CN202757884U - Triplet-triplet annihilation up-conversion luminescence microscope - Google Patents
Triplet-triplet annihilation up-conversion luminescence microscope Download PDFInfo
- Publication number
- CN202757884U CN202757884U CN 201220112052 CN201220112052U CN202757884U CN 202757884 U CN202757884 U CN 202757884U CN 201220112052 CN201220112052 CN 201220112052 CN 201220112052 U CN201220112052 U CN 201220112052U CN 202757884 U CN202757884 U CN 202757884U
- Authority
- CN
- China
- Prior art keywords
- triplet
- microscope
- triplet state
- conversion luminescence
- dichroscope
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Images
Abstract
The utility model relates to a triplet-triplet annihilation up-conversion luminescence microscope which comprises a microscope optical system. The microscope optical system comprises an object lens and an object stage. The triplet-triplet annihilation up-conversion luminescence microscope is characterized in that steady laser beams generated by a common continuous wave laser device with the central wavelength ranging from 500nm to 1000nm are guided into the microscope optical system via an optical fiber, a reversible excitation dichroscope for light reflection excitation and a galvanometer reflector are sequentially placed along an advancing direction of the laser beams, short-wavelength emitted light can be transmitted through the excitation dichroscope, the excitation dichroscope is placed properly so that an included angle of 45 degrees is formed between the excitation dichroscope and the laser beams, an optical axis is perpendicular to the laser beams and penetrates through the dichroscope, a confocal pinhole, gratings, a slit and a detector which is a photomultiplier are coaxially sequentially placed on the optical axis and positioned at the front of the dichroscope, and the object lens, a sample and the object stage are sequentially placed on the galvanometer reflector. The triplet-triplet annihilation up-conversion luminescence microscope can be used for directly imaging a material with a property of triplet-triplet annihilation up-conversion luminescence, and also can be used for bringing the material with the property of triplet-triplet annihilation up-conversion luminescence into cells, tissues or other matrixes for imaging the material.
Description
Technical field
The utility model belongs to the light microscope technique field, be specially the up-conversion luminescence microscope that a kind of triplet state-triplet state is buried in oblivion, this microscope uses common continuous wave laser (centre wavelength in the visible range or near-infrared region) as excitation source, adopt short logical dichronic mirror (exciting light reflection, short wavelength's utilizing emitted light sees through) to collect the signal in utilizing emitted light district.
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 the 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, such as sample easily by photobleaching, can not eliminate the interference of biological sample autofluorescence fully, use the 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 simultaneously the photobleaching of non-focal plane.Because the two-photon absorption performance of existing fluorescent material is very limited, the excitation source of Two Photon Fluorescence must use expensive femtosecond pulse laser, and (～$ 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 has high instantaneous power (peak power density is generally 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.
In recent years, the application of the up-conversion luminescent material of mesomeric state laser pump in bio-imaging caused more and more researchers' attention.Be typically the rare earth up-conversion luminescence nanometer crystal material, 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 (mainly being 980nm) to visible light.Adopt 980nm light as exciting light, can reduce exciting light to the damage of biological sample, improve imaging depth, and use cheap steady laser (～$ 2,000) to greatly reduce the instrument cost.In addition, because biological sample Intrinsic fluorescence 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, its low absorption cross section (is about 10 but the method is subject to the rare earth ion of material own
-21) and low quantum yield (＜1%), and the 980nm optical excitation causes causing the irradiation position of biological sample overheating effect to occur easily.
Recently, bury in oblivion (TTA) up-conversion luminescent material based on triplet state-triplet state and caused that people note.The process of TTA up-conversion luminescence is that sensitizer is excited behind a low-energy exciting light photon of long wavelength from the ground state transition to excited state, arrives excited triplet state by intersystem crossing (ISC) at once; Arrive again the excited triplet state of acceptor by the energy transmission (TTET) between sensitizer triplet state and acceptor (Acceptor) triplet state; Subsequently, triplet state-triplet state occuring between the acceptor molecule that two are excited bury in oblivion (TTA), make an acceptor be excited to the higher singlet of energy, finally get back to the emission that ground state realizes the short wavelength by radiation transistion, finishes the up-conversion luminescence process.Such TTA up-conversion luminescent material has absorption cross section and (is about 10 greatly
-18), the low (mW/cm of exciting power density
-2), quantum yield high (can reach 15%), sensitizer and acceptor (burying in oblivion agent) can select the outstanding advantages such as the large and upper conversion efficiency in space is high.Therefore, if triplet state-triplet state can be buried in oblivion up-conversion luminescent material and microtechnic combines, 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 purpose of this utility model is to provide a kind of triplet state-triplet state to bury in oblivion the up-conversion luminescence microscope, this microscopes directly carries out imaging to the material that triplet state-triplet state is buried in oblivion up-conversion luminescence character, also triplet state-triplet state can be buried in oblivion the up-conversion luminescent material incubated cell and carry out imaging.
Technical solution of the present utility model is: a kind of triplet state-triplet state is buried in oblivion the up-conversion luminescence microscope, comprise the microscope optical system that object lens and objective table form, it is characterized in that: the steady laser bundle that common continuous wave laser 6 (centre wavelength 500-1000nm) is produced imports in this microscope optical system by optical fiber 11, along placing successively turnover dichroic mirror 5 (the exciting light reflection that excites on this laser beam working direction, short wavelength's utilizing emitted light sees through), galvanometer mirror 4, wherein excite dichroic mirror 5 and the placement at 45 ° of this laser beam, perpendicular to this laser beam and pass on the optical axis of this dichroic mirror, be placed with successively coaxially the burnt pin hole 7 of copolymerization in this dichroic mirror the place ahead, grating 8, slit 9 and photomultiplier detector 10, above galvanometer mirror 4, be placed with successively object lens 3, sample 1 and objective table 2, sample 1 is positioned on the objective table 2.
The course of work of the present utility model is: the steady laser bundle passes through turnover dichroic mirror 5 (the exciting light reflection that excites, short wavelength's utilizing emitted light sees through), galvanometer mirror 4, be focused on the focus of object lens through object lens 3, the material that has the up-conversion luminescence character that triplet state-triplet state buries in oblivion in the sample 1 under the exciting of laser emission along the up-conversion luminescence of all directions, part up-conversion luminescence signal is collected by object lens 3 and is converted parallel beam to, then galvanometer mirror 4 is passed through in reflection again, turnoverly excite dichroic mirror 5 (exciting light reflection, the shortwave utilizing emitted light sees through), the burnt pin hole 7 of copolymerization, grating 8, again by slit 9, the signal of certain wavelength coverage is intercepted, and is then received by photomultiplier detector 10.
The utility model is compared with existing Induced Fluorescence Microscopy, have the following advantages: 1. because biological sample Intrinsic fluorescence material and organic fluorescent dye commonly used can not produce the upper switching emission that triplet state-triplet state is buried in oblivion, therefore the utility model has been eliminated the interference of the background fluorescences such as biological sample autofluorescence and organic fluorescent dye be luminous, is a kind of highly sensitive imaging technique.Since the up-conversion luminescent material buried in oblivion of triplet state-triplet state hardly by photobleaching, adopt the continuous wave laser of lower-wattage as excitation source, produce hardly thermal effect, so the utility model is a kind of imaging technique that can carry out to biological sample the long period Continuous Observation.3. the continuous wave laser of the utility model employing lower-wattage is compared with the femto-second laser that the two-photon fluorescence microscope adopts as excitation source, and is cheap, so the utility model is easy to popularize.
Description of drawings
The utility model is described in further detail below in conjunction with drawings and Examples.
Fig. 1 is basic structure synoptic diagram of the present utility model:
Identify among the figure: 1 sample, 2 liftable objective tables, 3 object lens, 4 galvanometer mirrors, 5 excite dichroic mirror, 6 continuous wave lasers, the burnt pin hole of 7 copolymerization, 8 gratings, 9 slits, 10 photomultiplier detector, 11 optical fiber.
Fig. 2 is the cell image of the up-conversion luminescent material mark buried in oblivion with triplet state-triplet state that embodiment of the present utility model obtains.(a) be the up-conversion luminescence image, 543nm excites, 420-480nm emission, and (b) the fluorescent emission image of burying in oblivion agent in the up-conversion luminescent material buried in oblivion of the light activated triplet state-triplet state of 405nm (c) is the image of a, b and light field stack.
Fig. 3 is up-conversion luminescent material and the commercial cell image that dyes nuclear reagent Hoechst 33258 common marks of burying in oblivion with triplet state-triplet state that an embodiment of the present utility model obtains.(a) being dying the blue-fluorescence emission of burying in oblivion agent in the up-conversion luminescent material that nuclear reagent and triplet state-triplet state bury in oblivion under the 405nm optical excitation, (b) is the upper conversion blue emission of the up-conversion luminescent material buried in oblivion of the light activated triplet state-triplet state of 543nm.
Fig. 4 is the up-conversion luminescent material buried in oblivion with triplet state-triplet state that embodiment of the present utility model obtains and the photobleaching experimental result of organic fluorescent dye DPA.
Embodiment
The below provides an embodiment preferably of the present utility model according to Fig. 1, in order to architectural feature of the present utility model and functional characteristics to be described, rather than is used for limiting scope of the present utility model.
As shown in Figure 1, in the present embodiment, continuous wave laser (centre wavelength is 543nm) 6, dichroic mirror 5 (543nm light is reflected, the following light transmission of 500nm), galvanometer mirror 4, object lens 3, sample stage 1, photomultiplier 10 etc. become light path to connect successively.Select the signal of certain wavelength coverage to have detecting device to receive by grating 8.Simultaneous computer is connected with scanning mirror with detecting device, realizes collection and the scan control of image.In the present embodiment, laser instrument center excitation wavelength is 543nm.(reflection wavelength is greater than the light of 515nm for dichroic mirror, see through wavelength less than the light of 515nm), microscope adopts OLYMPUS IX81 inverted microscope, the confocal scanning unit is OLYMPUS FV1000, detecting device is R6357 Enhanced model (HAMAMATSU), and image storage, analysis and scan control software are FV10-ASW (OLYMPUS).
As shown in Figure 2, the cell of the up-conversion luminescent material mark of triplet state-triplet state being buried in oblivion with present embodiment carries out two-dimensional imaging, can obtain clearly image, wherein (a) is the up-conversion luminescence image, 543nm excites, 420-480nm emission, (b) the fluorescent emission image of burying in oblivion agent in the up-conversion luminescent material buried in oblivion of the light activated triplet state-triplet state of 405nm (c) is the image of a, b and light field stack.
As shown in Figure 3, the up-conversion luminescent material that triplet state-triplet state is buried in oblivion and the commercial cell image that dyes nuclear reagent Hoechst 33258 common marks, the up-conversion luminescence microscopy that triplet state-triplet state is buried in oblivion can be eliminated common fluorescence and disturb.Under the 405nm optical excitation, the fluorescent emission of burying in oblivion agent DPA (420-480nm) of dying in the up-conversion luminescent material that nuclear reagent Hoechst 33258 and triplet state-triplet state bury in oblivion can both be excited out, can not effectively distinguish, such as figure a.Under the 543nm optical excitation, the upper conversion blue emission (420-480nm) of the up-conversion luminescent material that triplet state-triplet state is buried in oblivion is excited out, dye simultaneously nuclear reagent Hoechst 33258 and under this 543nm optical excitation, can't detect its fluorescent emission in blue channel (420-480nm), as scheme b, nuclear area does not have fluorescence signal.Contrast up-conversion luminescence microscopy that two groups of data can illustrate that triplet state-triplet state is buried in oblivion and can eliminate interference from the common fluorescent material of endogenous fluorescence or common mark fully.
As shown in Figure 4, up-conversion luminescence micro-imaging technique that triplet state-triplet state buries in oblivion and common Imaging-PAM have been investigated to the photobleaching situation of material DPA with present embodiment by the method for time series scanning imagery.At first investigate the up-conversion luminescence micro-imaging technique photobleaching luminous to material that triplet state-triplet state is buried in oblivion, be the 543nm laser irradiation 600 seconds of 0.57mW in output power, the up-conversion luminescence of the up-conversion luminescent material that triplet state-triplet state is buried in oblivion (430nm) still keeps 99% intensity.And be the 405nm light irradiation of 0.15mW after 600 seconds in output power, the fluorescence of organic dyestuff DPA (430nm) luminous intensity quickly falls to 1% of fluorescence intensity in fact.The light stability of the up-conversion luminescent material that correlation data explanation triplet state-triplet state is buried in oblivion is higher than organic fluorescent dye far away, and the up-conversion luminescence micro-imaging technique that triplet state-triplet state is buried in oblivion causes the up-conversion luminescent material photobleaching hardly.
Claims (1)
1. a triplet state-triplet state is buried in oblivion the up-conversion luminescence microscope, comprise the microscope optical system that object lens and objective table form, it is characterized in that: the steady laser bundle that the common continuous wave laser of centre wavelength 500-1000nm (6) is produced imports in this microscope optical system by optical fiber (11), along placing successively the turnover dichroic mirror (5) that excites on this laser beam working direction, galvanometer mirror (4), wherein excite dichroic mirror (5) and the placement at 45 ° of this laser beam, perpendicular to this laser beam and pass on the optical axis of this dichroic mirror (5), be placed with successively coaxially the burnt pin hole (7) of copolymerization in this dichroic mirror (5) the place ahead, grating (8), slit (9) and photomultiplier detector (10), be placed with successively object lens (3) in galvanometer mirror (4) top, sample (1) and objective table (2), sample (1) are positioned on the objective table (2); The described dichroic mirror that excites is the exciting light reflection, and short wavelength's utilizing emitted light sees through.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201220112052 CN202757884U (en) | 2012-03-22 | 2012-03-22 | Triplet-triplet annihilation up-conversion luminescence microscope |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201220112052 CN202757884U (en) | 2012-03-22 | 2012-03-22 | Triplet-triplet annihilation up-conversion luminescence microscope |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN202757884U true CN202757884U (en) | 2013-02-27 |
Family
ID=47737056
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 201220112052 Expired - Fee Related CN202757884U (en) | 2012-03-22 | 2012-03-22 | Triplet-triplet annihilation up-conversion luminescence microscope |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN202757884U (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104764729A (en) * | 2015-04-22 | 2015-07-08 | 华南师范大学 | Up-conversion-nanocrystal-based stimulated depletion super-resolution optical microscopic method and up-conversion-nanocrystal-based stimulated depletion super-resolution optical microscopic system |
| CN105445238A (en) * | 2014-08-25 | 2016-03-30 | 中国科学院大连化学物理研究所 | A singlet-oxygen microscopy imaging method on a confocal laser scanning microscopy |
-
2012
- 2012-03-22 CN CN 201220112052 patent/CN202757884U/en not_active Expired - Fee Related
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105445238A (en) * | 2014-08-25 | 2016-03-30 | 中国科学院大连化学物理研究所 | A singlet-oxygen microscopy imaging method on a confocal laser scanning microscopy |
| CN104764729A (en) * | 2015-04-22 | 2015-07-08 | 华南师范大学 | Up-conversion-nanocrystal-based stimulated depletion super-resolution optical microscopic method and up-conversion-nanocrystal-based stimulated depletion super-resolution optical microscopic system |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN201242612Y (en) | Conversion illuminating microscope on homeostasis laser pump | |
| CN102706846B (en) | Near-infrared laser scanning confocal imaging system | |
| CN105004704B (en) | The upper conversion nano crystalline substance new application of neodymium ion sensitization and high-resolution multi-photon microscopic system | |
| CN104764729A (en) | Up-conversion-nanocrystal-based stimulated depletion super-resolution optical microscopic method and up-conversion-nanocrystal-based stimulated depletion super-resolution optical microscopic system | |
| CN101294902A (en) | Conversion illuminating microscope on mesomeric state laser pump | |
| CN101435774B (en) | Method and device of dual-color single photon transverse super resolution imaging | |
| CN105467572B (en) | Single wavelength realizes multi-photon pulses STED-SPIM microscopic systems | |
| US9971136B2 (en) | Method and device to achieve spatially confined photointeraction at the focal volume of a microscope | |
| CN106645064B (en) | A kind of stimulated radiation loss method, super-resolution imaging method and microscopic imaging device | |
| WO2021243755A1 (en) | Fluorescence difference super-resolution imaging method and imaging system | |
| CN103439242A (en) | Microfluidic system and method for detecting and screening single beam biological cells | |
| CN107045187A (en) | Multi-photon super-resolution microscopic imaging device and method | |
| CN202757884U (en) | Triplet-triplet annihilation up-conversion luminescence microscope | |
| CN109633881A (en) | A kind of microscopical imaging system of stimulated emission depletion | |
| Yan et al. | Confocal Laser Scanning Microscopy Based on a Silicon Photomultiplier for Multicolor In Vivo Imaging in Near‐Infrared Regions I and II | |
| Bhawalkar et al. | Two‐photon laser scanning fluorescence microscopy‐from a fluorophore and specimen perspective | |
| CN106568754A (en) | Optical system used for measuring liquid sample multiphoton fluorescence spectrum | |
| CN105588826A (en) | Femtosecond time resolution multi-channel lock-phase fluorescence spectrophotometer based on optical parametric amplification | |
| CN213933595U (en) | High-voltage fluorescence service life detection system | |
| CN103885166A (en) | Ultrahigh-resolution nonlinear fluorescence excitation microscopic system based on Bragg diffraction crystals | |
| Kaneko et al. | Real-time two-photon microscopy and its application for in situ imaging | |
| CN101748207A (en) | DNA sequencing device with single wavelength | |
| CN202751381U (en) | Triplet-triplet annihilation based upconversion in-vivo imaging system | |
| CN214097162U (en) | Fluorescence emission spectrum and service life detection system | |
| CN201313902Y (en) | Single wavelength DNA sequencing device |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20130227 Termination date: 20160322 |