CN101294902A - Conversion illuminating microscope on mesomeric state laser pump - Google Patents
Conversion illuminating microscope on mesomeric state laser pump Download PDFInfo
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- CN101294902A CN101294902A CNA2008100388577A CN200810038857A CN101294902A CN 101294902 A CN101294902 A CN 101294902A CN A2008100388577 A CNA2008100388577 A CN A2008100388577A CN 200810038857 A CN200810038857 A CN 200810038857A CN 101294902 A CN101294902 A CN 101294902A
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Abstract
The invention belongs to the technology field of optical microscope, particularly to a steady-state laser pumping up-conversion luminescent microscope. The microscope comprises an optical microscope system composed of an object lens and an object stage. A stead-state laser beam generated by a semiconductor laser is led to the optical microscope system via optical fibers. An extender lens, a beam reshaping mirror and a short-pass dichroic mirror are sequentially placed along the forward direction of the laser beam. The short-pass dichroic mirror is positioned in such manner that the short-pass dichroic mirror and the laser beam form an angle of 45 DEG C. On an optical axis perpendicular to the laser beam and passing through the dichroic mirror, an emission optical filter and a detector are sequentially positioned coaxially above the dichroic mirror, and the object lens, a sample and the object stage are sequentially placed below the dichroic mirror. The microscope can directly develop imaging for a steady-state laser pumping up-conversion luminescent material, and can also lead the steady-state laser pumping up-conversion luminescent material to cells, tissue or other substrates for imaging.
Description
Technical field
The invention belongs to the light microscope technique field, be specifically related to a kind of conversion illuminating microscope on mesomeric state laser pump.
Background technology
Fluorescent microscope is an important tool in life science, medical science and the material science research.Wherein, based on the confocal fluorescent microscope of fluorescent material single photon process because characteristics such as its high resolving power, high sensitivity and high power have become indispensable research tool in the fields such as cytomorphology, (molecule) cell biology, neurology, pharmacology, science of heredity.But it also has intrinsic defective, as sample easily by photobleaching, can not eliminate the interference of biological sample autofluorescence fully, use short wavelengths' such as ultraviolet light and blue light laser to cause imaging depth limited (tens microns) as exciting light, short wavelength's light damages biological sample easily simultaneously.The two-photon fluorescence microscope that grows up subsequently adopt wavelength at the femtosecond laser in infrared light district as excitation source, reduced the optical damage of exciting light to biological sample, improved imaging depth (hundreds of micron), weakened the photobleaching of non-focal plane simultaneously.Because the two-photon absorption performance of existing fluorescent material is very limited, the microscopical excitation source of two-photon must use expensive femtosecond pulse laser (～$200,000), therefore, is difficult to popularize based on the two-photon fluorescence microscope of femtosecond laser technology.In addition, the femtosecond laser of pulse have high instantaneous power (peak power density-as greater than (10
11W/cm
2), so the bleaching at focus place is inevitable, limited the use of two-photon fluorescence microscope in the imaging of biological sample long-time continuous.
Recently, the application of the up-conversion luminescent material of mesomeric state laser pump in bio-imaging caused more and more researchers' attention.Compare with traditional fluorescent material, this class material has special up-conversion luminescence character, can absorb two or more lower energy photons and high-energy photon of radiation normally converts near infrared light to visible light.Adopt near infrared light as exciting light, can reduce the damage of exciting light, improve imaging depth, and use cheap steady laser (～$2,000) to greatly reduce the instrument cost biological sample.In addition, because biological sample endogenous fluorescent material can not be excited by the stable state near-infrared laser, use this class material can eliminate the interference of biological sample autofluorescence fully.Therefore, if the up-conversion luminescent material and the microtechnic of mesomeric state laser pump can be combined, develop a kind of novel microscope, then be expected to solve some problems that copolymerization is burnt and the two-photon fluorescence microscope exists, for life science, medical science and material science research provide a kind of new method.
Summary of the invention
The object of the present invention is to provide a kind of conversion illuminating microscope on mesomeric state laser pump, this microscope can directly carry out imaging to the material with mesomeric state laser pump up-conversion luminescence character, also the up-conversion luminescent material of mesomeric state laser pump can be introduced in cell, tissue or other matrix and be carried out imaging.
Technical solution of the present invention is: a kind of conversion illuminating microscope on mesomeric state laser pump comprises the microscope optical system that object lens and objective table are formed.(centre wavelength 〉=800nm) the steady laser bundle of generation imports in this microscope optical system by optical fiber by semiconductor laser, along being placed with extender lens successively on this laser beam working direction, beam shaping mirror and short logical dichronic mirror (near infrared light reflection, visible light transmissive), its short-and-medium reduction of fractions to a common denominator look mirror becomes 45 ℃ of angles to place with this laser beam, perpendicular to this laser beam and pass on the optical axis of this dichronic mirror, above this dichronic mirror, be placed with emission optical filter and detecting device coaxially successively, below this dichronic mirror, be placed with object lens coaxially successively, sample and objective table, sample is positioned on the objective table.
The course of work of the present invention is: (centre wavelength 〉=800nm) the steady laser bundle of generation passes through extender lens and beam shaping mirror to semiconductor laser, become a branch of bigger parallel beam, short logical dichronic mirror (near infrared light reflection, visible light transmissive) makes 90 ℃ of beam deflections, be focused on the focus of object lens through object lens, the material that has mesomeric state laser pump up-conversion luminescence character in the sample is launched the up-conversion luminescence along all directions under the exciting of laser, part up-conversion luminescence signal is collected by object lens and is converted parallel beam to, by this short logical dichronic mirror (near infrared light reflection, visible light transmissive), again by the emission optical filter, the signal of certain wavelength coverage is intercepted, and is received by detecting device then.
The present invention compares with existing fluorescent microscopic imaging technology, have the following advantages: 1. owing to biological sample endogenous fluorescent material and organic fluorescent dye commonly used can not be excited by the stable state near infrared laser, therefore the present invention has eliminated the interference of background fluorescences such as biological sample autofluorescence and organic fluorescent dye be luminous, is a kind of highly sensitive imaging technique.2. the up-conversion luminescent material owing to mesomeric state laser pump excites down hardly by photobleaching at the stable state near-infrared laser, and the stable state near-infrared laser is very weak to the photobleaching of organic fluorescent dye, damage to biological sample is little, so the present invention is a kind of imaging technique that can carry out continuous observation of long period to biological sample.3. the present invention adopts semiconductor laser as excitation source, compares with the femto-second laser that the two-photon fluorescence microscope adopts, and semiconductor laser domesticizes, and is cheap, so the present invention is easy to popularize.
Description of drawings
Fig. 1 is a basic structure synoptic diagram of the present invention.
Fig. 2 is the structural representation of an example of the present invention.
Fig. 3 is the cell image with the mesomeric state laser pump up-conversion luminescent material mark of one embodiment of the present of invention acquisition.(a) being the up-conversion luminescence image, (b) is the light field image.
Fig. 4 is the three-dimensional imaging image with the mesomeric state laser pump up-conversion luminescent material DOPED PMMA film of one embodiment of the present of invention acquisition.(a) being the two dimensional image that projects to the xy direction along 34 sections of z direction, (b) and (c) is respectively along the two dimensional image of (a) cathetus in yz and xz direction.
Fig. 5 is the photobleaching experimental result with mesomeric state laser pump up-conversion luminescent material, organic fluorescent dye DAPI and the DiI of one embodiment of the present of invention acquisition.(a) and (b) be respectively in high power 980nm steady laser (focal plane place power is about 19mW) and low-power 543,405nm steady laser (focal plane place power is about 15 and 0.8 μ W respectively) is shone down simultaneously, and the luminous intensity of organic fluorescent dye DAPI and DiI is curve over time.(c), be respectively (d) and (e) at high power 980,543, (focal plane place power is about 1.6 respectively to the 405nm steady laser, 0.13 and 19mW) irradiation simultaneously down, organic fluorescent dye DAPI and DiI, and the luminous intensity of mesomeric state laser pump up-conversion luminescent material curve over time.
Number in the figure: 1 is semiconductor laser, and 2 is extender lens, and 3 is the beam shaping mirror, 4 are short logical dichronic mirror, and 5 are the emission optical filter, and 6 is detecting device, 7 is that object lens, 8 are sample, and 9 is objective table, and 10 are the illumination pin hole, 11 is catoptron, 12 is condenser lens, and 13 is detecting pinhole, and 14 is grating, 15 is computing machine, and 16 is scanning mirror.
Embodiment
Provide an embodiment preferably of the present invention according to Fig. 2 below, in order to architectural feature of the present invention and functional characteristics to be described, rather than be used for limiting scope of the present invention.
As shown in Figure 2, the steady laser bundle that semiconductor laser 1 produces imports by optical fiber, along being placed with extender lens 2 successively on this laser beam working direction, beam shaping mirror 3, illumination pin hole 10 and short logical dichronic mirror 4, its short-and-medium reduction of fractions to a common denominator look mirror 4 becomes 45 ℃ of angles to place with this laser beam, perpendicular to this laser beam and pass on the optical axis of this dichronic mirror, below this dichronic mirror, become 45 ℃ to be placed with catoptron 11 with optical axis, on the working direction of the folded light beam of this catoptron, be placed with condenser lens 12 successively, detecting pinhole 13, grating 14 and photomultiplier detector 6, wherein photomultiplier detector is connected with computing machine 15.In addition, on optical axis vertical with laser beam above the dichronic mirror 4, be placed with scanning mirror 16, object lens 7, objective table 9 and sample 8 coaxially, wherein scanning mirror 16 is connected with computing machine 15.In the present embodiment, select the signal of certain wavelength coverage to receive by the mode of grating beam splitting by detecting device 6.In light path, introduce the confocal scanning unit that illumination pin hole 10, detecting pinhole 13 and scanning mirror 16 etc. are formed simultaneously.Computing machine 15 is connected with scanning mirror 16 with detecting device 6, realizes image storage and scan control.In addition, driving carrier platform 9 by step motor moves along the Z axle and can obtain 3-D view.In the present embodiment, it is 980nm that semiconductor laser adopts centre wavelength, output power is the adjustable homemade laser instrument of 0-600mW (the vast space Fibre Optical Communication Technology in a Shanghai company limited), short logical dichronic mirror is that reflection wavelength is greater than 850nm light, see through dichronic mirror (the Model 850DMSP of wavelength less than 850nm light, OMEGA), microscope adopts OLYMPUS IX81 inverted microscope, the confocal scanning unit is OLYMPUS FV1000, detecting device is R6357Enhanced model (HAMAMATSU), the image storage, analysis and scan control software are FV10-ASW (OLYMPUS).
The course of work of present embodiment is:
The laser beam that semiconductor laser (centre wavelength is 980nm) 1 sends is through extender lens 2 and beam shaping mirror 3, become the bigger parallel beam of a beam diameter, behind illumination pin hole 10, (reflection wavelength is greater than the light of 850nm for short logical dichronic mirror, see through the light of wavelength less than 850nm) 4 make 90 ° of beam deflections, be focused on the focus of object lens by object lens 7 through scanning mirror 16 backs, the material that has mesomeric state laser pump up-conversion luminescence character in the sample 8 is launched the up-conversion luminescence along all directions under the exciting of laser, part up-conversion luminescence signal is collected by object lens 7, through mirror 11 reflections that are reflected behind scanning mirror 16 and the short logical dichronic mirror 4, signal line focus lens 12 after the reflection, be focused at the focus place of focusing objective len, by the detecting pinhole 13 and the grating 14 at focus place, receive again by detecting device 6.By computing machine 15 gated sweep mirrors 16, utilize the mode of focal beam spot scanning samples to obtain a width of cloth two dimensional image then.On this basis, driving carrier platform 9 by step motor moves along the Z axle and can obtain 3-D view.
As shown in Figure 3, with present embodiment the cell of mesomeric state laser pump up-conversion luminescent material mark is carried out two-dimensional imaging, can obtain distinct image, wherein (a) excites the up-conversion luminescence image that obtains down in steady laser, (b) is the light field image.
As shown in Figure 4, with present embodiment mesomeric state laser pump up-conversion luminescent material DOPED PMMA film is carried out three-dimensional imaging, (a) being the image that projects to the XY direction along 34 two-dimensional slice image of Z direction, (b) and (c) is respectively along the two dimensional image of (a) cathetus in YZ and XZ direction.
As shown in Figure 5, investigated its photobleaching situation with present embodiment by the method for time series scanning imagery to mesomeric state laser pump up-conversion luminescent material, organic fluorescent dye DAPI and DiI.Ordinate is represented luminous intensity (after the normalization), horizontal ordinate express time among the figure.At first investigating the photobleaching of 980nm steady laser to organic fluorescent dye, (a) and (b) is respectively organic fluorescent dye DAPI and DiI luminous intensity curve over time under high power 980nm steady laser (focal plane place power is about 19mW) irradiation; This moment need be with 543, and the 405nm steady laser is controlled at very low power (focal plane place power is about 15 and 0.8 μ W respectively) to obtain DAPI and DiI image, avoids their bleachings to DAPI and DiI simultaneously as far as possible.By curve (a) and (b) as can be known, through the Continuous irradiation of 425s, the signal intensity of DAPI and DiI is respectively 84% and 90% of initial strength.Having only 543 of equal-wattage, the 405nm steady laser is shone and is not had in the control experiment of 980nm steady laser, and the signal intensity of DAPI and DiI is respectively 94% and 99% of initial strength.It is very weak to the photobleaching of organic fluorescent dye to contrast two groups of data declaration stable state near-infrared lasers.Investigate the light stability of material, organic fluorescent dye DAPI and DiI with mesomeric state laser pump up-conversion luminescence character then, (c), (d) and (e) be respectively at high power 980,543, (focal plane place power is about 1.6 respectively to the 405nm steady laser, 0.13 and 19mW) irradiation simultaneously down, organic fluorescent dye DAPI and DiI, and the luminous intensity of the up-conversion luminescent material of mesomeric state laser pump curve over time.By curve (c), (d) and (e) as can be known, through after the Continuous irradiation of 425s, the luminous intensity of up-conversion luminescent material still is 96% of an initial value.And the luminous intensity of DAPI and DiI decays very soon, and through the Continuous irradiation of 400s, the luminous intensity of DAPI and DiI decays to 1% and 2.5% of initial value respectively.The light stability that contrasts two groups of data declaration mesomeric state laser pump up-conversion luminescent materials is higher than organic fluorescent dye far away, and it excites down hardly by photobleaching at the stable state near-infrared laser.
Claims (1)
1, a kind of conversion illuminating microscope on mesomeric state laser pump, it is characterized in that by extender lens, the beam shaping mirror, short logical dichronic mirror, the emission optical filter, detecting device, object lens and objective table are formed, the steady laser bundle that is produced by semiconductor laser imports in this microscope optical system by optical fiber, along being followed successively by extender lens on this laser beam working direction, beam shaping mirror and short logical dichronic mirror, its short-and-medium reduction of fractions to a common denominator look mirror becomes 45 ℃ of placements with this laser beam, perpendicular to this laser beam and pass on the optical axis of this dichronic mirror, above this dichronic mirror, place emission optical filter and detecting device coaxially successively, below this dichronic mirror, place object lens coaxially successively, sample and objective table, sample is positioned on the objective table.
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102623286A (en) * | 2012-04-24 | 2012-08-01 | 上海交通大学 | Coaxial optical pumping device and manufacturing method thereof |
| CN103616359A (en) * | 2013-12-04 | 2014-03-05 | 中国检验检疫科学研究院 | Fluorescent quantitative detection method for green fluorescent protein (GFP) |
| CN105548099A (en) * | 2015-12-04 | 2016-05-04 | 西北大学 | Cultural relic lossless three-dimensional imaging and component identification method based on two-photon excitation fluorescence |
| CN105866937A (en) * | 2016-06-12 | 2016-08-17 | 福建师范大学 | Method for determining microscope objective lens focus for visible light through laser conversion |
| CN106596497A (en) * | 2017-01-16 | 2017-04-26 | 浙江大学 | Shortwave infrared fluorescence microimaging method |
| CN108333151A (en) * | 2017-01-19 | 2018-07-27 | 北京大学 | A kind of super-resolution micro imaging system and imaging method based on femtosecond pulse shaping |
| CN108387519A (en) * | 2018-05-03 | 2018-08-10 | 上海市质子重离子临床技术研发中心 | Microscopic system is just set in up-conversion luminescence near-infrared and the multi-functional wide field of colour imaging |
| CN108828770A (en) * | 2018-06-05 | 2018-11-16 | 广东欧谱曼迪科技有限公司 | A kind of bias light removing method and system suitable near infrared imaging |
| CN114659461A (en) * | 2020-12-23 | 2022-06-24 | 中国科学院微电子研究所 | Pulse laser-based through-focus scanning optical microscopic imaging device and method |
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2008
- 2008-06-12 CN CNA2008100388577A patent/CN101294902A/en active Pending
Cited By (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102623286A (en) * | 2012-04-24 | 2012-08-01 | 上海交通大学 | Coaxial optical pumping device and manufacturing method thereof |
| CN102623286B (en) * | 2012-04-24 | 2014-08-20 | 上海交通大学 | Coaxial optical pumping device and manufacturing method thereof |
| CN103616359A (en) * | 2013-12-04 | 2014-03-05 | 中国检验检疫科学研究院 | Fluorescent quantitative detection method for green fluorescent protein (GFP) |
| CN105548099B (en) * | 2015-12-04 | 2018-07-27 | 西北大学 | The lossless three-dimensional imaging of historical relic based on two-photon fluorescence excitation and Components identification method |
| CN105548099A (en) * | 2015-12-04 | 2016-05-04 | 西北大学 | Cultural relic lossless three-dimensional imaging and component identification method based on two-photon excitation fluorescence |
| CN105866937A (en) * | 2016-06-12 | 2016-08-17 | 福建师范大学 | Method for determining microscope objective lens focus for visible light through laser conversion |
| CN106596497A (en) * | 2017-01-16 | 2017-04-26 | 浙江大学 | Shortwave infrared fluorescence microimaging method |
| CN108333151A (en) * | 2017-01-19 | 2018-07-27 | 北京大学 | A kind of super-resolution micro imaging system and imaging method based on femtosecond pulse shaping |
| CN108333151B (en) * | 2017-01-19 | 2021-01-26 | 北京大学 | Super-resolution microscopic imaging system and method based on femtosecond pulse shaping |
| CN108387519A (en) * | 2018-05-03 | 2018-08-10 | 上海市质子重离子临床技术研发中心 | Microscopic system is just set in up-conversion luminescence near-infrared and the multi-functional wide field of colour imaging |
| CN108828770A (en) * | 2018-06-05 | 2018-11-16 | 广东欧谱曼迪科技有限公司 | A kind of bias light removing method and system suitable near infrared imaging |
| CN108828770B (en) * | 2018-06-05 | 2024-02-23 | 广东欧谱曼迪科技有限公司 | Background light elimination method and system suitable for near infrared imaging |
| CN114659461A (en) * | 2020-12-23 | 2022-06-24 | 中国科学院微电子研究所 | Pulse laser-based through-focus scanning optical microscopic imaging device and method |
| CN114659461B (en) * | 2020-12-23 | 2024-02-09 | 中国科学院微电子研究所 | Over-focus scanning optical microscopic imaging device and method based on pulse laser |
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Open date: 20081029 |