the present disclosure, each component or each step can be disassembled and / or recombined, and such disassembly and / or recombination should be considered as an equivalent aspect of the present disclosure.

[0083] The above description of the disclosed aspects is provided to enable any person skilled in the art to make or use the present disclosure. Various modifications to these aspects will be apparent to those skilled in the art, and the generic principles defined herein may be applied to other aspects without departing from the scope of the present disclosure. Thus, the present disclosure is not intended to be limited to the aspects shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

[0084] The foregoing description has been provided for purposes of illustration and description. It is not intended to limit the embodiments of the present disclosure to the precise form disclosed. While several exemplary aspects and embodiments have been described above, those skilled in the art may contemplate several variations, modifications, variations, additions, and subcombinations thereof.

Claims

1. An optical instrument for use in a miniature multiphoton microscope, the optical instrument having a fluorescence excitation light path and a fluorescence collection light path, the optical instrument including a focusing lens; the condenser lens includes a first lens group and a second lens group spaced apart from each other, the first lens group being located in the fluorescence collection optical path, the second lens group being located in the fluorescence excitation optical path and the fluorescence collection optical path, and the second lens group being in close contact with the microscope objective lens; optical equipment.

2. the optical device further includes a third lens group located in the fluorescence excitation light path, and in the fluorescence excitation light path, the third lens group and the second lens group are arranged to form a tube lens.

10. The optical instrument according to claim 1.

3. the optical instrument further includes a spectroscopic optical element located in the fluorescence excitation light path and the fluorescence collection light path, the spectroscopic optical element having a function of transmitting or reflecting the light beam depending on the wavelength of the light beam; 3. The optical instrument according to claim 2.

4. The spectroscopic optical element includes a dichroic mirror.

4. The optical instrument according to claim 3.

5. the first lens group and the second lens group are separated by the dichroic mirror, and the second lens group and the third lens group are separated by the dichroic mirror; 5. The optical instrument according to claim 4.

6. The optical instrument further includes a femtosecond laser device located in the fluorescence excitation light path, and the femtosecond laser device is used to simultaneously absorb at least two photons in a detection target sample mounted on the optical instrument.

6. The optical instrument according to claim 5.

7. the optical instrument further includes a collimating lens, a galvanometer mirror, and a scanning lens located in the fluorescence excitation light path; the collimating lens is used to collimate the received initial laser light beam and emit a collimated light beam; the galvanometer mirror is used to reflect and scan the collimated light beam; the scanning lens is used to focus the collimated light beam reflected by the galvanometer mirror onto a focal plane of the scanning lens to obtain a focused light beam, and emit the focused light beam to the third lens group; The optical device according to any one of claims 2 to 6.

8. The collimating lens is composed of one negative lens and one cemented doublet lens.

8. The optical instrument according to claim 7.

9. the objective lens includes an infinity objective lens, and the objective lens is located in the fluorescence excitation light path and the fluorescence collection light path. The optical device according to any one of claims 1 to 8.

10. The length of the infinity objective lens is 4.34 mm or less, and the physical diameter of the optical lens of the infinity objective lens is 3 mm or less.

10. The optical instrument according to claim 9.

11. In the fluorescence excitation light path, the second lens group is a tube lens.

10. The optical instrument according to claim 1.

12. the optical device further includes a fourth lens group, and in the fluorescence excitation light path, the fourth lens group and the second lens group are arranged as a tube lens, and the fourth lens group and the second lens group are closely contacted groups.

10. The optical instrument according to claim 1.

13. the second lens group is used to focus the fluorescent beam emitted by the objective lens and emit a first focused beam; The optical device according to any one of claims 1 to 12.

14. the first lens group is used to emit a second focused beam based on the first focused beam, and the diameter of the second focused beam is smaller than the diameter of the first focused beam; 14. The optical instrument according to claim 13.

15. 1. An optical imaging system comprising: The optical instrument according to any one of claims 1 to 14, which is used to collect an optical signal of a sample to be detected; a signal processing module used to convert the optical signals collected by the optical instrument into an image. Optical imaging system.

16. 1. An imaging method comprising: determining an imaging target area of ​​the sample to be detected; detecting the optical signal of the imaged target area by an optical instrument according to any one of claims 1 to 14, so as to generate an image corresponding to the imaged target area based on the optical signal; Imaging method.

Images