CN107688014A - A kind of cell imaging method - Google Patents
A kind of cell imaging method Download PDFInfo
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- CN107688014A CN107688014A CN201710831105.5A CN201710831105A CN107688014A CN 107688014 A CN107688014 A CN 107688014A CN 201710831105 A CN201710831105 A CN 201710831105A CN 107688014 A CN107688014 A CN 107688014A
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/645—Specially adapted constructive features of fluorimeters
- G01N21/6456—Spatial resolved fluorescence measurements; Imaging
- G01N21/6458—Fluorescence microscopy
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
- G01N21/39—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using tunable lasers
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6402—Atomic fluorescence; Laser induced fluorescence
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N2021/6417—Spectrofluorimetric devices
- G01N2021/6419—Excitation at two or more wavelengths
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N2021/6417—Spectrofluorimetric devices
- G01N2021/6421—Measuring at two or more wavelengths
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N2021/6417—Spectrofluorimetric devices
- G01N2021/6423—Spectral mapping, video display
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks
Abstract
The present invention relates to a kind of method of cell and imaging of tissue, methods described, comprise the following steps：1) biological cell or tissue sample are prepared；The sample can be it is living or dead (such as：It is fixed) cell or tissue；2) light and reflected light (Scattered and reflected light, SRL) IMAQ, processing and analysis are scattered to cell and tissue sample using the various functions of various microscopes (by taking laser scanning co-focusing microscope as an example).
Description
Technical field
The present invention relates to a kind of cell and imaging of tissue method, and more particularly to cell and tissue sample are carried out using LSCM
SRL images individually and the collection with fluorescence or transmitted light combination image.
Background technology
Cell is the basic 26S Proteasome Structure and Function unit of organism, it is known that all biologies in addition to virus are by cell institute group
Into, but viral life activity must could also embody in cell.Now, the basis in almost all of life science is ground
Study carefully and key difficulties are all closely bound up with cell.Using cell as research object, the various change that it occurs is studied, very
An important link is exactly imaging observation.Mainly have to the method for cell imaging at present：Light microscope technique includes common
Compound light microscope, fluorescence microscope, laser scanning microscope are (including single photon or Multi-photon Laser Scanning Microscopy, super
High resolution microscope etc.)；Electron Microscopy includes ESEM, transmission electron microscope.However, Electron Microscopy is gone back at present
Living cells imaging is cannot be directly used to, and transmission electron microscope can only observe the ultra-thin section of cell, can not observe complete cell, sweep
The surface topography of cell can only then be observed by retouching Electronic Speculum.LSCM then can carry out Real Time Observation to living cells, can be determined with real-time tracing
Position biomone or chemicals are by living cells or the process of tissue intake, the number of more fluorescence channel quantitative analysis biomones
Amount and fluorescence intensity, different pseudo-colours, and multi-planar image synthesis three can be selected in different passages in an image
Tie up stereogram etc..
Mainly application is that the fluorescence signal for gathering sample is imaged to LSCM at present, and cell is tracked in three dimensions
Change, if however, cell does not have fluorescence, a piece of dark in the visual field of fluorescence microscope, it is difficult to navigate to cell, cause
This method runs into cell three-dimensional orientation problem in practice：
Current solution method has
1 using transmiting photoimaging, this method While it can be seen that cell position, but transmit photoimaging be difficult to determine it is accurate
Focal plane, therefore, it is difficult to realize the function of three-dimensional localization.
2 utilize the autofluorescence of cell.The autofluorescence of most cells is very low, too faint, it is difficult to shows that cell is determined
Position
3 make cell carry fluorescent staining using fluorescent labelling techniques.Cell is marked using fluorescence probe：It is in general, first
First, dyed using specific fluorescent dye to biological specimen.Then, the exciting light of specific wavelength is recycled to biological sample
It is irradiated point by point, so as to by exciting the fluorescent dye in biological specimen, produce fluorescence.Shortcoming:First, fluorescence labeling operation
Complex to waste time and energy, it is necessary to grope condition, cost is high；Second, after cell is marked by fluorescent material, it can take certain
Excite and transmission channel, restrict the range of choice of other fluorescence labeling materials in experiment；3rd, the problem of photobleaching be present：By
In most fluorescent materials by for a long time, strong laser irradiation after fluorescence intensity can substantially weaken, it is impossible to when long
Between detected under LSCM, therefore imaging, it is quantitative when be inevitably generated systematic error；4th, taken the photograph in Continuous Observation cell
When thing dynamic process one kind of getting it filled is tested, the medicine that Real Time Observation is fluorescently labeled enters before cell, is not fluorescently labeled
Cell can not be focused on accurately；And when cell is fluorescently labeled, fluorescence probe can have certain influence to cell state, unavoidably
Ground produces experimental error.
The present invention provides a kind of cell and imaging of tissue method, and the cell of non-fluorescent label and tissue are carried out using LSCM
SRL is imaged, and can accurately track focal plane, solves the technical barrier that non-fluorescent label cell and tissue can not focus on；Increase
The selectable range of fluorescence labeling material in big multi-fluorescence labelling experiment；Carry out SRL and fluoroscopic image gathers simultaneously, break through
Be formerly only available collection single image, realize to cell and tissue structure, cell and tissue in biochemical information while supervise
Survey.
Therefore, the invention provides a kind of non-fluorescent label, the cell precisely focused on and imaging of tissue method to meet pair
The demand of cell and imaging of tissue.
The content of the invention
It is an object of the invention to provide a kind of method to cell and imaging of tissue, cell and tissue can be carried out continuous
, prolonged real-time tracing and be positioned to picture.
The present invention provides a kind of cell and imaging of tissue method, comprises the following steps：
1) cell and tissue sample are prepared；The sample can be it is living or dead (such as：It is fixed) cell or tissue；
2) using the various functions of laser scanning co-focusing microscope cell and tissue sample are carried out SRL IMAQs,
Processing and analysis.
The cell of the present invention and the method for imaging of tissue, the step 2) are to thin using laser scanning co-focusing microscope
Born of the same parents and tissue sample carry out SRL and fluoroscopic image collection or using laser scanning co-focusing microscope to cell and tissue simultaneously
Sample carries out SRL, fluorescence and transmitted light images collection, processing and analysis simultaneously.Above-mentioned fluorescence outside including cell and tissue, thoroughly
Penetrate light and SRL is acquired.
Cell and imaging of tissue method of the present invention, wherein the cell and be organized as culture cell and tissue or
The cell and tissue directly separated from body, such as tumour cell, normal cell, liver organization etc..
Cell and imaging of tissue method of the present invention, wherein the wavelength of the laser is 200nm to 2000nm scopes
Interior any wavelength；Such as conventional optical maser wavelength：405nm, 488nm, 514nm, 543nm, 561nm and 633nm.
Cell and imaging of tissue method of the present invention, wherein the gatherer process of the LSCM is：By cell and tissue
It is imaged by the SRL passages of laser scanning co-focusing microscope, fluorescence channel and transmission optical channel；Utilize excitation
Sample, corresponding SRL, fluorescence and transmitted light are gathered, according to experiment demand, choose whether to need to adjust the temperature of sample, 5%
CO2, humidity and oxygen content.
Cell and imaging of tissue method of the present invention, SRL images can be strengthened using DIC.
Cell and imaging of tissue method of the present invention, SRL images can be strengthened using hydrogel.
Cell and imaging of tissue method of the present invention, wherein dynamic sequential image acquisition, method are as follows：After focusing,
The collection of image is carried out to sample；Then, keep condition it is constant, wait baseline stability, add tested material, monitoring cellular uptake by
Try thing overall process.Or fluorescence labeling first is carried out to cell and tissue, then carrying out SRL, fluorescence and transmitted light images simultaneously adopts
Collection.Such as fluorescence labeling is carried out to nucleus, can combine SRL images, detection tested material whether continue after entrance cell into
Enter nucleus.
Cell and imaging of tissue method of the present invention, specifically by taking A549 cells as an example, is described in detail below：
First, cell sample is prepared：By A549 cells kind in being copolymerized in burnt glass capsule, treat cell attachment culture to properly
Density, it is placed under LSCM.
Secondly, by taking spectral scan as an example, SRL images and light intensity-wavelength curve are obtained.Comprise the following steps that：Open
Tetra- groups of excitation source passages of LSCM, send the laser of four kinds of wavelength：405nm, 488nm, 561nm, 633nm, detect wavelength of fluorescence
Scope is set to 400nm to 680nm, step pitch 5nm.
Again, exemplified by tracking the tested material dynamic experiment of cellular uptake FITC marks, the SRL figures under time series are obtained
Picture and single fluorescence labeling image.Its cell when starting does not have fluorescence labeling, adds FITC fluorescence labelings tested after cellular uptake
There is fluorescence after thing.Comprise the following steps that：First, condition is set：SRL gathers cellular informatics, fluorescence channel collection FITC marks
Tested material information.Wherein SRL passages are excited using 633nm, and pseudo-colours selection is red；Fluorescence channel is excited using 488nm, pseudo-
Colour selection green；Dynamic acquisition total time is 30min, every 5s collections once.Second, using SRL passages, focus on, to sample
Product are imaged；3rd, keep condition constant, carry out IMAQ, wait baseline stability；4th, after baseline stability, add
The tested material of FITC marks, dynamic monitoring cellular uptake tested material overall process.Finally, the tested material marked with cellular uptake FITC
Exemplified by cytoplasm or apoptotic nueleolus experiment, SRL images and multi-fluorescence mark image are obtained.Comprise the following steps that：First,
Carry out fluorescence labeling：Nucleus is marked using Hoechst33342, is placed under LSCM mirrors and detects.Second, add FITC
The tested material of mark, wait the cellular uptake tested material.3rd, after reaching the experiment scheduled time, progress SRL is with fluorescence and thoroughly
Light image collection is penetrated, whether detection tested material goes successively to nucleus after cell is entered.
Those listed above A549 examples explanation is only visually to explain the present invention, and they are not with to limit this hair
Bright protection domain, all equivalent embodiments done without departing from skill spirit of the present invention or change, combination, segmentation such as feature
Or repeat, it should be included in the scope of the protection.
It is the explanation of title term of the present invention below：
Prepare cell sample：Cell is pre-processed before experiment, such as in specific embodiment 5, A549 cells put
Enter and be copolymerized in burnt capsule, treat that cell is completely adherent, growth conditions are preferable.A549 cells：Human lung carcinoma cell, a kind of tumour cell.
Fluorescence probe marks：Using some can send fluorescence material covalent bond or physical absorption in the life to be studied
In thing sample on the ion of specific function, functional group or large biological molecule, studied object is provided using its fluorescent characteristic
Information.
LSCM：Laser scanning co-focusing microscope, it is a kind of for IMAQ and the large-scale precision instrument of analysis.
FITC passages, SRL passages and transmission photoimaging：The signal for being utilized respectively each passage collection is converted to image.
Pseudo-colours：It is gray level image that copolymerization Jiao, which obtains image, is changed into coloured image in end processing sequences, and it is colored
For post transition, therefore claim pseudo- color.
Tested material：The material used in experiment, such as medicine, nano material etc..
Cellular uptake tested material：Tested material enters the process of cell.
The method to cell imaging of the present invention has the advantage that：
1st, SRL imagings are carried out to the cell of non-fluorescent label using LSCM.
1) cell in sample is imaged, it is not necessary to carry out any dyeing, keep cell kilter；
2) when the cell in testing sample is imaged, the wavelength of the SRL under laser irradiation is identical with its excitation wavelength,
Show that SRL intensity peak is narrow and sharp in spectrum, signal to noise ratio is high, and imaging effect is good；
3) imaging that the cell in testing sample can be stablized under the SRL of laser, including three-dimensional imaging, xyz/xzy sections
Imaging etc., and show preferable photostability in photobleaching experiment；
2nd, SRL is carried out to cell using LSCM and fluoroscopic image gathers.
1) cell in testing sample, SRL have no effect on other fluorescence labelings of cell, and can be glimmering with other marks
Light is imaged altogether, common location, three-dimensional imaging, xyz/xzy sections imaging；
2) after the cell in testing sample is fixed, then other fluorescence labelings are carried out to cell, laser remains in the SRL of cell
It is enough be imaged altogether with fluorescence probe, common location, three-dimensional imaging, the imaging of xyz/xzy sections；
3) cell in test sample product is after strong laser Continuous irradiation, and the image of its SRL passage is still high-visible, light
It is strong almost unchanged, and the fluorescence intensity from other fluorescent markers then substantially weakens, it was confirmed that SRL images hardly light is floated
White influence, there is good photostability；
3rd, fluorescence labeling is not being carried out in advance, and after needing plus in the cell experiment of fluorescence labeling tested material, before solving
Technology starts can not fine-focused problem.Realize the accurate overall process of monitoring tested material and cytosis in real time.
Simultaneously for the multi-fluorescence labelling experiment for needing display cell outline, increasing other needs fluorescence labeling material
Alternative, simplify experimental implementation, reduce the influence to cell.
Brief description of the drawings
The following drawings only does schematic illustration and explanation to the present invention, not delimit the scope of the invention.
Fig. 1 400 to 680nm wave-length coverages under LSCM in spectrogram.(a) A549 cells, (b) quilt are not marked by DiI
DiI marks A549 cells, (c) DiI solution
Imaging of Fig. 2 LSCM SRL passages and fluorescence channel light path schematic diagram and A549 cells under above-mentioned passage.
Fig. 3 (a) be A549 cells after fluorescence labeled cell core and cell membrane, LSCM collection cell fluorescence folded with SRL
Synthesized image figure.(b) be (a) enlarged drawing, (c) is the xzy rip cutting images of (b).
Fig. 4 be A549 cells after fluorescence labeling, the image of each confluent monolayer cells sample is continuously scanned along z-axis.(a)
Hoechst33342 fluorescence channels, (b) DiI fluorescence channels, (c) SRL passages, (d) a, b, c stacking chart.
Fig. 5 be photobleaching before and after each channel image, (a) Hoechst33342 fluorescence channels, (b) DiI fluorescence channels,
(c) SRL passages, (d) a, b, c stacking chart.
Fig. 6 be photobleaching region real-time dynamic monitoring image, (a) DiI Hoechst33342 fluorescence channels, (b) DiI
Fluorescence channel, (c) SRL passages, (d) a, b, c stacking chart.
Fig. 7 is the quantitation curves of each passage luminous intensity of photobleaching region, red：DiI fluorescence channels, green：SRL leads to
Road.
Fig. 8 be A549 cellular uptake fluorescence labeling tested materials real-time dynamic monitoring image, (a) SRL passages, (b) FITC
Fluorescence channel, (c) a, b stacking chart.
Fig. 9 is the quantitation curves of each passage luminous intensity of A549 cellular uptake fluorescence labeling tested materials
Figure 10 presents live body heart tissue SRL 2D imagings, sample M is acquired, wherein b1 figures are histocyte
Mitochondrial fluorescence imaging, c1 figures are histocyte SRL imagings, and a1 figures are b1 and c1 overlapping figure；Therefrom can clearly it see
Observe, SRL can be to imaging of tissue.Sample N is acquired, wherein b2 figures are histocyte mitochondrial fluorescence imaging, and c2 schemes
It is imaged for histocyte profile SRL, a2 figures are b2 and c2 overlapping figure；It is that SRL schemes with fluorescence overlapping in histocyte to scheme d2, figure
E2 is d2 enlarged drawing, and f2 is e2 enlarged drawing；Therefrom it can clearly observe that SRL is preferable to the imaging effect of tissue, and
It can be used cooperatively with fluorescence channel.
Figure 11 presents live body heart tissue SRL3D imagings, to sample N using XYZ dimensional scan patterns in LSCM to group
Tissue samples are scanned, wherein distance is 1 μm between two neighboring optical section, i.e. Zn-Zn-2=2 μm；And total distance is 20 μm.
And we can clearly observe the 3D rendering of tissue sample by Figure 11
Figure 12 presents fixed heart tissue SRL2D imagings, sample M is acquired, wherein b1 figures are in histocyte
Mitochondria fluorescence imaging, c1 figures are histocyte SRL imagings, and a1 figures are b1 and c1 overlapping figure；Therefrom can clearly it observe
Arrive, SRL can be to imaging of tissue.Sample N is acquired, wherein b2 figures are histocyte mitochondrial fluorescence imaging, and c2 schemes
It is imaged for histocyte profile SRL, a2 figures are b2 and c2 overlapping figure；It is that SRL schemes with fluorescence overlapping in histocyte to scheme d2,
Scheme the enlarged drawing that e2 is d2, f2 is e2 enlarged drawing；Therefrom it can clearly observe that SRL is preferable to the imaging effect of tissue,
And it can be used cooperatively with fluorescence channel.
Figure 13 presents fixed heart tissue SRL3D imagings, to sample N using XYZ dimensional scan patterns in LSCM to group
Tissue samples are scanned, wherein distance is 1 μm between two neighboring optical section, i.e. Zn-Zn-2=2 μm；And total distance is 20 μm.
And we can clearly observe the 3D rendering of tissue sample by scheming N
Figure 14 presents live body brain tissue SRL2D imagings, and sample P is acquired, and wherein b1 figures are thin in histocyte
Karyon fluorescence imaging, c1 figures are histocyte SRL imagings, and a1 figures are b1 and c1 overlapping figure；Therefrom can clearly it observe
Arrive, SRL can be to imaging of tissue.
Sample Q is acquired, wherein b2 figures are the imaging of histocyte inner cell nuclear fluorescence, and c2 figures are histocyte wheel
Wide SRL imagings, a2 figures are b2 and c2 overlapping figure；Figure d2 is tissue
Figure 15 presents live body brain tissue SRL3D imagings, to sample N using XYZ dimensional scan patterns in LSCM to tissue
Sample is scanned, wherein distance is 1 μm between two neighboring optical section, i.e. Zn-Zn-2=2 μm；And total distance is 20 μm.And
And we can clearly observe the 3D rendering of tissue sample by Figure 15
Figure 16 presents fixed brain tissue SRL2D imagings, and sample P is acquired, and wherein b1 figures are thin in histocyte
Karyon fluorescence imaging, c1 figures are histocyte SRL imagings, and a1 figures are b1 and c1 overlapping figure；Therefrom can clearly it observe
Arrive, SRL can be to imaging of tissue.
Sample Q is acquired, wherein b2 figures are the imaging of histocyte inner cell nuclear fluorescence, and c2 figures are histocyte wheel
Wide SRL imagings, a2 figures are b2 and c2 overlapping figure；It is that SRL schemes with fluorescence overlapping in histocyte to scheme d2, and figure e2 is putting for d2
Big figure, f2 are e2 enlarged drawing；Therefrom can clearly observe SRL it is preferable to the imaging effect of tissue, and can and fluorescence
Passage is used cooperatively.
After Figure 17 presents fixation, brain SRL3D imagings, to sample N using XYZ dimensional scan patterns in LSCM to group
Tissue samples are scanned, wherein distance is 1 μm between two neighboring optical section, i.e. Zn-Zn-2=2 μm；And total distance is 20 μm.
And we can clearly observe the 3D rendering of tissue sample by Figure 17
Embodiment
In order to which the technical characteristic of invention, purpose and effect are more clearly understood, illustrate in conjunction with following examples
The embodiment of the present invention.
The laser scanning co-focusing microscope (LSCM) used in following examples is Leica TCS SP8.
Embodiment 1：The cell not being fluorescently labeled is detected with LSCM and spectral scan.
By A549 cells kind in being copolymerized in burnt glass capsule, treat that cell attachment culture to proper density, is placed under LSCM mirrors.
Tetra- groups of excitation source passages of LSCM are opened during spectral scan, send the laser of four kinds of wavelength：UV-405nm, Ar-
488nm, Kr-561nm, HeNe-633nm, Detection wavelength scope are set to 400nm to 680nm, step pitch 5nm, draw light intensity-ripple
Long curve.Testing result is as shown such as (a) in Fig. 1, is the launching light spectrogram in the receiver wavelength range of A549 cells.It is thin in A549
There is a sharp peak at 405,488,561,633nm on born of the same parents' spectrogram, peak width only has about 10nm, and these peaks are at it and swashed
Send out the position of wavelength.
Embodiment 2：By the cell of DiI fluorescence labelings LSCM detections and spectral scan.
By A549 cells kind in being copolymerized in burnt glass capsule, cell attachment culture is treated to proper density, using DiI to cell
After film carries out fluorescence labeling, it is placed under LSCM mirrors and detects.With DiI solution as a control group.Opened during spectral scan
Tetra- groups of excitation source passages of LSCM, send the laser of four kinds of wavelength：UV-405nm, Ar-488nm, Kr-561nm, HeNe-
633nm, Detection wavelength scope are set to 400nm to 680nm, step pitch 5nm, draw light intensity-wavelength curve.Testing result such as Fig. 1
In shown in (b) and (c), the respectively hair of DiI fluorescence labeled cells and DiI solution in 400-680nm receiver wavelength range
Penetrate spectrogram.Wherein (b) represents DiI fluorescence labeled cells, and (c) represents DiI solution.On DiI fluorescence labeled cell spectrograms
There is a sharp peak at 405,488,561,633nm, peak width only has about 10nm, and these peaks are at its excitation wavelength
Position.In DiI fluorescence labeled cell spectrograms, except this four groups of peaks, there is a very wide peak at 570nm, this group of peak
Location and shape are consistent with the peak in the spectrum of DiI solution, are DiI emission spectrum (DiI is excited at 561nm).
Fig. 2 be LSCM SRL passages and DiI fluorescence channel light path schematic diagrams, and embodiment 1 and 2 in it is not glimmering by DiI
Signal cell (a) and the imaging by DiI fluorescence labeled cells (b) under above-mentioned passage.When being irradiated with a laser, not by DiI
Fluorescence labeled cell can use the SRL wavelength of laser to be imaged, without any fluorescence under DiI fluorescence channels；It is thin by DiI fluorescence labelings
Born of the same parents can either detect SRL images in exciting light opening position, and and can shows DiI photoluminescent properties and obtains fluoroscopic image；And DiI is molten
Fluoroscopic image is only presented in liquid.
Embodiment 3：Detected by the cell of DiI and Hoechst3332 fluorescence labelings with LSCM
By A549 cells kind in being copolymerized in burnt glass capsule, cell attachment culture is treated to proper density, using DiI to cell
Film carries out fluorescence labeling, reuses after nucleus is marked Hoechst3332, is placed under LSCM mirrors and detects.
Fig. 3 is by the cell of DiI and Hoechst33342 fluorescence labelings, and planar imaging is carried out using LSCM, wherein：(b)
It is the partial enlargement image of (a), (c) is the xzy rip cutting images of (b).SRL passages can be carried out clearly to cell as shown in the figure
Imaging, and there is common location with DiI.
Fig. 4 is the image for continuously scanning each confluent monolayer cells sample under LSCM along z-axis, wherein：(a) Hoechst33342 fluorescence
Passage, (b) DiI fluorescence channels, (c) SRL passages, (d) a, b, c stacking chart.As illustrated, SRL passages and fluorescence channel exist
Sample can be clearly imaged in the sectional view picture along each aspect of Z axis, and SRL passages can combine the fluorescence of cell each several part
Passage positions cell jointly.These sections can be combined into three-dimensional image, be tested for subsequent cell.
Embodiment 4：Tested by the cell photobleaching of DiI and Hoechst33342 fluorescence labelings
Photobleaching phenomenon is the defects of one of fluorescent marker is inevitable, therefore be cannot be used under LSCM with sharp
Light is continuous, irradiates for a long time, is unfavorable for tracing and positioning fluorescence signal.But according to the image-forming principle of SRL technologies, SRL signals
With good photostability.
Operating procedure：Prepare cell sample by embodiment 3.Ready cell sample is imaged, drawn a circle to approve in cell sample
A certain region (A specific region of interest, ROI) improves the intensity of the excitation wavelength of detection, continuous to shine
Penetrate ROI 160s and real-time dynamic monitoring luminous intensity change and take pictures, and with LSCM DiI fluorescence channels, SRL passages,
Hoechst33342 fluorescence channels are imaged simultaneously and recording light intensity.
Fig. 5 to Fig. 7 is the contrast of the present embodiment SRL signals and fluorescence signal photostability.Fig. 5 is each before and after photobleaching
Channel image；Fig. 6 is the real-time dynamic monitoring image of photobleaching region；Fig. 7 is determining for each passage luminous intensity of photobleaching region
Measure curve, (a) Hoechst33342 fluorescence channels, (b) DiI fluorescence channels, (c) SRL passages, (d) a, b, c stacking chart.Such as
Shown in figure, after Continuous irradiation selection area, the fluorescence probe DiI fluorescence intensities on cell are marked substantially to weaken, and SRL is adopted
The form of cell is presented in the image of collection with being still apparent from, and intensity hardly changes.Fluorescence intensity-time plot of standardization
In quantitatively embody above-mentioned trend, after 160s laser Continuous irradiations, fluorescence intensity have dropped 70%, and SRL intensity only declines
20%.SRL signal intensities are influenceed small by continuous laser irradiation in a word, illustrate that it has extraordinary photostability.
Embodiment 5：With the dynamic process of LSCM detection cellular uptake tested materials.
1st, operating method：
By A549 cells kind in LSCM glass dishes, treat that cell is completely adherent, be placed in LSCM37 DEG C of 5%CO2Saturated humidity
Thermal station under, pass through fluorescence channel, SRL passages and transmission photoimaging；This method using LSCM gather image condition be,
Exciting light：405nm, 488nm, 514nm, 543nm or 561nm, 633nm：Gather corresponding SRL and fluorescence.With one group of exciting light
SRL exemplified by, first, SRL passages are excited using 633nm, and pseudo-colours selection is red.Fluorescence channel is excited using 488nm, pseudo- color
Color sorting selects green.Dynamic acquisition total time is 30min, every 5s collections once.Secondly, focus on and sample progress image is adopted
Collection；Then, keep condition constant, wait baseline stability, add tested material, monitor cellular uptake tested material overall process.
2nd, testing result：
Fig. 8 presents the real time imagery figure of cellular uptake tested material, wherein：(a) SRL passages, (b) fluorescence channel.SRL
Image shows cell imaging, and cellular uptake tested material is positioned.The extension with incubation time can be clearly observed, more
It is distributed in come more preparations in cytoplasm.Fig. 9 is the quantitation curves of each passage luminous intensity corresponding to Fig. 8.The fluorescence of standardization
Strength-duration curve chart is bright, and with the extension of time, the fluorescence intensity of fluorescence channel gradually strengthens, and the fluorescence of SRL passages
Intensity kept in balance.This explanation SRL can be imaged to living cells, cell be positioned, and can clearly observe cell
Absorb the dynamic process of preparation.
Embodiment 6：The tissue that is not fluorescently labeled and glimmering by acridine orange AO or mitochondrial membrane potential fluorescence probe JC-1
The tissue of signal is detected with LSCM
(1) experimental rat is anaesthetized, quickly removes whole rat heart, cut into slices；Take wherein M, N two panels, histotomy
M is placed in the burnt capsule of copolymerization, directly uses confocal microscopy；Histotomy N is placed in the burnt capsule of copolymerization, adds 10mM lines
Mitochondrial membrane potential fluorescence probe JC-1 1ml fluorescence probes, which are put in 37 DEG C of deposited casees, dyes 2h；Observed with LSCM.Led to by fluorescence
Road, SRL passages and transmission photoimaging, wherein mitochondrial membrane potential fluorescence probe JC-1 are imaged to mitochondria, and SRL is to whole
Individual cell outline is imaged.This method is exciting light using the LSCM conditions for gathering image：405nm, 488nm, 514nm,
543nm or 561nm, 633nm：Gather corresponding SRL and fluorescence.By taking the SRL of one group of exciting light as an example：SRL passages use
633nm is excited, pseudo-colours selection pink.Fluorescence channel is excited using 488nm, pseudo-colours selection green.
(2) (1) cardiac is cut into slices and fixed using 4% paraformaldehyde, it is corresponding to pass through fluorescence channel, SRL passages and thoroughly
Photoimaging is penetrated, gathers image.
(3) quick cranium of opening takes out rat whole brain, section；Wherein P is taken, Q two panels, histotomy P is placed on the burnt capsule of copolymerization
In, directly use confocal microscopy；Histotomy Q is placed in the burnt capsule of copolymerization, adds AO 1ml fluorescence probes to put 37 DEG C
Apply in case and dye 2h；Use confocal microscopy.By fluorescence channel, SRL passages and transmission photoimaging, wherein, AO enters
Row nuclei images, SRL are imaged to whole cell outline.This method is exciting light using the LSCM conditions for gathering image：
405nm, 488nm, 514nm, 543nm or 561nm, 633nm：Gather corresponding SRL and fluorescence.Using the SRL of one group of exciting light as
Example：SRL passages are excited using 633nm, and pseudo-colours selection is red.Fluorescence channel is excited using 488nm, pseudo-colours selection green.
(4) brain section in (3) is fixed using 4% paraformaldehyde, it is corresponding to pass through fluorescence channel, SRL passages and transmission
Photoimaging, gather image.
Although in above example, when irradiating testing sample with laser, the wavelength of the laser used is 405nm, 488nm,
561nm or 633nm, but not limited to this, the laser of other wavelength also can be as the light source of laser of the present invention irradiation.
Claims (10)
1. a kind of method of cell and imaging of tissue, it is characterised in that comprise the following steps：
1) biological cell or tissue sample are prepared；The sample can be it is living or dead (such as：It is fixed) cell or tissue；
2) light and reflected light image collection, processing are scattered to cell and tissue sample using microscopical various functions and is divided
Analysis.
2. a kind of method of cell and imaging of tissue, it is characterised in that the step 2) is micro- using laser scanning co-focusing
Mirror carries out SRL simultaneously to cell and tissue sample and fluoroscopic image gathers；Or using laser scanning co-focusing microscope to thin
Born of the same parents and tissue sample carry out SRL, fluorescence and transmitted light images collection simultaneously, including above-mentioned glimmering outside various biological cells and tissue
Light, transmitted light and SRL are acquired.
3. method as claimed in claim 1 or 2, it is characterised in that wherein described cell and be organized as various biological cells
And tissue.
4. method as claimed in claim 1 or 2, it is characterised in that the wavelength of wherein described SRL laser or other light sources is
Any wavelength in the range of 200nm to 2000nm.
5. method as claimed in claim 1 or 2, it is characterised in that the wavelength of wherein described laser is selected from：405nm, 488nm,
514nm, 543nm, 561nm and 633nm.
6. method as claimed in claim 1 or 2, it is characterised in that the collection of wherein described laser scanning co-focusing microscope
Process is：By cell and tissue by SRL passages, fluorescence channel and the light microscopic passage of laser scanning co-focusing microscope into
Picture；Using excitation sample, corresponding SRL, fluorescence and transmitted light are gathered, according to experiment demand, chooses whether to need to adjust
Save temperature, 5% CO of sample2, humidity and oxygen content.
7. method as claimed in claim 1 or 2, it is characterised in that also strengthen SRL images including the use of DIC.
8. method as claimed in claim 1 or 2, it is characterised in that also including the use of the various reinforcing agent enhancing SRL such as hydrogel
Image.
9. method as claimed in claim 1 or 2, it is characterised in that dynamic sequential image acquisition, method are as follows：It is right after focusing
Sample carries out the collection of image；Then, keep condition constant, wait baseline stability, add tested material, monitor cell and tested material
Act on overall process.
10. method as claimed in claim 1 or 2, it is characterised in that fluorescence labeling, Ran Houjin first are carried out to cell and tissue
Row SRL, fluorescence and transmitted light images collection.
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6287790B1 (en) * | 1998-11-30 | 2001-09-11 | The Regents Of The University Of California | Utilization of nuclear structural proteins for targeted therapy and detection of proliferative and differentiation disorders |
| CN101738462A (en) * | 2008-11-14 | 2010-06-16 | 华南师范大学 | Method for imaging live nucleus and cytoplasm and application thereof in monitoring live nucleus and cytoplasm signal pathway |
| CN102621117A (en) * | 2012-03-09 | 2012-08-01 | 福建师范大学 | Living cell laser scanning co-focusing microscope imaging system |
| CN103389287A (en) * | 2013-07-17 | 2013-11-13 | 福建师范大学 | A high resolution optical system suitable for the imaging of a liver surface in vivo |
| CN104122228A (en) * | 2014-07-14 | 2014-10-29 | 江苏大学 | A microscopic imaging system for analysis of integrated light interference and scattering information and a method |
| CN104316497A (en) * | 2014-10-31 | 2015-01-28 | 上海交通大学 | Cell imaging method based on nanogolds and LSCM (laser scanning confocal microscope) reflected light mode |
-
2017
- 2017-09-15 CN CN201710831105.5A patent/CN107688014B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6287790B1 (en) * | 1998-11-30 | 2001-09-11 | The Regents Of The University Of California | Utilization of nuclear structural proteins for targeted therapy and detection of proliferative and differentiation disorders |
| CN101738462A (en) * | 2008-11-14 | 2010-06-16 | 华南师范大学 | Method for imaging live nucleus and cytoplasm and application thereof in monitoring live nucleus and cytoplasm signal pathway |
| CN102621117A (en) * | 2012-03-09 | 2012-08-01 | 福建师范大学 | Living cell laser scanning co-focusing microscope imaging system |
| CN103389287A (en) * | 2013-07-17 | 2013-11-13 | 福建师范大学 | A high resolution optical system suitable for the imaging of a liver surface in vivo |
| CN104122228A (en) * | 2014-07-14 | 2014-10-29 | 江苏大学 | A microscopic imaging system for analysis of integrated light interference and scattering information and a method |
| CN104316497A (en) * | 2014-10-31 | 2015-01-28 | 上海交通大学 | Cell imaging method based on nanogolds and LSCM (laser scanning confocal microscope) reflected light mode |
Non-Patent Citations (7)
| Title |
|---|
| LITTLEJOHN ET AL.: "Anupdate:improvements in imaging perfluorocarbon-mounted plant leaves with implications for studies of plant pathology, physiology,development and cell biology", 《FRONTIERS IN PLANT SCIENCE》 * |
| SHANSHAN ZHAO ET AL.: "A Novel and Facile Approach to Imaging Nanoparticles Transport across Transwell Filter Grown Cell Monolayer in Real-Time and in Situ under Confocal Laser Scanning Microscopy", 《BIOLOGICAL AND PHARMACEUTICAL BULLETIN》 * |
| W.S. CHIU ET AL.: "Drug delivery into microneedle-porated nails from nanoparticle reservoirs", 《JOURNAL OF CONTROLLED RELEASE》 * |
| 何磊等: "基于多通道信息融合的激光扫描共聚焦显微镜图像分割", 《计算机应用研究》 * |
| 李容林主编: "《现代口腔设备技术与应用》", 31 December 2007, 广州：中山大学出版社 * |
| 李怀业等: "光、磁分子探针在脑组织间隙内的扩散分布规律", 《北京大学学报》 * |
| 陈涛等: "相干拉曼散射显微术", 《中国科学》 * |
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