CN110794563A

CN110794563A - High fluorescence collection rate hand-held type microscope

Info

Publication number: CN110794563A
Application number: CN201911268411.8A
Authority: CN
Inventors: 徐英舜
Original assignee: Jiangsu Deen Medical Technology Co Ltd
Current assignee: Jiangsu Deen Medical Technology Co Ltd
Priority date: 2019-03-18
Filing date: 2019-12-11
Publication date: 2020-02-14
Also published as: CN210166558U; CN210155400U; CN111722391A; CN210155406U; CN111722388A; CN210155405U; CN111722389A; CN210155402U; CN210572988U; CN211086789U; CN210166556U; CN111722390A; CN210155403U

Abstract

The invention relates to the technical field of miniature microscopes, in particular to a handheld microscope with high fluorescence collection rate, which comprises a microscope body, wherein the microscope body is arranged on the microscope body, the microscope body is connected with a photoelectric detector used for converting fluorescence photons into electric signals, the photoelectric detector is annular and is sleeved on the microscope body, the photoelectric detector comprises an inner ring, an outer ring and a side surface connected with the inner ring and the outer ring, and the side surface is obliquely arranged; the microscope further comprises a shell for holding, and the shell covers the microscope body. By adopting the scheme, the fluorescence photons which can not be received by the objective lens body can be collected, so that the fluorescence collection efficiency is improved, the signal-to-noise ratio of imaging is improved, and the imaging depth in a high-scattering medium is improved.

Description

High fluorescence collection rate hand-held type microscope

Technical Field

The invention relates to the technical field of miniature microscopes, in particular to a handheld microscope with high fluorescence collection rate.

Background

In nonlinear optical imaging microscopes, in particular multiphoton fluorescence microscopes, near-infrared laser pulses are focused by a microscope objective and excite isotropically emitted fluorescence photons in a sample. Biological tissue generally exhibits optical properties of strong absorption and high scattering. For epi-illumination (Epifluorescence) fluorescence detection, the same microscope objective is used to both focus the laser and collect the fluorescence photons. The intensity of the fluorescence photons collected by the microscope Objective depends on the numerical Aperture of the microscope Objective and the Objective Front Aperture (OFA). The larger the numerical aperture of the microscope objective and the aperture in front of the objective, the greater the intensity of the fluorescence photons that the microscope objective can collect. For a microscope objective with a numerical aperture of 0.8 and a magnification of 40X, which is common in two-photon fluorescence microscopes, only less than 10% of the fluorescence in the solid angle of the highly scattered sample is collected by the microscope objective.

Therefore, in recent years, many techniques have been developed for collecting fluorescence photons that cannot be collected by a microscope objective, for example, by arranging 5 to 8 high-na optical fibers around the microscope objective to collect fluorescence that cannot be collected by the microscope objective, 2-fold fluorescence collection efficiency enhancement can be obtained at the high-na microscope objective and 20-fold fluorescence collection efficiency enhancement can be obtained at the low-na microscope objective. For example, a compatible commercial two-photon fluorescence microscope that appeared in 2016, achieved a 2.75 fold increase in fluorescence collection efficiency at a high numerical aperture microscope objective using a total emission detection technique using a quarter ellipsoid mirror.

The above techniques for enhancing fluorescence collection efficiency all employ additional optical elements to collect fluorescence photons that cannot be collected by the microscope objective. Due to the fact that the scattering angle of the fluorescence photons is very large in discreteness, after the fluorescence photons enter the extra collection light path, the multiple reflection path of the fluorescence photons is complex and large in loss, and therefore the actual collection efficiency of the extra optical element is limited. In addition, the shape of the additional optical element is complex, the processing difficulty is high, the cost is high, the size of an instrument using the fluorescence collection efficiency enhancing technology is too large, the imaging area can be shielded, and meanwhile, the electrophysiological experiment operation is hindered.

Therefore, a microscope capable of collecting fluorescence photons that cannot be received by the objective lens body, thereby improving fluorescence collection efficiency, improving the signal-to-noise ratio of imaging, and improving the imaging depth in a high-scattering medium is needed.

Disclosure of Invention

The present invention is directed to a high fluorescence collection rate handheld microscope to solve the above-mentioned problems.

The basic scheme provided by the invention is as follows: the utility model provides a high fluorescence collection rate hand-held type microscope, includes the microscope mirror body, be equipped with the microscope mirror body on the microscope mirror body, the microscope mirror body is connected with and is used for turning into the photoelectric detector of the signal of telecommunication with fluorescence photon.

The basic scheme has the working principle and the beneficial effects that: laser signals pass through the microscope objective lens body and reach an experimental sample, light paths of the laser signals are in different angles due to back reflection and back scattering, the laser signals after back reflection and back scattering contain fluorescence photons, one part of the laser signals enter the microscope objective lens body through the microscope objective lens body, after passing through related elements, the microscope objective lens body converts the fluorescence photons in the received laser signals into electric signals, the other part of the laser signals pass through the photoelectric detector, the fluorescence photons in the laser signals are collected by the photoelectric detector and converted into electric signals, and therefore microscopic imaging is achieved according to the electric signals. Fluorescence photons which cannot be received by the microscope body of the microscope are collected through the photoelectric detector, so that the fluorescence collection efficiency is improved, the signal-to-noise ratio of imaging is improved, and the imaging depth in a high-scattering medium is improved.

The photoelectric detector directly collects laser signals which are subjected to back reflection and back scattering of the experimental sample, the laser signals do not need to be reflected for many times, loss of the laser signals is avoided, and collected fluorescence photons are reduced. Compared with the traditional electronic scanning microscope (namely, the microscope with the function of converting fluorescence photons in laser signals passing through the microscope objective body into electric signals), the fluorescence photons which cannot be received by the microscope objective body are collected through the photoelectric detector, so that the fluorescence collection efficiency is improved, the signal-to-noise ratio of imaging is improved, and the imaging depth in a high-scattering medium is improved.

Furthermore, the photoelectric detector is annular and is sleeved on the microscope body of the microscope objective. Has the advantages that: the laser signal passing through the microscope objective body, the laser signal reflected and scattered back by the experimental sample is concentrated near the microscope objective body and gradually decreases outwards by taking the microscope objective body as a center. The photoelectric detector is sleeved on the microscope objective body and is close to the microscope objective body, so that the photoelectric detector can collect more fluorescence photons.

Further, the photodetector includes a filter layer for filtering out back-reflected and back-scattered laser signals and passing through fluorescence photons. Has the advantages that: the filter layer is arranged to filter laser signals with long wavelength, so that fluorescent photons with short wavelength penetrate through the filter layer, and the fluorescent photons can be conveniently collected subsequently.

Further, the photodetector further includes a photosensitive layer for converting the fluorescence photons passing through the filter layer into an electrical signal. Has the advantages that: the photosensitive layer is located the one side of keeping away from the microscope mirror body, and the setting of photosensitive layer will see through the fluorescence photon conversion of filter layer and become the signal of telecommunication, is convenient for follow-up data transmission that carries on.

Further, the photosensitive layer comprises a plurality of photoelectric sensitive units which are circumferentially distributed by taking the microscope body as a center. Has the advantages that: the plurality of circumferentially distributed photoelectric sensitive units are arranged to collect the fluorescence photons, so that the fluorescence photons which cannot be received by the objective body are collected, and the fluorescence collection efficiency is improved.

Further, the photosensitive layer includes a photo sensitive unit that is shape matched to the photo detector. Has the advantages that: the photoelectric sensitive units are matched with the photoelectric detector in shape, and compared with the arrangement of a plurality of photoelectric sensitive units, gaps do not exist among the photoelectric sensitive units, so that more fluorescence photons are collected, and the fluorescence collection efficiency is improved.

Further, the photoelectric detector also comprises a protective layer used for transmitting light, and the protective layer wraps the filter layer and the photosensitive layer. Has the advantages that: the setting of protective layer keeps apart the experimental sample, avoids experimental sample and filter layer contact, keeps apart photosensitive layer and experimental sample, microscope mirror body simultaneously, avoids when required high voltage to cause the injury to experimental sample, microscope mirror body, operating personnel etc. when the photosensitive layer is avalanche photodiode etc. and has the component of internal gain.

Further, one side of the photoelectric detector is flush with one side of the microscope objective body. Has the advantages that: one side of the photoelectric detector is flush with one side of the microscope objective body, namely, the photoelectric detector is embedded in the microscope objective body, so that abrasion caused by the protrusion of the photoelectric detector is avoided.

Further, the photoelectric detector comprises an inner ring, an outer ring and a side surface connected with the inner ring and the outer ring, wherein the side surface is obliquely arranged. Has the advantages that: the photodetector has a certain angle, and compared with the level of the photodetector, the angle of the fluorescence photons collected by the photodetector is larger, namely the range of collecting the fluorescence photons is larger, so that more fluorescence photons are collected, and the fluorescence collection efficiency is improved.

Further, still include the casing that is used for handheld, the casing cladding microscope mirror body. Has the advantages that: the setting of casing is convenient for hand, convenient to use, and the setting of casing can protect the microscope mirror body simultaneously.

Drawings

FIG. 1 is a schematic structural diagram of a high fluorescence collection rate handheld microscope according to a first embodiment of the present invention;

FIG. 2 is a schematic diagram of a photodetector according to an embodiment of the present invention;

FIG. 3 is a schematic diagram of a second photodetector of an embodiment of a high fluorescence collection rate handheld microscope according to the present invention;

FIG. 4 is a schematic structural diagram of a third embodiment of a high fluorescence collection rate handheld microscope according to the present invention.

Detailed Description

The following is further detailed by way of specific embodiments:

reference numerals in the drawings of the specification include: the device comprises a microscope body 1, a photoelectric detector 2, a microscope body 3, an experimental sample 4, an external computer 5, a protective layer 21, a filter layer 22 and a photoelectric sensitive unit 23.

Example one

The utility model provides a high fluorescence collection rate hand-held type microscope, as shown in figure 1, includes microscope body 1, and the objective mounting hole has been seted up to microscope body 1's bottom, and the mounting hole is used for imbedding microscope objective lens body 3. The microscope body 3 is connected with a photoelectric detector 2 for converting fluorescence photons into electric signals, the photoelectric detector 2 is annular and is sleeved on the microscope body 3, and the photoelectric detector 2 is embedded in the bottom of the microscope body 1. The photoelectric detector 2 comprises an inner ring, an outer ring and a side surface connecting the inner ring and the outer ring, the bottom side of the photoelectric detector 2 is flush with the bottom of the microscope body, namely, the distance between the inner side of the photoelectric sensitive unit 23 of the photoelectric detector 2 opposite to the microscope objective lens body 3 and the focal plane of the microscope objective lens body 3 is equal to the distance between the outer side of the photoelectric sensitive unit 23 of the photoelectric detector 2 opposite to the microscope objective lens body 3 and the focal plane of the microscope objective lens body 3.

As shown in fig. 2, the photodetector 2 includes a connection filter layer 22 and a photosensitive layer, and a protective layer 21 wrapping the filter layer 22 and the photosensitive layer. The protective layer 21 is used for isolating the experimental sample 4, the liquid for liquid immersion, the filter layer 22, and also for electrical isolation, so as to prevent the high voltage of the photosensitive layer from damaging the experimental sample 4, a microscope, and an operator; the protective layer 21 also serves to transmit laser signals containing fluorescent photons. The protective layer 21 is an optical element made of a light-transmitting insulating material, and in the present embodiment, the light-transmitting insulating material is preferably optical glass. The thickness of the protective layer 21 is greater than 100 micrometers, and in the present embodiment, the thickness of the protective layer 21 is preferably 50010 micrometers. When the photosensitive layer is an avalanche diode, the thickness of hundreds of microns is enough to bear the high driving voltage of the avalanche diode, so as to avoid the damage to the experimental sample 4, the microscope and the operator, and the technicians in the field can select the protective layer 21 with different thicknesses according to the requirements.

Filter layer 22 is used to filter out the laser signals that are back-reflected and back-scattered and transmit the fluorescence photons, i.e., filter out the laser signals with long wavelength and transmit the fluorescence photons with short wavelength. The filter layer 22 is an optical filter made of an insulating material that transmits visible light, and the dielectric strength of the optical filter is greater than 5 MV/mm. In the present embodiment, the dielectric strength of the optical filter is 1.2MV/mm, and those skilled in the art can select optical filters with different dielectric strengths according to actual situations.

The photosensitive layer, which is used to convert the fluorescence photons that pass through the filter layer 22 into electrical signals, comprises a photo sensitive unit 23, which photo sensitive unit 23 is shape matched to the photo detector 2. The photo-sensitive unit 23 is one of an avalanche diode, a photocoupler, a metal semiconductor oxide device, a photomultiplier device, a single photon counting device, and a hybrid device based on any of the above photoelectric conversion principles, and in the present embodiment, the photo-sensitive unit 23 is preferably an avalanche diode. In the present embodiment, the Avalanche diode is an optical element with an open central hole formed by mechanically drilling or etching a single Large-Area Avalanche diode (Large Area Avalanche photo diode, LAAPD), and in other embodiments, the Avalanche diode is an optical element with a central part capable of transmitting a laser signal, which is formed by making the single Large-Area Avalanche diode from a transparent material. The photo detector 2 further comprises a driving circuit for providing a high voltage and a driving signal to the photo sensitive unit 23 of the photo detector 2, and the driving circuit and its usage are prior art and thus will not be described in detail. Other optical elements for constructing the optical path in the microscope body 1 can be optical elements recorded in the existing microscope, which belong to the prior art, and therefore, the description thereof is omitted.

Specifically, the input end of the microscope objective body 3 is connected with the output end of the microscope objective body 1, one output end of the microscope objective body 3 is connected with the experimental sample 4, the output end of the experimental sample 4 is connected with the input end of the protection element of the photoelectric detector 2, the output end of the protection element of the photoelectric detector 2 is connected with the input end of the optical filter of the photoelectric detector 2, the output end of the optical filter of the photoelectric detector 2 is connected with the input end of the photoelectric sensitive unit 23 of the photoelectric detector 2, the output end of the photoelectric sensitive unit 23 of the photoelectric detector 2 is connected with the input end of the driving circuit of the photoelectric detector 2, and the output end of the driving circuit of the photoelectric detector 2 is connected with the external computer 5.

The microscope body 3 serves to collect fluorescence photons within the aperture, while the photodetector 2 is located at the bottom of the microscope body 3, i.e. the photodetector 2 is located on the side of the microscope body 1 close to the experimental sample 4. The photodetector 2 is annular and located around the front aperture of the microscope body 3, the protection element of the photodetector 2 is used to isolate the experimental sample 4, the liquid used for immersion, and the optical filter of the photodetector 2, and the protection element of the photodetector 2 is also used to electrically isolate, so as to prevent the high voltage of the photo sensitive unit 23 of the photodetector 2 (especially when the photodetector 2 is an element with internal gain such as an avalanche photodiode) from causing danger to the experimental sample 4 and the operator. In other embodiments, the surface of the protective element of photodetector 2 is also coated with an anti-reflective optical coating to improve transmission of fluorescent photons. The optical filter of the photodetector 2 is used for filtering back-reflected and back-scattered laser signals, the photoelectric sensitive unit 23 of the photodetector 2 is used for converting fluorescence photons passing through the optical filter into electrical signals, the driving circuit of the photodetector 2 is used for providing high voltage and driving signals for the photoelectric sensitive unit 23 of the photodetector 2 and is connected with the external computer 5, the driving circuit and the connection of the driving circuit and the external computer 5 are the prior art, and therefore description is omitted, and the photodetector 2 is used for collecting fluorescence photons which cannot be collected by the microscope objective.

In this embodiment, a commonly used liquid immersion objective with a numerical aperture of 0.8 and a magnification of 40X is taken as an example, and the fluorescence emission half-angle of the liquid immersion objective is arcsin (0.8/1.33) ═ 30 degrees, so that it can be calculated, in this scheme, the annular photodetector 2 with a width of 1mm is arranged around the front aperture of the same type of microscope objective, and can collect fluorescence photons with a fluorescence emission half-angle of 30 degrees to 60 degrees, which is equivalent to having an excitation numerical aperture of 0.8 and a collection numerical aperture of 1.0, so that the fluorescence collection efficiency is greatly improved, the signal-to-noise ratio of imaging is improved, and the imaging depth in a high scattering medium is improved.

Example two

The difference between the present embodiment and the first embodiment is: as shown in fig. 3, the number of the Photo-sensitive units 23 is multiple, in this embodiment, the number of the Photo-sensitive units 23 is eight, the Photo-sensitive units 23 are Avalanche photodiodes (Avalanche Photo diodes), and the Photo-sensitive units 23 are circumferentially distributed with the microscope body 3 as a center to form an annular array. In the present embodiment, the photosensitive layer is an optical element with an opening in the middle portion, which is made by mechanical drilling or etching, and in other embodiments, the photosensitive layer is an optical element which is made of a transparent material and has a middle portion capable of transmitting a laser signal. A plurality of generally sized avalanche diodes are used to receive fluorescence photons that are not received by the microscope body 3.

EXAMPLE III

The present embodiment differs from the above embodiments in that: as shown in fig. 4, the shape of the photodetector 2. The photoelectric detector 2 comprises an inner ring, an outer ring and a side surface connecting the inner ring and the outer ring, wherein the side surface comprises a top side and a bottom side, the top side is far away from the experimental sample 4, and the bottom side is obliquely arranged, namely the distance between the inner side of the photoelectric sensitive unit 23 of the photoelectric detector 2 opposite to the microscope objective lens body 3 and the focal plane of the microscope objective lens body 3 is larger than the distance between the outer side of the photoelectric sensitive unit 23 of the photoelectric detector 2 opposite to the microscope objective lens body 3 and the focal plane of the microscope objective lens body 3. That is, the photodetector 2 is formed in a horn shape as a whole.

Example four

The present embodiment differs from the above embodiments in that: the microscope is characterized by further comprising a shell used for holding, wherein the shell covers the microscope body 1 and is used for protecting the microscope body 1 and facilitating use of an operator.

The foregoing is merely an example of the present invention, and common general knowledge in the field of known specific structures and characteristics is not described herein in any greater extent than that known in the art at the filing date or prior to the priority date of the application, so that those skilled in the art can now appreciate that all of the above-described techniques in this field and have the ability to apply routine experimentation before this date can be combined with one or more of the present teachings to complete and implement the present invention, and that certain typical known structures or known methods do not pose any impediments to the implementation of the present invention by those skilled in the art. It should be noted that, for those skilled in the art, without departing from the structure of the present invention, several changes and modifications can be made, which should also be regarded as the protection scope of the present invention, and these will not affect the effect of the implementation of the present invention and the practicability of the patent. The scope of the claims of the present application shall be determined by the contents of the claims, and the description of the embodiments and the like in the specification shall be used to explain the contents of the claims.

Claims

1. The utility model provides a high fluorescence collection rate hand-held type microscope, includes the microscope mirror body, be equipped with the microscope mirror body on the microscope mirror body, its characterized in that: the microscope body is connected with a photoelectric detector for converting fluorescence photons into an electric signal.

2. The high fluorescence collection rate hand-held microscope of claim 1, wherein: the photoelectric detector is annular and is sleeved on the microscope body of the microscope.

3. The high fluorescence collection rate hand-held microscope of claim 1, wherein: the photodetector includes a filter layer for filtering out back-reflected and back-scattered laser signals and passing through fluorescence photons.

4. A high fluorescence collection rate hand-held microscope according to claim 3, wherein: the photodetector further includes a photosensitive layer for converting fluorescent photons passing through the filter layer into an electrical signal.

5. The high fluorescence collection rate hand-held microscope of claim 4, wherein: the photosensitive layer comprises a plurality of photoelectric sensitive units which are circumferentially distributed by taking a microscope body as a center.

6. The high fluorescence collection rate hand-held microscope of claim 4, wherein: the photosensitive layer comprises a photoelectric sensitive unit which is matched with the shape of the photoelectric detector.

7. The high fluorescence collection rate hand-held microscope of claim 4, wherein: the photoelectric detector also comprises a protective layer used for transmitting light, and the protective layer wraps the filter layer and the photosensitive layer.

8. The high fluorescence collection rate hand-held microscope of claim 1, wherein: one side of the photoelectric detector is flush with one side of the microscope objective body.

9. The high fluorescence collection rate hand-held microscope of claim 2, wherein: the photoelectric detector comprises an inner ring, an outer ring and a side face connected with the inner ring and the outer ring, wherein the side face is obliquely arranged.

10. The high fluorescence collection rate hand-held microscope of claim 1, wherein: the microscope further comprises a shell for holding, and the shell covers the microscope body.