CN105629453A - Fluorescence microscope - Google Patents
Fluorescence microscope Download PDFInfo
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- CN105629453A CN105629453A CN201610136068.1A CN201610136068A CN105629453A CN 105629453 A CN105629453 A CN 105629453A CN 201610136068 A CN201610136068 A CN 201610136068A CN 105629453 A CN105629453 A CN 105629453A
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- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/06—Means for illuminating specimens
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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
- G01N21/645—Specially adapted constructive features of fluorimeters
- G01N21/6456—Spatial resolved fluorescence measurements; Imaging
- G01N21/6458—Fluorescence microscopy
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- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/0004—Microscopes specially adapted for specific applications
- G02B21/002—Scanning microscopes
- G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
- G02B21/0052—Optical details of the image generation
- G02B21/0076—Optical details of the image generation arrangements using fluorescence or luminescence
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Abstract
The invention provides a fluorescence microscope, which comprises a laser source, a laser gathering module, a dichroic beam combiner module and a filter module, wherein the laser source is provided with N different output modes, and in each output mode, the laser source outputs laser of one wavelength; the laser gathering module is used for receiving incident laser, gathering the laser on a sample to excite the sample to generate fluorescence whose wavelength is related to that of the laser; the dichroic beam combiner module is located between the laser source and the laser gathering module and comprises N dichroic beam combiners, and each dichroic beam combiner is used for reflecting the laser of one wavelength to the laser gathering module and transmitting the fluorescence whose wavelength is related to that of the laser; the filter module is used for filtering the fluorescence transmitted by the dichroic beam combiner module; and the filter module comprises N filters, and each filter is used for transmitting the fluorescence of one wavelength. Thus, better observation effects can be obtained.
Description
Technical field
It relates to optical technical field, it is specifically related to a kind of fluorescent microscope.
Background technology
Fluorescent microscope is the pointolite utilizing a high-luminous-efficiency, the light sending certain wavelength through colour filter system is as excitation light source, after exciting the fluorescence that the fluorescent substance in testing sample launches various different colours, then observed by the amplification of object lens and eyepiece. Like this strong under lining background, even if fluorescence is very faint is also highly susceptible to identification, susceptibility height, it is possible to for the research of cellularstructure and function, biochemical composition etc.
The mercury lamp that the excitation light source of traditional fluorescent microscope is all-wave length excites, all fluorescent substances in this excitation wavelength range are all excited, when needing the fluorescence excitation seeing certain specific fluorescent substance, need by a colour filter, this colour filter through the fluorescence of a kind of wavelength, then can only observe the target fluorescence excitation through colour filter by eyepiece.
As main drawback during excitation light source, mercury lamp is that all-wave length excites, except the specific fluorescent material of excited sample generates except specific fluorescent, also can generate other the assorted fluorescence that some we do not need by other fluorescent substance in excited sample; Fluorescence is through colour filter, colour filter not only weakens or prevents other assorted fluorescence, also weaken the fluorescence intensity that we need simultaneously, and by colour filter only within the scope of one, the exciting light of the fluorescent substance that such wavelength is close also can pass through colour filter, is just impacted by real fluorescent signal during observation.
It should be noted that, in information disclosed in above-mentioned background technology segment only for strengthening the understanding to background of the present disclosure, therefore can comprise the information not formed prior art known to persons of ordinary skill in the art.
Summary of the invention
Object of the present disclosure is to provide a kind of fluorescent microscope, for overcoming one or more problem caused due to restriction and the defect of correlation technique at least to a certain extent.
Other characteristics of the present disclosure and advantage become clear by by detailed description below, or the acquistion partially by practice of the present disclosure.
According to one side of the present disclosure, it is provided that a kind of fluorescent microscope, described fluorescent microscope comprises:
LASER Light Source, has N number of different output pattern, and under each described output pattern, described LASER Light Source exports the laser of a kind of wavelength;
Light-collecting module, for receiving incident described laser and be gathered on sample to excite described sample to generate the wavelength fluorescence relevant to the wavelength of described laser;
Dichroscope module, between described LASER Light Source and light-collecting module and comprise N number of dichroscope, each described dichroscope for the described laser that reflects a kind of wavelength to the fluorescence relevant to the wavelength of described laser of wavelength described in described light-collecting module and transmission;
Color filter module, for filtering the fluorescence of described dichroscope module transmission; Described color filter module comprises N number of color filter, and each described color filter is used for the described fluorescence through a kind of wavelength;
Detection module, for receiving the described fluorescence that described color filter module passes through.
In the exemplary embodiment of one of the present disclosure, described fluorescent microscope also comprises:
Laser beam expanding module, is arranged between described LASER Light Source and described dichroscope module, inputs to described dichroscope module for after the laser beam expanding that described LASER Light Source exported.
In the exemplary embodiment of one of the present disclosure, the power density after described laser beam expanding is 30Mw/cm2To 110Mw/cm2��
In the exemplary embodiment of one of the present disclosure, described fluorescent microscope also comprises:
Drive module, with described dichroscope module and color filter model calling, for when described LASER Light Source exports the laser of a kind of wavelength, driving corresponding dichroscope and color filter to move in the light path of described laser and fluorescence.
In the exemplary embodiment of one of the present disclosure, described fluorescent microscope also comprises:
Control module, with described LASER Light Source and drive model calling, for the output pattern of LASER Light Source described in the Attributions selection according to described sample and control dichroscope corresponding to described driving module drive and color filter moves to the light path of described laser and fluorescence.
In the exemplary embodiment of one of the present disclosure, described laser is 45 degree relative to the incident angle of described dichroscope module and described fluorescence relative to the incident angle of described dichroscope module.
In the exemplary embodiment of one of the present disclosure, described detection module comprises CCD sensor devices, and described CCD sensor devices is for being converted to electrical signal by the described fluorescent signal received and transfer to a treatment system.
In the exemplary embodiment of one of the present disclosure, the wavelength corresponding relation of described laser and the wavelength fluorescence relevant to the wavelength of described laser comprise following one or more:
520nm/492nm��570nm/554nm��615nm/588nm��565nm/555nm��520nm/490nm��565nm/552nm��667nm/650nm��620nm/596nm��
In the exemplary embodiment of one of the present disclosure, described dichroscope comprises the separating layer become by multi-layer dielectric film stack.
In the exemplary embodiment of one of the present disclosure, described color filter is the interference color filter become by multi-layer dielectric film stack.
Fluorescent microscope in example embodiment of the present disclosure, can so that often kind of fluorescent substance of sample with the laser of its single excitation wavelength as excitation light source, fluctuate up and down between �� 1nm, can greatly reduce other materials target substance is excited generate fluorescence interference, reduce the generation of assorted fluorescence, the light intensity of the laser that LASER Light Source produces is strong simultaneously, such target fluorescent material excites the fluorescent brightness of generation greatly to increase, and the fluorescent brightness observed through color filter also can increase greatly. Further, the present invention is provided with the excitation light source of different wave length, the dichroscope for different wave length laser and different fluorescence, the color filter for different fluorescence, fluorescent substance according to different sample, carry out selecting switching different LASER Light Source, dichroscope, color filter, thus realize the observation to different fluorescent material, and the observation to fluorescent substance different in same sample.
Should be understood that, it is only exemplary and explanatory that above general description and details hereinafter describe, and can not limit the disclosure.
Accompanying drawing explanation
Accompanying drawing herein is by being incorporated in specification sheets and forms the part of this specification sheets, shows and meets embodiment of the present disclosure, and is used from specification sheets one and explains principle of the present disclosure. It is appreciated that the accompanying drawing in the following describes is only embodiments more of the present disclosure, for those of ordinary skill in the art, under the prerequisite not paying creative work, it is also possible to obtain other accompanying drawing according to these accompanying drawings.
Fig. 1 is the structural representation of a kind of fluorescent microscope in disclosure example embodiment.
Description of reference numerals:
10 LASER Light Source
20 light-collecting modules
30 dichroscope modules
31 dichroscopes
40 color filter modules
41 color filter
50 detection modules
60 laser beam expanding modules
70 light collecting lenss
80 samples
81 Stage microscopes
Embodiment
Referring now to accompanying drawing, exemplary embodiment is more fully described. But, exemplary embodiment can be implemented in a variety of forms, and should not be understood to be limited to enforcement mode set forth herein; On the contrary, it is provided that these enforcement modes make the disclosure comprehensively with complete, and will pass on the design of exemplary embodiment to the technician of this area comprehensively. In the drawings, in order to clear, exaggerate, be out of shape or simplify shape size. Reference numeral identical in the drawings represents identical or similar structure, thus will omit their detailed description.
In addition, described feature, structure or step can be combined in one or more embodiment in any suitable manner. In the following description, it is provided that many details thus provide fully understanding embodiment of the present disclosure. But, it will be appreciated by persons skilled in the art that, it is possible to that puts into practice technical scheme of the present disclosure and do not have in described specific detail is one or more, or can adopt other method, step, structure etc.
This example embodiment provides a kind of fluorescent microscope. In this example embodiment, described fluorescent microscope can comprise LASER Light Source, light-collecting module, dichroscope module, color filter module and detection module. Wherein, described LASER Light Source has N number of different output pattern, and under each described output pattern, described LASER Light Source exports the laser of a kind of wavelength. Described light-collecting module is for receiving incident described laser and be gathered on sample the fluorescence to excite described sample generation wavelength relevant to the wavelength of described laser. Described dichroscope module is between described LASER Light Source and light-collecting module and comprises N number of dichroscope, each described dichroscope for the described laser that reflects a kind of wavelength to the fluorescence relevant to the wavelength of described laser of wavelength described in described light-collecting module and transmission. Described color filter module is for filtering the fluorescence of described dichroscope module transmission; Described color filter module comprises N number of color filter, and each described color filter is used for the described fluorescence through a kind of wavelength. The described fluorescence that described detection module passes through for receiving described color filter module.
By the fluorescent microscope in this example embodiment, can so that often kind of fluorescent substance of sample with the laser of its single excitation wavelength as excitation light source, fluctuate up and down between �� 1nm, can greatly reduce other materials target substance is excited generate fluorescence interference, reduce the generation of assorted fluorescence, the light intensity of the laser that LASER Light Source produces is strong simultaneously, such target fluorescent material excites the fluorescent brightness of generation greatly to increase, and the fluorescent brightness observed through color filter also can increase greatly. Further, the present invention is provided with the excitation light source of different wave length, the dichroscope for different wave length laser and different fluorescence, the color filter for different fluorescence, fluorescent substance according to different sample, carry out selecting switching different LASER Light Source, dichroscope, color filter, thus realize the observation to different fluorescent material, and the observation to fluorescent substance different in same sample.
Hereinafter, with reference to figure 1, the fluorescent microscope in this example embodiment is described in detail.
Shown in figure 1, described LASER Light Source 10 can comprise 5 kinds of different output patterns, and under each described output pattern, described LASER Light Source 10 exports the laser of a kind of wavelength. Citing, the LASER Light Source 10 in this example embodiment can provide 5 kinds that wavelength is respectively in 520nm, 570nm, 615nm, 565nm, 520nm, 565nm, 667nm, 620nm respectively; But it should be noted that, not as limit in this example embodiment, according to actual demand, described LASER Light Source 10 can also the corresponding laser providing other wavelength. In actually operating, it is possible to as required by the output pattern of switched laser light source 10 thus different wave length laser is provided, and open laser source every time and only radiate the laser of single wavelength. The switching of output pattern can by PC control such as computers, it is also possible to realize the switching of output pattern by arranging output pattern switching part in LASER Light Source 10. Described LASER Light Source 10 can be Solid State Laser light source (crystal and neodymium glass), gas laser light source (comprising atom, ion, molecule, quasi-molecule), liquid LASER Light Source (including organic dye, inorganic liquid, inner complex) and semiconductor laser light resource etc., and this does not do in this example embodiment special restriction.
Shown in Fig. 1, the described laser reflected from dichroscope 31 is received by light-collecting module 20 and is gathered in sample 80, thus the fluorescent substance exciting the described sample 80 being positioned on Stage microscope 81 generates the wavelength fluorescence relevant to the wavelength of described laser. The laser of different wave length can generate the fluorescence of specific wavelength by specific fluorescent material in excited sample 80; Citing, the corresponding relation that in the laser of different wave length and its excited sample 80, specific fluorescent material generates between the fluorescence of specific wavelength can comprise following one kind of multiple: 520nm/492nm, 570nm/554nm, 615nm/588nm, 565nm/555nm, 520nm/490nm, 565nm/552nm, 667nm/650nm, 620nm/596nm; But it should be noted that, not as limit in this example embodiment, according to actual demand, those skilled in the art can other corresponding relations of corresponding selection, it belongs to protection domain of the present disclosure equally.
Shown in Fig. 1, described dichroscope module 30 is between described LASER Light Source 10 and light-collecting module 20 and comprises 5 dichroscopes 31, each described dichroscope 31 for the described laser that reflects a kind of wavelength to the fluorescence relevant to the wavelength of described laser of wavelength described in described light-collecting module 20 and transmission. Optical maser wavelength that in this example embodiment, 5 dichroscopes 31 can reflect and can be respectively in 520nm/492nm, 570nm/554nm, 615nm/588nm, 565nm/555nm, 520nm/490nm, 565nm/552nm, 667nm/650nm, 620nm/596nm 5 kinds of the corresponding relation between the optical maser wavelength of transmission. But it should be noted that, not as limit in this example embodiment, according to actual demand, those skilled in the art can also select the dichroscope 31 of other reflections and transmission parameters. Laser described in this exemplary embodiment can be 45 degree relative to the incident angle of described dichroscope module 30 and described fluorescence relative to the incident angle of described dichroscope module 30. The separating layer that described dichroscope 31 can comprise glass substrate and be positioned on described glass substrate; Separating layer described in this example embodiment can be become by multi-layer dielectric film stack; Different reflections and transmission parameters can be realized by the thickness and the number of plies adjusting dielectric membranous layer. But it is understood that other available dichroscopes 31 belong to protection domain of the present disclosure equally.
Shown in Fig. 1, described color filter module 40 is between described dichroscope module 30 and detection module 50. The module of color filter described in this example embodiment 40 can comprise 5 color filter 41, and each described color filter 41 is for the described fluorescence through a kind of wavelength, and therefore described color filter 41 can eliminate assorted fluorescence. The specific wavelength fluorescence one_to_one corresponding that described color filter 41 and laser excitation sample 80 generate, the wavelength of fluorescence that 5 described color filter 41 can pass through can be five kinds in 492nm, 554nm, 588nm, 555nm, 490nm, 552nm, 650nm, 596nm. It should be noted that, passing through wavelength in this example embodiment is ��21It can transmission peak wavelength be �� that the fluorescence color filter 41 of (such as 492nm) is positioned at21In the light path of the emergent light of the dichroscope 31 of (such as 492nm); It is �� through wavelength22It can transmission peak wavelength be �� that the fluorescence color filter 41 of (such as 554nm) is positioned at22In the light path of the emergent light of the dichroscope 31 of (such as 554nm) ..., i.e. one_to_one corresponding between the two. In this exemplary embodiment, described color filter 41 can interference color filter 41 for becoming by multi-layer dielectric film stack; Mode but it should be noted that, color filter 41 described in this example embodiment can also be the color filter 41 of other types being made up of Tinted optical or coloured chemical glued membrane, cited by being not limited in this example embodiment.
Fluorescent microscope described in this example embodiment can also comprise driving module (not shown). Module is driven to be connected with described dichroscope module 30 and color filter module 40, for when described LASER Light Source 10 exports the laser of a kind of wavelength, driving corresponding dichroscope 31 and color filter 41 to move in the light path of described laser and fluorescence; Such as, when LASER Light Source 10 output wavelength is ��11During the laser of (such as 520nm), module drive reflection wavelength is driven to be ��11The laser of (such as 520nm) and transmission peak wavelength are ��21The dichroscope 31 of the fluorescence of (such as 492nm) and transmission peak wavelength are ��21The color filter 41 of the fluorescence of (such as 492nm) moves in the light path of described laser and fluorescence. Described driving module such as can comprise micromotor and power supply device etc.
Further, in order to obtain better Detection results, sample by driving the movement of the optical elements such as light-collecting module 20, dichroscope module 30, color filter module 40 described in module drive to make light beam move according to preset algorithm thus realize the fluorescence excitation collection of scan mode, and then can more comprehensively can also be detected by this example embodiment. In this example embodiment, described scan mode can be such as comb wave scanning or square wave scanning etc. Sample more comprehensively can be detected by above-mentioned scanning. But it should be noted that, in other exemplary embodiments of the present disclosure, it is also possible to by driving moving thus realizing the fluorescence excitation collection of scan mode of module drive Stage microscope 81, this is not done by this example embodiment special restriction.
In addition, fluorescent microscope described in this example embodiment can also comprise control module (not shown). Control module and described LASER Light Source 10 and drive model calling, for the output pattern of LASER Light Source 10 described in the Attributions selection according to described sample 80 and control dichroscope 31 corresponding to described driving module drive and color filter 41 moves to the light path of described laser and fluorescence. Described control module can be such as the upper computer such as computer, PLC (programmable logic controller). In other exemplary embodiments of the present disclosure, it is also possible to be carry out ACTIVE CONTROL as required by operator, this is not done special restriction by this example embodiment.
Shown in Fig. 1, the described fluorescence that described detection module 50 passes through for receiving described color filter module 40. Detection module 50 described in this exemplary embodiment can comprise CCD (ChargeCoupledDevice, Charge Coupled Device (CCD)) sensor devices, described CCD sensor devices, for the described fluorescent signal received is converted to electrical signal, then converts electrical signal to numerary signal by analog to digital converter and transfers to a treatment system; Described treatment system can be such as computer. In addition, in this example embodiment, it is also possible to utilize eyepiece to observe the fluorescence through described color filter 41 by naked eyes. In other exemplary embodiments of the present disclosure, described detection module 50 can also be the detection module of other types such as photomultiplier; In addition, described detection module 50 is except being used for realizing other purposes such as spectroscopic analysis for imaging; This is not all done special restriction by this example embodiment.
Shown in Fig. 1, owing to the laser beam of the outgoing of LASER Light Source 10 own is relatively thin, optical power density is higher, on the one hand, it is possible to causes surveyed area to diminish, affects detected result; On the other hand, it is easy to cause detection module 50 saturated, directly have influence on the quality of imaging; Another further aspect, laser energy is too high may be affected the work-ing life of other parts or sample is caused damage. Based on this, fluorescent microscope described in this example embodiment can also comprise laser beam expanding module 60. Described laser beam expanding module 60 is arranged between described LASER Light Source 10 and described dichroscope module 30, inputs to described dichroscope module 30 for after the laser beam expanding that described LASER Light Source 10 exported; In this example embodiment, described laser beam expanding module 60 can be such as beam expanding lens. It should be noted that, if laser power density is too low, the fluorescent signal that cell produces can not overcome background noise, and image quality is poor. Therefore power density after laser beam expanding is comparatively important, and in this example embodiment, the power density after described laser beam expanding can be 30Mw/cm2To 110Mw/cm2��
Those skilled in the art are it is understood that the fluorescent microscope in this example embodiment can also arrange other more parts as required; Such as, it is also possible to light collecting lens 70 etc. is set between color filter module 40 and detection module 50; Again such as, it is also possible between color filter module 40 and detection module 50, pin hole etc. is set. Therefore, it is not limited with this example embodiment.
In sum, by the fluorescent microscope in this example embodiment, can so that often kind of fluorescent substance of sample with the laser of its single excitation wavelength as excitation light source, fluctuate up and down between �� 1nm, can greatly reduce other materials target substance is excited generate fluorescence interference, reduce the generation of assorted fluorescence, the light intensity of the laser that LASER Light Source produces is strong simultaneously, such target fluorescent material excites the fluorescent brightness of generation greatly to increase, and the fluorescent brightness observed through color filter also can increase greatly. Further, the present invention is provided with the excitation light source of different wave length, the dichroscope for different wave length laser and different fluorescence, the color filter for different fluorescence, fluorescent substance according to different sample, carry out selecting switching different LASER Light Source, dichroscope, color filter, thus realize the observation to different fluorescent material, and the observation to fluorescent substance different in same sample.
The disclosure is described by above-mentioned related embodiment, but above-described embodiment is only enforcement example of the present disclosure. Must being pointed out that, the embodiment disclosed does not limit the scope of the present disclosure. On the contrary, not departing from spirit and scope of the present disclosure the change and retouching done, scope of patent protection of the present disclosure is all belonged to.
Claims (10)
1. a fluorescent microscope, it is characterised in that, described fluorescent microscope comprises:
LASER Light Source, has N number of different output pattern, and under each described output pattern, described LASER Light Source exports the laser of a kind of wavelength;
Light-collecting module, for receiving incident described laser and be gathered on sample to excite described sample to generate the wavelength fluorescence relevant to the wavelength of described laser;
Dichroscope module, between described LASER Light Source and light-collecting module and comprise N number of dichroscope, each described dichroscope for the described laser that reflects a kind of wavelength to the fluorescence relevant to the wavelength of described laser of wavelength described in described light-collecting module and transmission;
Color filter module, for filtering the fluorescence of described dichroscope module transmission; Described color filter module comprises N number of color filter, and each described color filter is used for the described fluorescence through a kind of wavelength;
Detection module, for receiving the described fluorescence that described color filter module passes through.
2. fluorescent microscope according to claim 1, it is characterised in that, described fluorescent microscope also comprises:
Laser beam expanding module, is arranged between described LASER Light Source and described dichroscope module, inputs to described dichroscope module for after the laser beam expanding that described LASER Light Source exported.
3. fluorescent microscope according to claim 2, it is characterised in that, the power density after described laser beam expanding is 30Mw/cm2To 110Mw/cm2��
4. fluorescent microscope according to claim 1, it is characterised in that, described fluorescent microscope also comprises:
Drive module, with described dichroscope module and color filter model calling, for when described LASER Light Source exports the laser of a kind of wavelength, driving corresponding dichroscope and color filter to move in the light path of described laser and fluorescence.
5. fluorescent microscope according to claim 4, it is characterised in that, described fluorescent microscope also comprises:
Control module, with described LASER Light Source and drive model calling, for the output pattern of LASER Light Source described in the Attributions selection according to described sample and control dichroscope corresponding to described driving module drive and color filter moves to the light path of described laser and fluorescence.
6. fluorescent microscope according to claim 1, it is characterised in that, described laser is 45 degree relative to the incident angle of described dichroscope module and described fluorescence relative to the incident angle of described dichroscope module.
7. fluorescent microscope according to claim 1, it is characterised in that, described detection module comprises CCD sensor devices, and described CCD sensor devices is for being converted to electrical signal by the described fluorescent signal received and transfer to a treatment system.
8. fluorescent microscope according to claim 1, it is characterised in that, the wavelength corresponding relation of described laser and the wavelength fluorescence relevant to the wavelength of described laser comprise following one or more:
520nm/492nm��570nm/554nm��615nm/588nm��565nm/555nm��520nm/490nm��565nm/552nm��667nm/650nm��620nm/596nm��
9. fluorescent microscope according to claim 1��8 any one, it is characterised in that, described dichroscope comprises the separating layer become by multi-layer dielectric film stack.
10. fluorescent microscope according to claim 1��8 any one, it is characterised in that, described color filter is the interference color filter become by multi-layer dielectric film stack.
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| CN107561676A (en) * | 2017-09-07 | 2018-01-09 | 江苏斯托利仪器仪表有限公司 | Multichannel fluorescent lighting device |
| WO2021129588A1 (en) * | 2019-12-25 | 2021-07-01 | 上海睿钰生物科技有限公司 | Fluorescent lighting device and microscopy imaging system |
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