CN110888228A - Fluorescent microscopic illumination method adopting deep ultraviolet light source - Google Patents
Fluorescent microscopic illumination method adopting deep ultraviolet light source Download PDFInfo
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- CN110888228A CN110888228A CN201911169494.5A CN201911169494A CN110888228A CN 110888228 A CN110888228 A CN 110888228A CN 201911169494 A CN201911169494 A CN 201911169494A CN 110888228 A CN110888228 A CN 110888228A
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- 238000005286 illumination Methods 0.000 title claims abstract description 30
- 238000000034 method Methods 0.000 title claims abstract description 16
- 230000005284 excitation Effects 0.000 claims abstract description 33
- 238000000799 fluorescence microscopy Methods 0.000 claims description 9
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 3
- 238000001914 filtration Methods 0.000 abstract description 6
- 230000003287 optical effect Effects 0.000 abstract description 2
- 238000003384 imaging method Methods 0.000 description 5
- 238000010586 diagram Methods 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- 230000000903 blocking effect Effects 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 230000000149 penetrating effect Effects 0.000 description 1
- 230000035515 penetration Effects 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
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- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/16—Microscopes adapted for ultraviolet illumination ; Fluorescence microscopes
Abstract
The invention discloses a fluorescent microscopic illumination method adopting a deep ultraviolet light source. The system consists of an ocular lens, a deep ultraviolet-impermeable objective lens, a deep ultraviolet excitation light source and a light condensing system, wherein the deep ultraviolet excitation light source and the light condensing system are arranged around the objective lens and form dark field illumination, the dark field illumination is irradiated on a specimen to excite the specimen to generate fluorescence, and the fluorescence is collected by the objective lens and then imaged to the ocular lens. The invention can use the objective lens with short working distance, thereby avoiding the loss of light energy and having higher optical efficiency; the color filtering block is not needed, the structure is simple, and the cost is reduced.
Description
Technical Field
The invention relates to the technical field of microscopes, in particular to a fluorescence micro-excitation illumination technology.
Background
The fluorescence microscope has the advantages of high contrast, high detection capability, small stimulation to cells, capability of performing multiple staining and the like, and can be widely applied to researches of biology, hematology, histology, pathology, pharmaceutical chemistry and the like and clinical tests. The fluorescence microscope mainly comprises a fluorescence excitation source, a light condensing system, a color filtering block (consisting of an excitation color filter, a light splitting color separation block and a blocking color filter) and a microscope, generally adopts an epi-illumination structure, and has the following specific principle: the light emitted by the fluorescence excitation source after being electrified is imaged on the back focal plane of the objective lens through a light-gathering and excitation color filter (only can transmit light with a certain wavelength) and a light-splitting color-splitting sheet (the light with a wavelength smaller than the certain wavelength is reflected and the light with a wavelength larger than the certain wavelength is transmitted), and forms a Korea illumination system with the objective lens, so that uniform epi-illumination can be obtained on a sample; the sample is excited by the falling radiation to generate fluorescence, the wavelength of the fluorescence is larger than that of light which can be transmitted by the light splitting color separation sheet, and the fluorescence directly transmits through the light splitting color separation sheet and the blocking color filter (only light with a wavelength larger than a certain wavelength can be transmitted), and reaches an ocular lens or a camera device for observation.
Because the objective lens simultaneously acts as the light gathering and imaging functions, the structure is simple, and the device is widely applied. However, for specific wavelengths such as deep ultraviolet (below 350 nm), since conventional fluorescent objectives, light-gathering systems, can only transmit wavelengths above ultraviolet, this approach is not suitable unless totally redesigned, but is expensive.
The prior patent CN 107003242A discloses a system and method for controlling the imaging depth in tissue by using a fluorescence microscope under the condition of ultraviolet excitation after dyeing by using a fluorescent agent, and adopts an oblique illumination mode, i.e. illumination focusing and imaging part are independent, thus solving the problem that the objective lens can not transmit deep ultraviolet, but for a high power objective lens, because the working distance is short (such as about 1 mm), the utilization rate of oblique illumination is very low, even can not be used.
The prior patent CN 205091263U, CN105092550A discloses a fluorescence microscopic imaging method and device, which includes a light source device, a sample placing table, an objective lens, an emission and filtering module and an image acquisition device, wherein the light source device includes a plurality of monochromatic fluorescence excitation light sources and a control system electrically connected with the plurality of monochromatic fluorescence excitation light sources, the plurality of monochromatic fluorescence excitation light sources are arranged around a central axis of an imaging light path formed by the objective lens and the image acquisition device, and the monochromatic fluorescence excitation light emitted by each monochromatic fluorescence excitation light source intersects with the central axis of the imaging light path at a preset position of the sample placing table, and the control system lights at least one monochromatic fluorescence excitation light source with the same color as a target light source in the plurality of monochromatic fluorescence excitation light sources according to experimental requirements; the sample placing table is arranged at the intersection position of the monochromatic fluorescence excitation light emitted by the plurality of monochromatic fluorescence excitation light sources. However, the comparison document is used for transmitting fluorescence, and needs a color filtering block, so that the cost is higher.
Disclosure of Invention
The invention provides a fluorescent micro-lighting method adopting a deep ultraviolet light source. The technical scheme includes that a deep ultraviolet excitation light source and a light condensing system thereof are arranged around an objective lens, light emitted by the excitation light source forms a dark field after passing through the light condensing system to illuminate on a specimen surface, the specimen is excited to emit fluorescence, and the fluorescence is collected by the objective lens and then imaged on an eyepiece.
The fluorescence excitation light source is an ultraviolet solid-state light source, and the wavelength of the ultraviolet solid-state light source is less than 400 nm. Such as an LED, a solid state laser, preferably an LED, mounted on a ring-shaped circuit board.
The ultraviolet solid-state light source is an LED.
The number of the fluorescence excitation light sources may be one or more.
The wavelength may be a single wavelength or a combination of wavelengths.
The light condensing system is any one or combination of a lens, an off-axis paraboloid and an off-axis curved surface.
The light condensing system is used for forming dark field illumination by light emitted by the excitation light source and converging the dark field illumination on the specimen plane to be superposed with the focal plane of the objective lens.
The lens must be transparent to deep ultraviolet light, such as quartz glass.
The fluorescence microscope in the prior art patent uses filter blocks, the wavelength of the fluorescence microscope is changed by the filter blocks, and each emission filter area has a passing waveband which is a waveband of fluorescence emitted by particles in a sample plate excited by a monochromatic fluorescence excitation light source of one color. The invention can change the wavelength through the fluorescence excitation light source without using a color filtering block, thereby achieving the purpose of exciting the fluorescence of the sample. The invention can use the objective lens with short working distance, thereby avoiding the loss of light energy and having higher optical efficiency; meanwhile, the invention does not need color filtering blocks, and has simple structure and lower cost.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention and not to limit the invention. In the drawings:
FIG. 1 shows a schematic view of a curved mirror alone according to the present invention.
FIG. 2 is a schematic diagram of deep ultraviolet penetration according to the present invention.
FIG. 3 is a schematic diagram of the deep ultraviolet ray penetrating microscope of the present invention.
Fig. 4 is a schematic diagram of the deep ultraviolet transmitting structure of the present invention.
Detailed Description
To further illustrate the various embodiments, the invention provides the accompanying drawings. The accompanying drawings, which are incorporated in and constitute a part of this disclosure, illustrate embodiments of the invention and, together with the description, serve to explain the principles of the embodiments. Those skilled in the art will appreciate still other possible embodiments and advantages of the present invention with reference to these figures. Elements in the figures are not drawn to scale and like reference numerals are generally used to indicate like elements.
As shown in fig. 1, 2, 3, and 4, the invention provides a fluorescence micro-lighting method using a deep ultraviolet light source, which comprises a specimen 1, a specimen 2, an off-axis paraboloid 3, a light source 4, an objective lens 5, an eyepiece lens 6, a lens array 7, a head 8, a dark field objective lens 9, an objective table 10, a frame 11, an objective lens liner 12, a wire buckle 13, and a housing, wherein the deep ultraviolet excitation light source 3 is a solid-state light source such as an LED, a solid-state laser, preferably an LED, and the light source 3 is fixed on an annular circuit board. The number of the light sources 3 may be single or plural. The wavelength may be a single wavelength or a combination of wavelengths.
The deep ultraviolet excitation light source 3 and the condensing system thereof are arranged around the objective lens, light emitted by the excitation light source 3 forms a dark field illumination after passing through the condensing system and irradiates the surface of the specimen 1, the specimen 1 is excited to emit fluorescence, and the fluorescence is collected by the objective lens 4 and then imaged on the ocular lens 5. The light-gathering system is any one or combination of a lens array 6, an off-axis paraboloid 2 and an off-axis curved surface.
The light condensing system is used for forming dark field illumination by the light emitted by the excitation light source 3 and converging the dark field illumination on the surface of the specimen 1, and the dark field illumination is superposed with the focal plane of the objective 4. The light-gathering system can be composed of a ring lens, a lens array 6 and a curved reflector, as shown in fig. 1, a single curved reflector such as an off-axis parabolic reflector can be used, and the lens can transmit deep ultraviolet rays such as quartz glass.
While the invention has been particularly shown and described with reference to a preferred embodiment, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.
Claims (9)
1. A fluorescent microscopic illumination method adopting a deep ultraviolet light source comprises an ocular lens, an objective lens, a fluorescent excitation light source and a light condensing system, and is characterized in that: the fluorescence excitation light source and the light condensing system are arranged around the objective lens, light emitted by the excitation light source forms dark field illumination through the light condensing system and irradiates on the specimen, the specimen is excited to emit fluorescence, and the fluorescence is collected by the objective lens and then imaged on the eyepiece.
2. The fluorescence microscopy illumination method adopting deep ultraviolet light source as claimed in claim 1, wherein: the fluorescence excitation light source is an ultraviolet solid-state light source, and the wavelength of the ultraviolet solid-state light source is less than 400 nm.
3. The fluorescence microscopy illumination method adopting deep ultraviolet light source as claimed in claim 2, characterized in that: the wavelength is a single wavelength or a combination of multiple wavelengths.
4. The fluorescence microscopy illumination method adopting deep ultraviolet light source as claimed in claim 2, characterized in that: the ultraviolet solid-state light source is a deep ultraviolet LED.
5. The fluorescence microscopy illumination method adopting deep ultraviolet light source as claimed in claim 1, wherein: the fluorescence excitation light source is one or more than one.
6. The fluorescence microscopy illumination method adopting deep ultraviolet light source as claimed in claim 1, wherein: the light-gathering system is any one or combination of a lens, an off-axis paraboloid and an off-axis curved surface.
7. The fluorescence microscopy illumination method adopting deep ultraviolet light source as claimed in claim 1, wherein: the light condensing system is used for forming dark field illumination by light emitted by the excitation light source and converging the dark field illumination on the specimen plane to be superposed with the focal plane of the objective lens.
8. The fluorescence microscopy illumination method adopting deep ultraviolet light source as claimed in claim 1, wherein: the lens of the condensing system must be transparent to deep ultraviolet.
9. The fluorescence microscopy illumination method adopting deep ultraviolet light source as claimed in claim 8, wherein: the lens is made of quartz glass.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
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| CN201911169494.5A CN110888228A (en) | 2019-11-26 | 2019-11-26 | Fluorescent microscopic illumination method adopting deep ultraviolet light source |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201911169494.5A CN110888228A (en) | 2019-11-26 | 2019-11-26 | Fluorescent microscopic illumination method adopting deep ultraviolet light source |
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| CN110888228A true CN110888228A (en) | 2020-03-17 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113534430A (en) * | 2021-06-08 | 2021-10-22 | 浙江工商职业技术学院 | Design method and device of dark field condenser for metallographic microscope |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113534430A (en) * | 2021-06-08 | 2021-10-22 | 浙江工商职业技术学院 | Design method and device of dark field condenser for metallographic microscope |
| CN113534430B (en) * | 2021-06-08 | 2023-10-10 | 浙江工商职业技术学院 | Design method and device of dark field condenser for metallographic microscope |
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