CN103513411A - Device and method for focusing in fluorescence microscope - Google Patents
Device and method for focusing in fluorescence microscope Download PDFInfo
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- CN103513411A CN103513411A CN201310450223.3A CN201310450223A CN103513411A CN 103513411 A CN103513411 A CN 103513411A CN 201310450223 A CN201310450223 A CN 201310450223A CN 103513411 A CN103513411 A CN 103513411A
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Abstract
The invention provides a device and method for focusing in a fluorescence microscope. According to the device and method for focusing in the fluorescence microscope, the focusing time can be shortened, the system speed can be increased, and undesired photobleaching can be avoided. According to the fluorescence microscope, part of exciting light is used for forming an image of a sample so that the focal plane of fluorescence imaging can be determined. Due to the fact that the intensity of the part of exciting light is far greater than the intensity of fluorescence, the exposure time for forming a focusing target image is greatly shortened. The fluorescence microscope can conduct prefocusing and multi-path focusing.
Description
Technical field
The present invention relates to a kind of fluorescent microscope, be specifically related to a kind of focalizer for fluorescent microscope and method.
Background technology
Fluorescent microscope is a kind of optical microscope that uses fluorescence and phosphorescence synthetic image.Sample is used by the irradiation of at least one specific wavelength of fluorescent absorption, makes them launch more long wavelength's light.Typical fluorescent microscope comprises: as the light source of mercury-arc lamp, exciter filter, dichroic beamsplitter and transmitting light filter.Select light filter and dichroic beamsplitter, with the excitation of spectra and the emission characteristics of the fluorophore with for mark sample, match.Most of fluorescent microscopes in use are all epifluorescence microscopes, and wherein exciting with the detection of fluorescence of fluorophore all undertaken by same object lens.These microscopes are widely used in biology and medical science.
In order to obtain high-quality image in fluorescence microscope system, focusing is vital factor.By convention, microscope focusing algorithm depends on image itself.Inspection is at a series of images of different focal planes, and selects the image with maximum details correctly to focus on.For example, focusing system is estimating that focal position obtains a series of images around.For each image, this system log (SYSLOG) Ta position, and calculate the focusing score that characterizes each image sharpness.Finally, this system-computed focuses on the position of score maximum, and the position of choosing calculating is as focal position.
US7,141,773 provide a kind of imaging device, have for obtaining the automatic focusing mechanism of focusedimage.This device comprises: object lens, for changing the focus controller of distance between object lens and sample, for find the target view finder of the target centroid that closes and the measurement luminous intensity measurement unit of the light intensity level of definite target centroid that closes thus in sample.Focus control has changed sample-object lens distance, so that the light intensity level of measuring is maximum, thereby arrives focal position.The target centroid that closes can be by filtering out general objective and determining by covering background area alternatively.
WO2007138369 discloses a kind of for having method and the fluorescence imaging system of the sample of fluorescent target point on digitized processing slide.The method is used this fluorescence microscope system, for detection of with scanning fluorescent target point, and comprise step: by the position of sample on visable indicia toolmark slide glass, to define, comprise sample and fluorescent target Dian sample area; With the first optical amplifier multiplying power, catch the bright field image of at least a portion slide glass that comprises sample area, and determine by visable indicia instrument the position of dropping on the field of regard in sample area; With the field of regard of using the second optical amplifier multiplying power scanned sample district higher than the first optical amplifier multiplying power, wherein scanning comprises and at least focusing on a part of field of regard.
Above-mentioned prior art is used the focusing based on fluorescence, and it can high precision focus on.Yet low fluorescence intensity needs time exposure to obtain the image with high s/n ratio, therefore very slow based on many image ratios focus search.In addition, away from focal plane fluorescence intensity, reduce rapidly, thereby make to start focus search away from focal plane, become difficulty.The focusing time is long, needs lasting exciting, and this may cause photobleaching.Focus on especially serious for the wide visual field of scanning fluorescence microscopy application problem slowly.Finally, when focusing spended time is too many, system speed step-down.
In order to shorten focal time and to avoid photobleaching, also can use non-fluorescence to focus.As the additional source of light of halogen bulb and fluorescent microscope integrate, for focusing on.In most cases, in order to obtain high-quality focusing, need to use other optical technology, as phase contrast (PC) and differential (DIC).
Phase contrast is light microscope technique, and the brightness that its phase shifting by the light through transparent sample becomes in image changes.Phase shift itself is invisible, but when being shown as brightness variation, just becomes visible.In phasecontrast microscope method, making the visible ultimate principle of phase transformation is that this has made up prospect details, and differently handles them from the separated illumination of sample scattered light bias light.In order to carry out such technology, need to use special-purpose optical device, as phase grommet, condenser and phase plate.
Differential interference contrast microscope method is also a kind of optical microscope lighting engineering of contrast of, transparent sample pure for strengthening.DIC is with interferometric principle work, to obtain the information about the optical path length of sample, to see otherwise sightless feature.DIC is by being separated into polarized light source the Coherent Part of two mutually orthogonal polarizations, and they are spatial displacement (shear) on sample surfaces, and recombinates before observation.Polarizer, DIC prism, condenser, that analyser is realized DIC technology is essential.
US5,790,710 have instructed a kind of phase contrast for fluorescent microscope automatically to focus.
US6,674,574 provide a kind of for microscopical focusing system, comprising: object lens; Sample stage; Reflective illumination system for generation of the fluorescence from sample; Propagate illuminator, the optical imagery of propagating with seizure for illumination light on sample; One group of optical element, is used for, according to the phase information that is included in the light of propagating by sample, forming the optical imagery of propagating; The optical element that is used for making the optical imagery of fluoroscopic image and propagation to separate; For catching the sensor of the optical imagery of the propagation being separated by the optical element for minute opening the light; Be used for, according to the signal from this sensor output, detecting the focus detection part of the focusing grade of the optical imagery of propagating; And driver, according to focusing grade, at least one of object lens and objective table moved to focus on sample.
US5,790,710 and US6,674,574 is all the focusing method using based on non-fluorescence.They have the fireballing advantage of focusing.Yet their shortcoming is that all fluorescence channels only provide a focus, microscopical complex structure, and the considerable special-purpose optical device of needs.
Therefore, for focalizer and the method for fluorescent microscope, especially wide visual field scanning fluorescent microscope application, the whole slide glass imaging of picture and digital pathology, there is a unsatisfied demand, rapid focus under simple structure need to be provided, and do not use too many special-purpose optical device.
Summary of the invention
Therefore, invention required for protection is to provide a kind of focalizer for fluorescent microscope and method, and it can reduce focal time.
First aspect of the present invention is to provide a kind of device focusing on for fluorescent microscope.
According to the first embodiment of invention required for protection, a kind of focalizer for fluorescent microscope, comprising: excitation source, for launching exciting light with excited sample; Beam splitter, for reflecting exciting light by object lens to sample, and the fluorescence that sent by sample of transmission; And focus module, it is between excitation source and beam splitter, and wherein focus module comprises reflective optical device and the focal imaging camera for light reflection, wherein in the path of the exciting light of reflective optical device between excitation source and beam splitter; Wherein sample is to beam splitter reflection, scattering or reflection and scattering first exciting light backward backward; and beam splitter is to reflective optical device reflection first exciting light, and reflective optical device is to focal imaging camera reflection second portion exciting light; Wherein be focused at least one excitation light image that the second portion exciting light of catching as camera is formed for focusing on the sample of fluorescence imaging.
According to the second embodiment of invention required for protection, a kind of focalizer for fluorescent microscope, comprising: excitation source, for launching exciting light with excited sample; Beam splitter, for reflecting exciting light by object lens to sample, and the fluorescence that sent by sample of transmission; And focus module, be positioned at below sample, wherein focus module comprises convergent lens and the focal imaging camera for converging light, wherein convergent lens is between sample and focal imaging camera; Wherein a part of exciting light transmission is by sample, and this part exciting light propagates to convergent lens, and assembles to focal imaging camera by convergent lens; And be wherein focused at least one excitation light image that the second portion exciting light of catching as camera is formed for focusing on the sample of fluorescence imaging.
A second aspect of the present invention is to provide a kind of method focusing on for fluorescent microscope.
According to invention required for protection embodiment, the focus method for fluorescent microscope, comprising: by object lens, send exciting light and be used for irradiating sample, wherein exciting light is reflected towards sample by beam splitter; By sample to beam splitter reflection, scattering or reflection and scattering first exciting light backward backward; by beam splitter, to reflective optical device, reflect first's exciting light, and to focal imaging camera, reflect second portion exciting light by reflective optical device; And by being used for focusing on the focal imaging camera of fluoroscopic image, with second portion exciting light, form at least one excitation light image.
According to another embodiment of invention required for protection, the focus method for fluorescent microscope, comprising: by object lens, send exciting light, be used for irradiating sample, wherein exciting light is reflected towards sample by beam splitter; Sample is passed through in the transmission of part exciting light, and assembles this part exciting light to focal imaging camera; With this part exciting light, form at least one excitation light image with the focal imaging camera by being used for focusing on fluoroscopic image.
Owing to having used a part of exciting light to focus on, compare with the focusing system of tradition based on fluorescence, in order to focus on this part exciting light while forming detail image, carrying much higher intensity, thus accurately focusing in can be between short-term, and avoided undesirable photobleaching.
Different from traditional focusing system based on non-fluorescence, fluorescent microscope of the present invention only needs a light source.In addition, do not need the special-purpose optical system as PC or DIC, and fluorescence is not subject to special-purpose optical system and for any interference of the non-fluorescence that focuses on.
The more important thing is, focalizer of the present invention is to carry out that multichannel focuses on and prefocus simultaneously, and focusing system based on fluorescence and non-fluorescence all can not be done like this.
Accompanying drawing explanation
Hereinafter, with reference to accompanying drawing, embodiment of the present invention will be described in more detail, wherein
Fig. 1 shows having in reflection and/or utilizing backward the schematic diagram of fluorescent microscope of the focalizer of a part of exciting light under backscatter mode according to the first embodiment of invention required for protection;
Fig. 2 shows having in reflection and/or utilizing backward the schematic diagram of fluorescent microscope of the focalizer of a part of exciting light under backscatter mode according to the second embodiment of invention required for protection;
Fig. 3 be illustrate according to inventive embodiment required for protection in reflection and/or utilize backward the process flow diagram of step of focus method of the fluorescent microscope of a part of exciting light under backscatter mode;
Fig. 4 shows according to the schematic diagram of the fluorescent microscope with the focalizer that utilizes a part of exciting light under transmission mode of inventive embodiment required for protection;
Fig. 5 is the process flow diagram illustrating according to the step of the focus method of the fluorescent microscope that utilizes a part of exciting light under transmission mode of inventive embodiment required for protection;
Fig. 6 is the process flow diagram illustrating according to the step of the focus method that passes through change lens and sample distance of inventive embodiment required for protection;
Fig. 7 is the process flow diagram illustrating according to the step of the prefocus method of the scanning fluorescent microscope of inventive embodiment required for protection;
Fig. 8 A is according to the excitation light image of the sample being obtained by a part of exciting light of inventive embodiment required for protection; With
Fig. 8 B is the fluoroscopic image of the sample that obtained by fluorescence.
Embodiment
In the following description, focalizer and the method for fluorescent microscope as preferred embodiment, have been listed.For a person skilled in the art obviously, without departing from the scope and spirit of the present invention, can carry out some and revise, comprise and add and/or replace.Concrete details can be omitted, so as not to making the present invention not obvious; Yet its disclosure is written into, so that those skilled in the art put into practice instruction herein, and without too much experiment.
Fig. 1 shows having in reflection and/or utilizing backward the schematic diagram of fluorescent microscope of the focalizer of a part of exciting light under backscatter mode according to the first embodiment of invention required for protection.This fluorescent microscope comprises: excitation source 1, focus module 2a, collector lens 3, exciter filter 4, dichroic beamsplitter 5, object lens 6, sample 7, objective table 8, transmitting light filter 9, tube lens 10 and fluorescence imaging camera 11.Focus module 2a, between excitation source 1 and collector lens 3, and comprises beam splitter 12 and focal imaging camera 13.Beam splitter 12 is on the exciting light path between excitation source 1 and collector lens 3, and focal imaging camera 13 is positioned on the top of beam splitter 12.
Exciting light, through beam splitter 12, collector lens 3 and exciter filter 4, then arrives dichroic beamsplitter 5.Then exciting light is reflected by dichroic beamsplitter 5, by object lens 6, is finally focused on sample 7.Arrow 101 shows the path of exciting light.
After exciting light irradiates sample 7, first's exciting light is reflected and/or scattering backward by sample 7, and is collected by object lens 6 and dichroic beamsplitter 5.Dichroic beamsplitter 5 is towards exciter filter 4, collector lens 3, beam splitter 12 reflection first exciting lights afterwards.Beam splitter 12 is towards focal imaging camera 13 reflection second portion exciting lights, the excitation light image focusing on to be formed for fluorescence imaging.Therefore, second portion exciting light is a part for first's exciting light.Arrow 102a and 102b show respectively the path of the first and second part exciting lights.
According to the various embodiment of invention required for protection, beam splitter 12 can be replaced by any following reflective optical device: can reflect wholly or in part first exciting light towards focal imaging camera 13 and during transmission, minimize reducing from the excitating light strength of excitation source 1 simultaneously.
According to another embodiment of invention required for protection, beam splitter 12 can be able to be replaced towards the catoptron of focal imaging camera 13 total reflection first exciting lights.Therefore, second portion exciting light is identical with first exciting light.Preferably, catoptron is less than the xsect in the exciting light path of location catoptron, makes to excite optical transmission not to be subject to very large impact.
Under the irradiation of exciting light, the fluorescence that sample 7 emission wavelengths are longer than exciting light.Fluorescence is by object lens 6, dichroic beamsplitter 5, transmitting light filter 9 and tube lens 10, and the final fluorescence imaging camera 11 that is used to form fluoroscopic image that arrives.Arrow 103 shows the path of fluorescence.
Use focal imaging camera 13 to form the excitation light image of sample 7 with second portion exciting light.When second portion excites light intensity far above glimmering light intensity, there is even in some cases the difference of several orders of magnitude, the excitation light image of sample also can form at short notice, because greatly reduced the time of storage for the light of the q.s of imaging.Then, formed excitation light image is further used for finding out sample for the focal plane of fluorescence imaging.
Compare with the classic method of using fluorescence to focus on, the present invention can shorten focal time, thereby has fundamentally improved the speed of system, and has avoided undesirable fluorescence photobleaching.In addition, compare with the focus method based on non-fluorescence, the architecture of fluorescent microscope of the present invention is simply too much, because only need a light source, and also not for the special-purpose optical device of PC or DIC.The more important thing is, owing to not needing special-purpose optical device in the present invention, can avoid fluorescence to be interfered by the special-purpose optical device in light path.And due to simple system architecture, avoided being used for focusing on and the PC/DIC of imaging and the complicated signal in fluorescence combination microscope is synchronous and mechanical motion control.
Fig. 2 shows having in reflection and/or utilizing backward the schematic diagram of fluorescent microscope of the focalizer of a part of exciting light under backscatter mode according to the second embodiment of invention required for protection.This fluorescent microscope comprises excitation source 1, focus module 2b, collector lens 3, exciter filter 4, dichroic beamsplitter 5, object lens 6, sample 7, objective table 8, transmitting light filter 9, tube lens 10 and fluorescence imaging camera 11.Focus module 2b, between collector lens 3 and exciter filter 4, and comprises beam splitter 12, tube lens 10' and focal imaging camera 13.In the path of the exciting light of beam splitter 12 between collector lens 3 and exciter filter 4, and tube lens 10 ' between beam splitter 12 and focal imaging camera 13.
According to the various embodiment of method required for protection, beam splitter 12 can be replaced by any following reflective optical device: can reflect wholly or in part first's exciting light simultaneous minimization reducing from the excitating light strength of excitation source 1 towards focal imaging camera 13.
According to another embodiment of invention required for protection, beam splitter 12 can be able to be replaced towards the catoptron of focal imaging camera 13 total reflection first exciting lights.Therefore, second portion exciting light is identical with first exciting light.Preferably, catoptron is less than the xsect in the exciting light path of location catoptron, makes to excite optical transmission not to be subject to very large impact.
Fig. 3 be according to inventive embodiment required for protection in reflection and/or utilize backward the process flow diagram of step of focus method of the fluorescent microscope of a part of exciting light under backscatter mode.In step 301, exciting light, by focus module and collector lens, is launched towards dichroic beamsplitter from excitation source.Focus module comprises: reflective optical device and focal imaging camera, and in the path of the exciting light of reflective optical device between excitation source and dichroic beamsplitter.In step 302, exciting light is reflected towards the sample that scribbles fluorescent material by dichroic beamsplitter by object lens.At step 303Zhong， first exciting light, by object lens, by sample, towards dichroic beamsplitter, reflected and/or scattering backward.In step 304Zhong， first, exciting light is reflected towards reflective optical device by dichroic beamsplitter.In step 305, second portion exciting light is reflected optical device and reflects towards focal imaging camera.Second portion exciting light is a part for first's exciting light, or identical with first exciting light.In step 306, the excitation light image of sample forms by focal imaging camera with second portion exciting light.In step 307, according to the image of the exciting light forming, be identified for the focal plane of the sample of fluorescence imaging.
Fig. 4 shows according to the schematic diagram of the fluorescent microscope with the focalizer that utilizes a part of exciting light under transmission mode of inventive embodiment required for protection.Fluorescent microscope comprises excitation source 1, focus module 2c, collector lens 3, exciter filter 4, dichroic beamsplitter 5, object lens 6, sample 7, objective table 8, transmitting light filter 9, tube lens 10 and fluorescence imaging camera 11.Focus module 2c is positioned at objective table 8 belows, and comprises convergent lens 14 and focal imaging camera 13.Convergent lens 14, between objective table 8 and focus imaging video camera 13, and in this section in the path of exciting light, is used for this part exciting light to converge on focal imaging camera 13.
At sample 7, by the exciting light from excitation source 1 between the light period, a part of exciting light transmission is by sample 7.This part exciting light, by comprising the objective table 8 for light transmissive through hole, arrives convergent lens 14, then by convergent lens 14, is focused on imaging camera 13.Preferably, convergent lens 14 is object lens of long reach, the lens combination of the object lens of low range or formation high precision image.Arrow 104 shows the path of this part exciting light.
Similarly, this part exciting light under transmission mode is for forming the excitation light image of sample 7 by focal imaging camera 13.When this part excites light intensity far above glimmering light intensity, will greatly shorten and focus on the time shutter.
Fig. 5 is according to the process flow diagram of the step of the focus method of the fluorescent microscope that utilizes a part of exciting light under transmission mode of inventive embodiment required for protection.In step 501, exciting light is launched to dichroic beamsplitter by collector lens from excitation source.In step 502, exciting light, by object lens, is reflected towards the sample that scribbles fluorescent material by dichroic beamsplitter.In step 503, a part of exciting light passes through sample transmission towards focus module.This focus module comprises convergent lens and focal imaging camera, and convergent lens is between sample and focal imaging camera.In step 504, this part exciting light is converged lens and assembles towards focal imaging camera.In step 505, by focal imaging camera, with this part exciting light, form the excitation light image of sample.In step 506, according to the excitation light image forming, determine the focal plane of sample, for fluorescence imaging.
According to one embodiment of present invention, in order to determine the focal plane of sample, this fluorescent microscope also comprises central processing unit CPU.This CPU, be connected with focal imaging camera, objective table and fluorescence imaging camera, for analyzing the excitation light image of being taken by focal imaging camera, determine that fluoroscopic image is whether on focal plane, by moving stage, regulate the distance of lens and sample, once and sample in focus, just indicate fluorescence camera to take fluoroscopic image.
Fig. 6 is the process flow diagram illustrating according to the step of the focus method that passes through change lens and sample distance of inventive embodiment required for protection.In step 601, focal imaging camera is taken the excitation light image of the sample being formed by a part of exciting light.In step 602, check excitation light image, whether sample is in the focal plane for fluorescence imaging.If, on focal plane, do not change the distance of lens and sample in step 603, and repeating step 601 to 602.If on focal plane, in step 604, by fluoroscopic image camera, take fluoroscopic image.Preferably, by the linear movement of the objective table by motor control, change the distance of lens and sample.
Because the visual field of focal imaging camera is different from the visual field of fluorescence imaging camera, fluorescent microscope of the present invention can carry out also can carrying out the focus method of the focusing on next visual field during the photographs fluoroscopic image of prefocus ，Qi Shi visual field.Owing to taking fluoroscopic image and focusing on, be parallel generation, so prefocus can further reduce the fluorescence imaging time.
Fig. 7 is the process flow diagram illustrating according to the step of the prefocus method of the scanning fluorescent microscope of inventive embodiment required for protection.In step 701, by exciting light, irradiate sample, and utilize the fluorescence from sample, by fluorescence imaging camera at i visual field photographs fluoroscopic image.In step 702, use a part of exciting light, by focal imaging camera i+1Ge visual field photographs excitation light image.In step 703, according to excitation light image, determine the focal plane of i+1Ge visual field.In step 704, be used in focal plane definite in step 703, i+1Ge visual field photographs fluoroscopic image.
Traditional focusing based on fluorescence can not be carried out prefocus, because only have an imaging camera for fluorescence imaging and focal imaging, and that these two tasks can not be entered X by single camera is simultaneously capable.On the other hand, although the focusing based on non-fluorescence can be carried out prefocus, fluorescence imaging quality often worsens, because there is the two-beam from two Different Light in same light-path, thereby can cause two interference between light beam.On the contrary, the problems referred to above have been avoided in prefocus of the present invention, because only use a light source.
The present invention can carry out multichannel focusing, and this is used for finding the focal plane of different fluorescence excitations and the sample of transmitting channel.According to one embodiment of present invention, more than one exciter filter, dichroic beamsplitter and transmitting light filter are used to form various optical filtering combination optical pieces.The combination optical piece of this optical filtering can further be configured to form optical filtering piece turntable, and it can rotate under fluorescent microscope, to change the exciting light for the different-waveband of sample irradiation.Therefore, the focus method based on non-fluorescence, as PC or DIC, cannot carry out multichannel focusing, because only focused light source can not adapt to the exciting light of the frequency change of different-waveband.In contrast, the present invention can change for changing the light for focusing in the exciting light of fluorescence.
Fig. 8 A is according to the excitation light image of the sample being obtained by a part of exciting light of inventive embodiment required for protection.Fig. 8 B is the image of the sample that obtained by fluorescence.Although the time shutter of the excitation light image of Fig. 8 A is shorter than Fig. 8 B, Fig. 8 A is brighter than Fig. 8 B, this means by using this part exciting light to receive more light for imaging.
According to invention required for protection embodiment, when laser instrument or LED are during as excitation source, because LED can provide specific band, so may not need exciter filter.
According to invention required for protection embodiment, the transmission of being undertaken by beam splitter in focus module and the ratio of reflection are 80 to 20.
According to invention required for protection embodiment, exciter filter can be used as separated light filter change equipment (filter wheel or light filter slide block) and places away from light filter turntable.And so separated light filter changes equipment and can be placed between excitation source and focus module.
Embodiment disclosed herein can realize with universal or special computing equipment, computer processor or electronic circuit, includes but not limited to digital signal processor (DSP), special IC (ASIC), field programmable gate array (FPGA) and other programmable logic device (PLD) that configures or programme according to instruction of the present disclosure.Operate in computer instruction or software code in universal or special computing equipment, computer processor or programmable logic device (PLD), can easily by the working technician of software or electronic technology, be prepared according to instruction of the present disclosure.
In certain embodiments, the present invention includes and have computer instruction or software code is stored in computer-readable storage medium wherein, it can be used for programmed computer or microprocessor, to carry out any processing of the present invention.Storage medium can comprise, but be not limited to: floppy disk, CD, Blu-ray Disc, DVD, CD-ROM, magneto-optic disk, ROM, RAM, flash memory device or be applicable to stored medium or the device of any type of instruction, code and/or data.
For the purpose of illustration and description, provide description above of the present invention.Its object is not exhaustive or the present invention is defined as to disclosed precise forms.For this area working technician, obviously can carry out many modifications and variations.
In order to explain best principle of the present invention and practical application thereof, select and described these embodiment, thereby for various embodiment and be suitable for the various modifications of certain expected purposes, can make those skilled in the art understand the present invention.Object is that scope of the present invention is defined by claims and their equivalent.
Claims (20)
1. for a focalizer for fluorescent microscope, comprising:
Excitation source, for launching exciting light with excited sample;
The first beam splitter, for reflecting exciting light by object lens to sample, and the fluorescence that sent by sample of transmission; With
Focus module, between excitation source and the first beam splitter, wherein focus module comprises reflective optical device and the focal imaging camera for light reflection, wherein in the path of the exciting light of reflective optical device between excitation source and the first beam splitter;
Wherein sample to the first beam splitter reflect, scattering or reflection and scattering first exciting light backward backward; and the first beam splitter is to reflective optical device reflection first exciting light, and reflective optical device is to focal imaging camera reflection second portion exciting light; With
Wherein be focused at least one excitation light image that the second portion exciting light of catching as camera is formed for focusing on the sample of fluorescence imaging.
2. focalizer according to claim 1, wherein reflective optical device is the second beam splitter, and second portion exciting light is a part for first's exciting light.
3. focalizer according to claim 1, wherein reflective optical device is catoptron, and second portion exciting light is identical with first exciting light.
4. focalizer according to claim 1, further comprises:
Collector lens between the first beam splitter and reflective optical device.
5. focalizer according to claim 1, further comprises:
Collector lens between excitation source and reflective optical device.
6. focalizer according to claim 5, wherein focus module also comprises the tube lens between reflective optical device and focal imaging camera.
7. focalizer according to claim 1, further comprises:
Exciter filter between excitation source and the first beam splitter.
8. focalizer according to claim 7, further comprises:
More than one exciter filter and more than one the first beam splitter, to form more than one combination of filters optical block, to carry out multichannel focusing.
9. focalizer according to claim 1, further comprises:
CPU (central processing unit), for determining the focal plane of fluoroscopic image according to excitation light image.
10. for a focalizer for fluorescent microscope, comprising:
Excitation source, for launching exciting light with excited sample;
Beam splitter, for reflecting exciting light by the first object lens to sample, and the fluorescence that sent by sample of transmission; With
The focus module that is positioned at sample below, wherein focus module comprises convergent lens and the focal imaging camera for converging light, wherein convergent lens is between sample and focal imaging camera;
Wherein a part of exciting light transmission is by sample, and this part exciting light propagates to convergent lens, and assembles to focal imaging camera by convergent lens; And
Wherein be focused at least one excitation light image that this part exciting light of catching as camera is formed for focusing on the sample of fluorescence imaging.
11. focalizers according to claim 10, wherein convergent lens is the second object lens.
12. focalizers according to claim 10, further comprise:
Exciter filter between excitation source and beam splitter.
13. focalizers according to claim 12, further comprise:
More than one exciter filter and more than one beam splitter, to form more than one combination of filters optical block, carry out multichannel focusing.
14. focalizers according to claim 10, further comprise:
CPU (central processing unit), for determining the focal plane of fluoroscopic image according to excitation light image.
15. 1 kinds of focus methods for fluorescent microscope, comprising:
By object lens, send exciting light, be used for irradiating sample, wherein exciting light is reflected towards sample by the first beam splitter;
Carry out below any:
By sample to the first beam splitter reflect, scattering or reflection and scattering first exciting light backward backward; by the first beam splitter, to reflective optical device, reflect first's exciting light, and to focal imaging camera, reflect second portion exciting light by reflective optical device; Or
The transmission of third part exciting light is passed through to sample, and assemble third part exciting light to focal imaging camera; With
By the focal imaging camera that is used for focusing on fluoroscopic image, with second portion exciting light or third part exciting light, form at least one excitation light image.
16. focus methods according to claim 15, further comprise:
Change the distance between object lens and sample, and repeat above-mentioned steps, until determined the focal plane for the sample of fluorescence imaging.
17. focus methods according to claim 15, further comprise:
The fluorescence of launching to the transmission of fluorescence imaging camera from sample via the first beam splitter, is used to form at least one fluoroscopic image.
18. focus methods according to claim 15, further comprise:
The step while with forming excitation light image, catches at least one fluoroscopic image with the fluorescence sending from sample by fluorescence imaging camera, to carry out prefocus, wherein the visual field of fluoroscopic image and the visual field of excitation light image are different.
19. focus methods according to claim 15, wherein reflective optical device is the second beam splitter, and second portion exciting light is a part for first's exciting light.
20. focus methods according to claim 15, wherein reflective optical device is catoptron, and second portion exciting light is identical with first exciting light.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH11183381A (en) * | 1997-12-22 | 1999-07-09 | Bunshi Bio Photonics Kenkyusho:Kk | Fluorescence microscope evaluating method and device |
| US20050056767A1 (en) * | 2001-08-06 | 2005-03-17 | Eran Kaplan | Image focusing in fluorescent imaging |
| CN101151378A (en) * | 2005-03-30 | 2008-03-26 | 奥林巴斯株式会社 | Predermined site luminescence measuring method, predetermined site luminenscence measuring apparatus, expression amount measuring method, and measuring apparatus |
| US20130128025A1 (en) * | 2010-07-28 | 2013-05-23 | Leica Microsystems Cms Gmbh | Device and Method for Acquiring a Microscopic Image of a Sample Structure |
| US20130229493A1 (en) * | 2010-09-17 | 2013-09-05 | Japan Science And Technology Agency | Three-dimensional confocal microscopy apparatus and focal plane scanning and aberration correction unit |
-
2013
- 2013-09-27 CN CN201310450223.3A patent/CN103513411B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH11183381A (en) * | 1997-12-22 | 1999-07-09 | Bunshi Bio Photonics Kenkyusho:Kk | Fluorescence microscope evaluating method and device |
| US20050056767A1 (en) * | 2001-08-06 | 2005-03-17 | Eran Kaplan | Image focusing in fluorescent imaging |
| CN101151378A (en) * | 2005-03-30 | 2008-03-26 | 奥林巴斯株式会社 | Predermined site luminescence measuring method, predetermined site luminenscence measuring apparatus, expression amount measuring method, and measuring apparatus |
| US20130128025A1 (en) * | 2010-07-28 | 2013-05-23 | Leica Microsystems Cms Gmbh | Device and Method for Acquiring a Microscopic Image of a Sample Structure |
| US20130229493A1 (en) * | 2010-09-17 | 2013-09-05 | Japan Science And Technology Agency | Three-dimensional confocal microscopy apparatus and focal plane scanning and aberration correction unit |
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| CN111208106A (en) * | 2013-12-16 | 2020-05-29 | 克罗姆尼贡公司 | Microscope system and method for detecting fluorescence emitted from sample using microscope system |
| CN106164736A (en) * | 2014-05-30 | 2016-11-23 | 斯坦福大学托管董事会 | For big intact tissue sample being carried out the method and device of imaging |
| US10746981B2 (en) | 2014-05-30 | 2020-08-18 | The Board Of Trustees Of The Leland Stanford Junior University | Methods and devices for imaging large intact tissue samples |
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| JPWO2019111440A1 (en) * | 2017-12-06 | 2020-12-10 | 國昭 永山 | Microscopic observation method and transmission microscope device |
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| CN108152290A (en) * | 2017-12-24 | 2018-06-12 | 大连益盛达智能科技有限公司 | A kind of display module detection of particles camera mechanism |
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