CN210155406U - A three-dimensional head-mounted microscope - Google Patents

Abstract

The utility model relates to the technical field of optical microscopic imaging, in particular to a three-dimensional head-mounted microscope, comprising a lens assembly, an objective lens, and a scanner assembly, the objective lens is provided with a photoelectric detector, and the photoelectric detector Photosensitive unit, drive circuit. This scheme realizes the simplification of the relevant components for fluorescence collection, makes the volume of the relevant components for fluorescence collection smaller, and enables relevant experimental operations and detection.

Description

A three-dimensional head-mounted microscope

technical field

The utility model relates to the technical field of optical microscopic imaging, in particular to a three-dimensional head-mounted microscope.

Background technique

In nonlinear optical imaging microscopy, especially multiphoton fluorescence microscopy, a near-infrared laser pulse is focused by a microscope objective to excite an isotropically emitted fluorescence signal in the sample. Biological tissues usually exhibit strong absorption and high scattering optical properties. For epifluorescence detection, the same microscope objective is used to both focus the excitation light and collect the fluorescence signal. The intensity of the fluorescence signal collected by the microscope objective depends on the numerical aperture of the microscope objective and the Objective Front Aperture (OFA) (E.Beaurepaire, et.al, Applied Optics, vol.41, no.25, pp. 5376-5382, 2002). The larger the numerical aperture of the microscope objective and the front aperture of the objective, the greater the intensity of the fluorescent signal that the microscope objective can collect. For the 0.8 numerical aperture, 40X magnification microscope objective commonly found in two-photon fluorescence microscopy, less than 10% of the fluorescence within the solid angle of the highly scattering sample is collected by the microscope objective.

et.al,Optics Letters,vol.31,no.16,pp.2447-2449,2006)。2007年采用抛物面镜和2011年采用圆柱面镜的发射检测技术被提出，仿真获得了10倍荧光收集效率增强，实验获得了8.9倍荧光收集效率增强(C.A.Combs,et.al,Journal fMicroscopy,vol.228,no.3,pp.330-337,2007和V.Crosignani,et.al,Journal ofBiophotonics,vol.4,no.9,pp.592-599,2011)。之后，上述技术分别经过改进后被用于落射式荧光检测(C.A.Combs,et.al，Journalof Microscopy,vol.241,no.2,pp.153-161,2011和V.Crosignani,et.al,Journal of Biomedical Optics,vol.17,no.11,pp.116023,2012)。最近报道了一种可用于直立双光子显微镜的紧凑的全发射检测器件(C.A.Combs,et.al，Journal of Microscopy,vol.253,no.2,pp.83-92,2014)。此外通过在显微物镜周围安排5-8根高数值孔径的光纤来收集显微物镜收集不到的荧光，可以在高数值孔径显微物镜获得2倍荧光收集效率增强，在低数值孔径显微物镜获得20倍荧光收集效率增强(C.J.Engelbrecht,et.al,Optics Express,vol.17,no.8,pp.6421-6435,2009和J.D.McMullen,et.al,Journal of Microscopy,vol.241,no.2,pp.119–124,2011)。2016年一种兼容商用双光子荧光显微镜的采用四分之一椭球反射镜的全发射检测技术被提出，在高数值孔径显微物镜获得了2.75倍荧光收集效率增强(Y.Xu,et.al,IEEE PhotonicsJournal,Vol.8,Issue 5,6901109,2016)。In recent years, many techniques have emerged to collect fluorescent photons that cannot be collected by microscope objectives, and in 2006, a refracting microscope objective was proposed (D.

et.al, Optics Letters, vol. 31, no. 16, pp. 2447-2449, 2006). In 2007, the emission detection technology using a parabolic mirror and a cylindrical mirror was proposed in 2011. The simulation obtained 10 times the fluorescence collection efficiency enhancement, and the experiment obtained 8.9 times the fluorescence collection efficiency enhancement (CACombs, et.al, Journal fMicroscopy, vol. 228, no. 3, pp. 330-337, 2007 and V. Crosignani, et. al, Journal of Biophotonics, vol. 4, no. 9, pp. 592-599, 2011). Afterwards, the above techniques were respectively improved and used for epifluorescence detection (CACombs, et.al, Journal of Microscopy, vol.241, no.2, pp.153-161, 2011 and V.Crosignani, et.al, Journal of Biomedical Optics, vol. 17, no. 11, pp. 116023, 2012). A compact all-emission detection device that can be used in upright two-photon microscopy was recently reported (CACombs, et. al, Journal of Microscopy, vol. 253, no. 2, pp. 83-92, 2014). In addition, by arranging 5-8 high numerical aperture optical fibers around the microscope objective to collect the fluorescence that cannot be collected by the microscope objective, the fluorescence collection efficiency can be enhanced by 2 times in the high numerical aperture microscope objective, and in the low numerical aperture microscope The objective lens obtains a 20-fold increase in fluorescence collection efficiency (CJEngelbrecht, et.al, Optics Express, vol.17, no.8, pp.6421-6435, 2009 and JDMcMullen, et.al, Journal of Microscopy, vol.241, no. 2, pp. 119–124, 2011). In 2016, a full-emission detection technique using a quarter ellipsoid mirror compatible with commercial two-photon fluorescence microscopes was proposed, which achieved a 2.75-fold fluorescence collection efficiency enhancement in a high numerical aperture microscope objective (Y.Xu, et. al, IEEE Photonics Journal, Vol. 8, Issue 5, 6901109, 2016).

The above techniques for enhancing fluorescence collection efficiency all use additional optics to collect fluorescence photons that cannot be collected by microscope objectives. Due to the large dispersion of the scattering angle of fluorescent photons, the multiple reflection paths of fluorescent photons after entering the additional collection optical path are complex, and the loss is large, which limits the actual collection efficiency of the additional optical elements. In addition, the additional optical elements have complex shapes, which are difficult and expensive to process. Many of the above techniques for enhancing fluorescence collection efficiency are too bulky, which will block the imaging area and hinder simultaneous electrophysiological experiments.

In addition, in mouse experiments, microscopes are often used to detect the activities of experimental mice and some indicators of biological tissues of mice. However, according to the above, it can be seen that due to the current fluorescence collection related equipment and the volume of components Both are relatively large, so they cannot be worn on the head of the mouse, and cannot perform real-time detection of some indicators such as the activity and biological tissue of the mouse, which is very inconvenient to operate.

Utility model content

The purpose of the present invention is to provide a three-dimensional head-mounted microscope, which can simplify the relevant components of fluorescence collection, make the volume of the relevant components of fluorescence collection smaller, and can perform relevant experimental operations and detections.

In order to achieve the above purpose, the technical scheme of the present utility model is: a three-dimensional head-mounted microscope, comprising a lens assembly, an objective lens, and a scanner assembly, the objective lens is provided with a photodetector, and the photodetector includes a protection element, a filter, Photosensitive unit, drive circuit.

The working principle and effect of this solution are as follows: the scanner component in this solution is used to separate laser and nonlinear optical signals and output the nonlinear optical signal, and is also used to change the incident angle of the laser so that the laser can touch the interior of the living sample. Two-dimensional line scanning is performed on the plane of the tissue, and at the same time, the Z-axis scanning at the distal end can be performed to realize three-dimensional imaging. Any scanner that can realize the three-dimensional scanning function can be called a scanner assembly, and the scanner assembly can be based on different needs. Specific settings for different numbers and types of scanners. The objective lens is used to focus the laser light from the scanner assembly inside the living sample to excite the living sample to generate nonlinear optical signals and to output nonlinear optical signals. The lens assembly has the functions of collimating the laser output from the fiber, reducing the chromatic aberration of the laser with different frequencies, and focusing the laser. type of lens. Photodetectors are used to collect fluorescent photons that cannot be collected by the objective. The protection element is used to isolate the external experimental sample, the liquid used for immersion and the filter of the photodetector, and also used for electrical isolation to prevent the high voltage of the photosensitive unit of the photodetector from causing danger to the sample and the operator. The photodetector filter is used to filter out backreflected and backscattered excitation light. The photosensitive unit of the photodetector is used to convert the fluorescent photons passing through the filter into electrical signals, and the driving circuit of the photodetector is used to provide high voltage and driving signal to the photosensitive unit of the photodetector, and is used for external amplification. The circuit is connected to the computer.

Compared with the prior art, this solution is illustrated by taking a commonly used liquid immersion objective lens with a numerical aperture of 0.8 and a magnification of 40X as an example. It can be seen that the utility model arranges a ring photodetector with a width of 1 mm around the front aperture of the same microscope objective lens, which can collect fluorescent photons with a fluorescence emission half angle between 30 degrees and 60 degrees, which is equivalent to having an excitation numerical aperture of 0.8 , while the collection numerical aperture is 1.0, so the fluorescence collection efficiency is greatly improved, the signal-to-noise ratio of imaging is improved, and the imaging depth in high scattering media is improved.

In addition, in this scheme, the photodetector is installed around the objective lens to realize the collection of fluorescence. Compared with the existing fluorescence collection equipment and components, the volume is greatly reduced, which is convenient to be placed on the head of the animal and has no effect on the activity of the animal. The real-time detection of certain indicators such as biological tissue is simple and convenient to operate, and the cost is greatly reduced compared to the larger-volume fluorescence collection equipment and components.

Further, the lens assembly includes a combination of one or more lenses including a collimating lens, a cylindrical lens, a focusing lens, and a collecting lens; the collimating lens is used for collimating the laser light output from the laser input fiber and reducing the difference between the laser light of different frequencies. chromatic aberration and output laser signal; cylindrical lens, used to form a linear focus; focusing lens, used for laser focusing; collection lens, used to collect nonlinear optical signals and input the laser output fiber. In the actual production and manufacturing process, according to the needs of use, according to the different functions of each microscope and the specific differences of different microscopes, the lens assembly can include the above-mentioned different numbers and types of lenses.

Further, a wave plate is also included, and the wave plate is used to change the polarization direction of the laser light.

Further, the photosensitive unit is one of an avalanche diode, a photocoupler device, a metal semiconductor oxide device, a focal plane array device, a photomultiplier tube device, a single photon counting device, or a hybrid device including a plurality of the above devices. The above devices are all common photoelectric sensitive units, which are easy to purchase and configure, and have low cost.

Further, the scanner assembly includes a dichroic mirror scanner and a vertical dichroic mirror scanner. The dichroic mirror scanner is used for separating the laser light from the nonlinear optical signal and outputting the nonlinear optical signal, and also for changing the incident angle of the laser light to allow the laser light to perform two-dimensional line scanning on the plane of the internal tissue of the living sample. Vertical dichroic mirror scanner for remote Z-axis scanning for 3D imaging. In the aspect of brain imaging of active animals, this solution does not provide a scan lens (Scan lens) and a tube lens (Tube lens), instead of using a dichroic mirror scanner, a vertical dichroic mirror scanner and a lens assembly The dichroic mirror and the micro-electromechanical scanner in the prior art can greatly improve the imaging speed, optimize the internal structure, and reduce the weight under the premise of satisfying the imaging quality. On the premise of reducing the weight of the miniature optical probe, it is more convenient to wear it on the animal's head when collecting data from living samples, especially in the area of brain imaging of active animals, reducing the impact of weight on animal activities and further reducing detection error.

Further, the dichroic mirror scanner includes a dichroic mirror and a driver capable of driving the dichroic mirror to change the angle, and the dichroic mirror covers the driver. The driver is used to drive the dichroic lens to rotate to realize scanning.

The existing MEMS scanner includes a reflective sheet and a MEMS driver. The reflective sheet is covered on the MEMS driver, wherein there are several reflective sheets, and the MEMS driver can respectively drive the reflective sheet to change the angle. In this solution, the reflective mirror is replaced with a dichroic mirror, and the driver does not affect the transmission of nonlinear optical signals in this solution. In addition, the dichroic lens not only plays the role of the dichroic mirror in the prior art, but also achieves the effect of allowing the driver to change the laser reflection angle, and can also reduce the number of components, making the entire microscope smaller in size and weight. lighter. Through the above assembly method, the dichroic mirror scanner can be obtained at low cost.

Further, the backside of the wafer of the dichroic mirror scanner is provided with a transmission hole through which the nonlinear optical signal is transmitted. As a result, more mature products can be obtained quickly.

Further, when the photosensitive unit is one of a photocoupler device, a metal semiconductor oxide device, a focal plane array device, a photomultiplier tube device, a single photon counting device, or a hybrid device including the above devices, the protection of the photodetector The surface opposite to the photosensitive unit of the element is a microlens array. Therefore, through this solution, the fluorescence is focused on each pixel of the photosensitive unit, and the photosensitive efficiency is improved.

Further, it also includes a reflection mirror, and the reflection mirror includes a transmission surface and a reflection surface. The reflection will be used to adjust the direction of irradiation of the laser light, the angle of incidence, and the like. Through the design of the transmission surface and the reflection surface, better reflection and transmission effects can be achieved.

Further, the surface of the protection element is provided with an anti-reflection optical coating. Thereby, the transmittance of fluorescent photons is improved.

Description of drawings

Fig. 1 is a front sectional view of a three-dimensional head-mounted microscope in Example 1 (a laser roadmap is disclosed at the same time);

Fig. 2 is the enlarged schematic diagram of the objective lens of the microscope in Fig. 1;

3 is a schematic structural diagram of a photodetector;

4 is a schematic diagram of a three-dimensional line scan in Example 1;

5 is a schematic structural diagram of a dichroic mirror scanner;

Fig. 6 is the front sectional view that adopts the mode of etching to produce dichroic mirror scanner;

FIG. 7 is a schematic diagram of the three-dimensional head-mounted microscope being worn on the head of a mouse.

Detailed ways

The following is further described in detail by specific embodiments:

Reference numerals in the drawings include: collimating lens 10 , cylindrical lens 12 , mirror 20 , dichroic mirror scanner 30 , objective lens 40 , focusing lens 50 , wave plate 60 , vertical dichroic mirror scanner 70 , collection lens 80, laser input fiber 90, laser output fiber 91, housing 100, substrate 11, driver 22, dichroic mirror 33, photodetector 2, protection element 2.1, filter 2.2, photosensitive unit 2.3, Driver circuit 2.4.

Example 1

Basically as shown in FIG. 1: a three-dimensional head-mounted microscope, comprising a square housing 100, the housing 100 is a sealing structure made of high molecular polymer material, and the housing 100 is installed with a collimating lens 10, a cylindrical lens 12, a reflection Mirror 20, dichroic mirror scanner 30, objective lens 40, focusing lens 50, wave plate 60, vertical dichroic mirror scanner 70 and collecting lens 80. connection or welding. 5 and 6, the dichroic mirror scanner 30 in this embodiment includes 33 dichroic mirrors and a MEMS driver 22 that does not affect the transmission of nonlinear optical signals. The 33 dichroic mirrors cover the On the MEMS driver 22, the MEMS driver 22 can drive the dichroic mirror 33 to change the angle. In this embodiment, the objective lens 40 is an aspherical lens. As shown in FIG. 2 , the bottom of the objective lens 40 in this solution is fixed with the photodetector 2 by bonding. Of course, it can also be fixed by other methods, such as gluing , welding and snap connection, etc. 3, the photodetector 2 in this embodiment includes a protection element 2.1, a filter 2.2, a photosensitive unit 2.3 and a driving circuit 2.4, the filter 2.2 is located at the bottom, and the photosensitive unit 2.3 is located at the filter Above 2.2, the driving circuit 2.4 is located above the photosensitive unit 2.3. In this embodiment, the material of the protection element 2.1 of the photodetector 2 is an insulating material that can transmit visible light wavelengths, and the material of the optical filter 2.2 of the photodetector 2 is an insulating material that can transmit visible light wavelengths, and the dielectric strength is greater than 5MV/mm . The filter 2.2 of the photodetector 2 is used to filter out long-wavelength excitation light and transmit short-wavelength fluorescent photons. In this embodiment, the microscope objective lens 40 is attached to the protection element 2.1 of the photodetector 2, the protection element 2.1 of the photodetector 2 is attached to the filter 2.2 of the photodetector 2, and the filter of the photodetector 2 is attached. The chip 2.2 is attached to the photosensitive unit 2.3 of the photodetector 2. The output end of the photosensitive unit 2.3 of the photodetector is electrically connected to the input end of the drive circuit 2.4 of the photodetector 2. The drive circuit 2.4 of the photodetector 2 is electrically connected. The output end is electrically connected with the external amplifying circuit and the computer. In this embodiment, the objective lens 40 is used to collect fluorescent photons within the aperture, and the photodetector 2 is located at the end of the microscope objective lens 40 that is closer to the sample, and is annularly located around the front aperture of the microscope objective lens 40. Protection element 2.1 of 1 is used to isolate the sample, liquid for immersion and filter 2.2 of photodetector 2, protection element 2.1 of photodetector 2 is also used for electrical isolation, preventing the photosensitive unit of photodetector 2 The high voltage of 2.3 is dangerous to the sample and the operator. The surface of the protective element 2.1 of the photodetector 2 is also coated with an anti-reflection optical coating to improve the transmittance of fluorescent photons. The filter 2.2 of the photodetector 2 is used for The back-reflection and back-scattered excitation light is filtered out, the photosensitive unit 2.3 of the photodetector 2 is used to convert the fluorescent photons passing through the filter 2.2 into electrical signals, and the drive circuit 2.4 of the photodetector 2 is used to The photosensitive unit 2.3 of the photodetector 2 provides high voltage and driving signal, and is connected with an external amplifying circuit and a computer. The photodetector 2 is used to collect fluorescent photons that cannot be collected by the microscope objective lens. In this embodiment, the protection element 2.1, the optical filter 2.2, the photosensitive unit 2.3 and the driving circuit 2.4 can be fixed together by bonding, of course, they can also be fixed by other means, such as snap connection.

In this embodiment, the photosensitive unit 2.3 of the photodetector 2 is made of a single Large Area Avalanche Photo Diode (LAAPD) through mechanical drilling or corrosion processing or is made of transparent materials, and the central hole or transparent material is used for The remaining annular portion of the large-area photomultiplier tube is used to receive fluorescence photons that are not received by the microscope objective 40 through the excitation light transmitted through the microscope objective 40 . Since the avalanche diode needs to work in reverse bias mode, the cathode faces the liquid immersion liquid and biological samples, and the driving voltage is as high as several hundreds to 2000 volts, so the protection element 2.1 of the photodetector 2 can be made of light-transmitting insulating materials, such as Optical glass, etc., the thickness of the protective element 2.1 hundreds of microns is enough to withstand the high driving voltage of avalanche diodes to avoid damage to samples, microscopes and operators. In addition, the photosensitive unit 2.3 in this embodiment is one of a photocoupling device, a metal semiconductor oxide device, a focal plane array device, a photomultiplier tube device, a single photon counting device, or one based on any of the above multiple photoelectric conversion principles. When mixing devices, the surface of the protection element 2.1 of the photodetector 2 opposite to the photosensitive unit 2.3 is a microlens array, which is used to focus the fluorescence to each pixel of the photosensitive unit 2.3 to improve the photosensitive efficiency.

Referring to FIG. 1 , the collimating lens 10 in this embodiment is used to collimate the laser light output from the laser input fiber 90 , reduce the chromatic aberration between laser light of different frequencies, and output the laser signal to the mirror 20 . The housing 100 in this embodiment is provided with a hole through which the laser input optical fiber 90 passes, and the laser input optical fiber 90 is clamped on the hole. In addition, the number of the reflectors 20 in this embodiment is three, and the reflector 20 is provided with a reflecting surface, wherein the first reflector 20 is located below the collimating lens 10, and the reflecting surface faces the right side, and the second reflector 20 is located below the collimating lens 10. The reflecting mirror 20 is located on the right side of the first reflecting mirror 20, the reflecting surface of the second reflecting mirror 20 is opposite to the reflecting surface of the first reflecting mirror 20 towards the left, and the third reflecting mirror 20 is located at the second reflecting mirror 20, and the reflective surface of the third mirror 20 also faces to the left. The objective lens 40 of the aspherical lens in this embodiment is located on the bottom of the housing 100, and the bottom of the housing 100 is provided with a hole for installing the objective lens 40, and the curvature radius of the objective lens 40 changes with the central axis to improve the optical quality, Reduce optical components and reduce design costs.

In this embodiment, the lenticular lens 12 is located on the left side of the third reflecting mirror 20 , the lenticular lens 12 is located on the upper right of the objective lens 40 , and the dichroic mirror scanner 30 is located above the objective lens 40 and on the left side of the lenticular lens 12 , the cylindrical lens 12 is used to focus the collimated laser light into a linear focus in a certain direction (herein referred to as the X direction) on the surface of the dichroic mirror scanner 30, that is, a certain direction (X direction) of the cylindrical lens 12 The focal position of the dichroic mirror surface, and the focal position of the other direction (here referred to as the Y direction) orthogonal to a certain direction (X direction) of the lenticular lens 12 is not on the dichroic mirror surface.

The mirror 20 in the present embodiment is used for the irradiation angle of the laser light and reflects the laser light onto the dichroic mirror scanner 30 through the cylindrical lens 12 . In addition, the reflector 20 can be multiple pieces for translating the optical path, and the material is optical glass or high molecular polymer. The reflector 20 can also be provided with a transmission surface and a reflection surface. The surface has an optical coating that enhances the reflectivity, thereby improving the effect of laser reflection and transmission.

In this embodiment, the focusing lens 50 is located above the dichroic mirror scanner 30, and the dichroic mirror scanner 30 is used for separating the laser light from the nonlinear optical signal and outputting the nonlinear optical signal, and also for changing the laser light The dichroic mirror scanner 30 reflects the s-type linearly polarized laser light, and then the focusing lens 50 collimates the s-type linearly polarized laser light in the X direction and in another direction (Y direction) perpendicular to the X direction Focused into a linear shape, the s-type linearly polarized laser continues to pass through the wave plate 60 located above the focusing lens, the polarization direction of the s-type linear polarization is rotated by 45 degrees, and then the laser is focused in the Y direction on the vertical di-directional above the wave plate 60 On the surface of the chromatic mirror scanner 70, the vertical dichroic mirror scanner 70 reflects the laser light, the reflected and scattered laser light passes through the wave plate 60 again, and the polarization direction of the laser light is rotated 45 degrees in the same direction again, becoming p-type linearly polarized light , again through the focusing lens 50, the beam focused in the X direction and collimated in the Y direction is projected on the surface of the dichroic mirror scanner 30. The dichroic mirror scanner 30 transmits the p-type linearly polarized laser with the same wavelength, and the dichroic mirror scans The detector 30 is located at the back focal plane of the objective lens 40, the movable mirror in the dichroic mirror scanner 30 rotates along the rotation axis parallel to the X axis, and finally the p-type linearly polarized light passes through the objective lens 40 to form the X direction in the sample The linear focus is collimated and focused in the Y direction, and the linear focus is scanned along the X direction, thereby forming a two-dimensional scanning trajectory, enabling the laser to perform two-dimensional line scanning on the plane of the internal tissue of the living sample. When the dichroic mirror scanner 30 When a frame of two-dimensional line scan image is completed, the movable dichroic mirror on the vertical dichroic mirror scanner 70 moves a distance along the optical axis (Z direction). The two-dimensional line scanning plane of the internal tissue of the living sample also moves a distance along the optical axis, and the three-dimensional line scanning is realized by scanning in the Z direction on the vertical dichroic mirror scanner 70, and the nonlinear signal excited in the sample is captured by the objective lens 40 collects, passes through the dichroic mirror scanner 30 that transmits the wavelength of the nonlinear signal, the focusing lens 50, the wave plate 60, and is linearly focused on the surface of the vertical dichroic mirror scanner 70 in the Y direction, and the vertical dichroic mirror scanner 70 70 transmits the wavelength of the nonlinear signal, and then the collection lens 80 located above the vertical dichroic mirror scanner 70 linearly focuses the nonlinear signal on the surface of the laser output fiber 91 located on the collection lens 80 in the X direction, and finally transmits it to External photoelectric detection equipment, wherein, the laser input fiber 90 is a large-mode field single-mode fiber or a polarization-maintaining fiber or a photonic crystal fiber, and the laser output fiber 91 is a fiber bundle, and the schematic diagram of the three-dimensional line scan is shown in Figure 4; The top of 100 is provided with a hole for the laser input fiber 90, and the laser input fiber 90 is clamped on the hole.

In addition, due to the linear focus formed by the line scanning method adopted by the present invention, the fluorescence collected by the objective lens 40 is also linear, and moves parallel to the end face of the laser output fiber 91 with the rotation of the dichroic mirror scanner 30 , so the detection of the moving linear fluorescence is done by a scientific complementary metal oxide semiconductor (sCMOS) camera with a synchronizable rolling shutter technology, the position of the linear fluorescence corresponds to a certain line currently read out by the rolling shutter of the sCMOS camera The photoelectric detection units are strictly synchronized, enabling high-speed imaging.

Specific use: the collimating lens 10 in this embodiment uses an achromatic collimating lens 10 (#65-286, Edmund Optics Inc., Barrington, NJ, USA; diameter: 2mm, equivalent focal length: 3mm, special near- Infrared light), can collimate the output laser and reduce the chromatic aberration between different frequency components of the femtosecond laser, which is beneficial to improve the transmission efficiency (up to 50% from the laser source to the sample), beam focusing and excitation efficiency. Of course, it can also be designed for achromatic design. The design wavelength is any two wavelengths between 700nm and 1600nm, which can be 817nm and 1064nm, but not limited to these two wavelengths. The material is optical glass or high molecular polymer, and the surface has enhanced transmission. High-efficiency optical coatings for laser collimation.

The numerical aperture of the objective lens 40 is 0.7 (in water), the diameter of the movable dichroic mirror in the dichroic mirror scanner 30 is 2 mm, the package size is 5×5 mm ² , the first resonance frequency is 400 Hz, and its maximum optical scanning angle is It is ±15 degrees, and the Z direction moving range of the vertical dichroic mirror scanner is 300um. In addition, considering that the actual single-mode core of the laser output fiber 91 is at least 3um, the supported frame size is 512×512×100 and the maximum field of view is 400 × 400 × 300um ³ imaging, to achieve high-speed image acquisition, the Japanese Hamamatsu ORCA-FLASH 4.0 CMOS camera can reach 512 × 512 × 300@4fps.

The focusing lens 50 is achromatic design, the design wavelength is any two wavelengths between 350nm and 700nm, usually 408nm and 633nm, but not limited to these two wavelengths, the material is optical glass or high molecular polymer, and the surface has enhanced transmission High-efficiency optical coatings used to focus and couple the received nonlinear optical signal into the collection fiber.

The specific laser input fiber 90 large mode field single mode fiber or polarization maintaining fiber or photonic crystal fiber, the design wavelength is any wavelength between 700nm and 1600nm, the material is optical glass, quartz, plastic or polymer, used for transmission Laser generated by an external excitation light source.

In this embodiment, as shown in FIGS. 5 and 6 , the dichroic mirror scanner 30 is a uniaxial structure, and specifically includes a hollow substrate 11 , a driver 22 and a mirror surface, and the driver 22 is fixed on the On the substrate 11 made of high molecular polymer, the two ends of the driver 22 are provided with torsion beams, and the torsion beams are rotatably connected to the inner wall of the substrate 11. The driver 22 is used to change the angle of the mirror surface according to the instruction, and the mirror surface includes several dichroics. Mirror 33, the dichroic mirror 33 includes an ultra-thin sheet, and the ultra-thin sheet is coated with a dichroic film, and the material of the dichroic mirror 33 is optical glass or high molecular polymer, and is used for s-type reflection wavelengths of 700nm-1600nm. The polarized laser light transmits p-type polarized laser light with a wavelength of 700nm-1600nm and a nonlinear optical signal with a wavelength of 350nm-700nm. The driver 22 includes a number of mirrors for the nonlinear optical signal to pass through. The dichroic mirrors 33 are respectively fixed on On the surface of the mirror body, the mirror body is annular, and the shape of the mirror surface is disc. The driver 22 in this embodiment is generally driven by electrostatics. In the design of the driver 22, this embodiment uses the existing driver 22 design. Specifically, the surface micromachining process SOIMUMPs of MEMSCAP company can be used. This technology is the prior art, and details are not repeated here.

Vertical dichroic mirror scanner 70, the lens is 44 dichroic mirrors, the material of the 44 dichroic mirrors is optical glass or high molecular polymer, which is used to reflect laser light with a wavelength of 700nm-1600nm and transmit a wavelength of 350nm- 700nm nonlinear optical signal.

The objective lens 40 is achromatic design, the design wavelength is any two wavelengths between 700nm and 1600nm, usually 817nm and 1064nm, but not limited to these two wavelengths, the material is optical glass or polymer, and the surface has enhanced transmittance The structure of the optical coating can be a traditional refractive lens, a gradient index lens or a gradient index lens with a curved shape, which is used to focus the incident laser on the surface of a living sample (or human body) to excite nonlinear optical signals.

The mirror 20 is placed at 45 degrees for reflecting the laser light (laser signal) at 90 degrees to the dichroic mirror scanner 30 .

In this embodiment, the volume of the casing 100 is less than 5mm*5mm*5mm, and the utility model can be fixed on the top of the mouse’s head (as shown in FIG. 7 ) during specific use, or can be installed on the top of the head of other animals, such as Marmosets, rabbits, etc.

Through this embodiment, in this solution, the photodetector 2 can be installed around the objective lens 40 to realize the collection of the fluorescence of the detected object. The head of the animal is used for real-time detection of some indicators such as animal activity and biological tissue. The operation is simple and convenient, and the cost is greatly reduced compared with the equipment and components of the larger fluorescence collection. Compared with the prior art, the present embodiment has the following advantages: taking the commonly used liquid immersion objective lens 40 with a numerical aperture of 0.8 and a magnification of 40X as an example, the fluorescence emission half-angle of the liquid immersion objective lens 40 is arcsin (0.8/1.33 )=30 degrees, it can be calculated that the annular photodetector 2 with a width of 1 mm arranged around the front aperture of the same microscope objective lens 40 in the present invention can collect fluorescence photons with a fluorescence emission half angle between 30 degrees and 60 degrees. , which is equivalent to having an excitation numerical aperture of 0.8 and a collection numerical aperture of 1.0, thus greatly improving the fluorescence collection efficiency, improving the signal-to-noise ratio of imaging, and improving the imaging depth in high scattering media.

Example 2

In this embodiment, the photosensitive unit 2.3 of the photodetector 2 is composed of a plurality of ordinary size avalanche diodes (Avalanche Photo Diode, LAAPD) to form an annular array. The regular size avalanche diode of the sheet is used to receive fluorescence photons that are not received by the microscope objective.

Example 3

In this embodiment, the photosensitive unit 2.3 of the photodetector 2 is composed of a circular array of two-dimensional pixel photoelectric sensors, such as CCD (photocoupler) devices, CMOS (metal semiconductor oxide) devices, FPA (focal plane array) devices, PMT (photomultiplier tube) devices, single photon counting devices or hybrid devices based on any of the above multiple photoelectric conversion principles, such as Hamamatsu Hybrid Photodetectors (HPD), with a central hole or transparent material for transmission through the microscope objective The excitation light, a circular array of 2D pixel photosensors is used to receive fluorescence photons that are not received by the microscope objective.

The above are only the preferred embodiments of the present utility model. It should be pointed out that for those skilled in the art, some modifications and improvements can be made without departing from the concept of the present utility model. These should also be regarded as The protection scope of the present utility model will not affect the implementation effect of the present utility model and the practicability of the patent. The technologies, shapes and structural parts that are omitted from the description of the present invention are all known technologies.

Claims (10)

1. a three-dimensional head-mounted microscope, comprising lens assembly, object lens, scanner assembly, it is characterized in that: described object lens is provided with photoelectric detector, described photoelectric detector comprises protection element, filter, photosensitive unit ,Drive circuit. 2. A three-dimensional head-mounted microscope according to claim 1, wherein the lens assembly comprises a combination of one or more lenses of a collimating lens, a cylindrical lens, a focusing lens, and a collecting lens; The collimating lens is used for collimating the laser output from the laser input fiber and reducing the chromatic aberration between the lasers of different frequencies and outputting the laser signal; the cylindrical lens is used for forming a linear focus; the focusing lens is used for the laser Focusing; the collecting lens is used to collect nonlinear optical signals and input the laser output fiber. 3 . The three-dimensional head-mounted microscope according to claim 1 , further comprising a wave plate, wherein the wave plate is used to change the polarization direction of the laser light. 4 . 4. A three-dimensional head-mounted microscope according to any one of claims 1-3, wherein the photosensitive unit is an avalanche diode, a photocoupler, a metal semiconductor oxide device, a focal plane array device, a photoelectric One of a multiplier tube device, a single-photon counting device, or a hybrid device comprising a plurality of the above devices. 5 . The three-dimensional head-mounted microscope according to claim 1 , wherein the scanner assembly comprises a dichroic mirror scanner and a vertical dichroic mirror scanner. 6 . 6 . The three-dimensional head-mounted microscope according to claim 5 , wherein the dichroic mirror scanner comprises a dichroic lens and a driver that can drive the dichroic lens to change the angle, and the dichroic lens can change its angle. 7 . The chromatic lens covers the driver. 7 . The three-dimensional head-mounted microscope according to claim 5 , wherein the back of the wafer of the dichroic mirror scanner is provided with a transmission hole for transmitting the nonlinear optical signal. 8 . 8. A three-dimensional head-mounted microscope according to claim 4, characterized in that: when the photosensitive unit is a photoelectric coupling device, a metal semiconductor oxide device, a focal plane array device, a photomultiplier tube device, a single photon device When counting one of the devices or a hybrid device including the above multiple devices, the surface of the protection element of the photodetector opposite to the photosensitive unit is a microlens array. 9 . The three-dimensional head-mounted microscope according to claim 1 , further comprising a reflection mirror, and the reflection mirror comprises a transmission surface and a reflection surface. 10 . 10 . The three-dimensional head-mounted microscope according to claim 1 , wherein the surface of the protection element is provided with an anti-reflection optical coating. 11 .

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