WO2017000703A1 - 自动多通道类流式图像荧光分析系统 - Google Patents

自动多通道类流式图像荧光分析系统 Download PDF

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Publication number
WO2017000703A1
WO2017000703A1 PCT/CN2016/082770 CN2016082770W WO2017000703A1 WO 2017000703 A1 WO2017000703 A1 WO 2017000703A1 CN 2016082770 W CN2016082770 W CN 2016082770W WO 2017000703 A1 WO2017000703 A1 WO 2017000703A1
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light source
excitation light
fluorescent excitation
source device
sample
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PCT/CN2016/082770
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English (en)
French (fr)
Inventor
陈睿
颜海波
黄海清
罗浦文
夏浩涵
张莹莹
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上海睿钰生物科技有限公司
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Publication of WO2017000703A1 publication Critical patent/WO2017000703A1/zh

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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
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/06—Means for illuminating specimens
    • 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
    • G01N21/01—Arrangements or apparatus for facilitating the optical investigation
    • 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
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/16—Microscopes adapted for ultraviolet illumination ; Fluorescence microscopes
    • 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
    • G01N2021/6463—Optics
    • G01N2021/6471—Special filters, filter wheel
    • 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
    • G01N2021/6463—Optics
    • G01N2021/6478—Special lenses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2201/00—Features of devices classified in G01N21/00
    • G01N2201/06—Illumination; Optics
    • G01N2201/061—Sources
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2201/00—Features of devices classified in G01N21/00
    • G01N2201/06—Illumination; Optics
    • G01N2201/062—LED's
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/36—Microscopes arranged for photographic purposes or projection purposes or digital imaging or video purposes including associated control and data processing arrangements

Definitions

  • the invention relates to the field of fluorescence analysis technology, and more particularly to an automatic multi-channel flow image fluorescence analysis system.
  • Cell analysis is of great significance in basic biological research and clinical diagnostic applications. Cell analysis and research are subdivided into many directions and applications. From the perspective of quantity and nature, it can be divided into qualitative research and quantitative research.
  • Flow cytometry is based on this principle and is named after this.
  • the advantage of this method of forming a fast-flowing single-cell flow is to ensure that a large number of single-cell sample information is quickly acquired on the basis of obtaining individual cell information, and quantitative analysis is performed on this basis.
  • the shortcomings are also obvious. Due to the rapid flow of cells, it is difficult to achieve visual observation analysis by microscopy. Therefore, whether it is the Coulter principle or flow cytometry, electronic detection signals are collected for analysis. And statistics, the cell diameter and other indicators obtained in this way are calculated by the strength of the electrical signal and by the time. Whether this calculation is accurate or not, it is still consistent with the microscopic test to judge. Therefore, this analog signal is not intuitive, and because of this, the ability to eliminate impurity interference is limited. At the same time, only suspended cells can be detected by this method, and adherent cells cannot be detected by this method.
  • microscopic fluorescence observation and analysis methods are good at cell observation and qualitative research, but the quantitative analysis of classification, counting and other large samples is its weakness and difficulty.
  • the advantage of the flow fluorescence analysis method is to quickly realize the acquisition of a large number of cell samples and a variety of fluorescent characteristic signals, based on the principle of density function probability classification analysis, and finally achieve qualitative research on the sample.
  • the disadvantage of this method is that it cannot be obtained by microscopic observation. Under the imaging of a single cell, it is impossible to conduct in-depth observation and judgment of a single cell directly, and its judgment on the nature of the cell is a probabilistic judgment, and it is impossible to make an absolute judgment.
  • the present invention provides an automatic multi-channel flow image fluorescence analysis system capable of rapidly acquiring and analyzing imaging information of a large number of samples based on microscopic imaging technology, and analyzing by using a density function algorithm. At the same time, the morphological imaging analysis results of each cell and the stream-like data analysis results based on the density function of the large sample size are obtained.
  • An automatic multi-channel flow image fluorescence analysis system comprises: an integrated fluorescence excitation light source device, a sample stage device, a microscopic imaging device, an automatic control system and an analysis processing system; wherein:
  • the automatic control system is respectively connected to the integrated fluorescent excitation light source device, the sample stage device and the microscopic imaging device, and respectively controls the integrated fluorescent excitation light source device, the sample stage device and the microscopic imaging device;
  • the integrated fluorescent excitation light source device, the sample stage device and the microscopic imaging device constitute a microscopic optical system
  • the analytical processing system is coupled to the microscopic imaging device and analyzes the image acquired by the microscopic imaging device.
  • the integrated fluorescent excitation light source device comprises: a light source frame, a transmission bright field light source device vertically disposed at a center of the light source frame, and a fluorescent excitation light source device with two or more surrounding light source frames disposed obliquely on the light source frame ;
  • the light-emitting beam of the fluorescent excitation light source device and the light-emitting beam of the transmission bright-field light source device form a intersection at a certain position, and the fluorescent excitation light source device and the transmission bright-field light source device are connected to the automatic control system, and the transmission The bright field light source device and the fluorescent excitation light source device are controlled by the automatic control system to realize a combination of different light sources in which a single light source is sequentially opened and closed, a plurality of light sources are simultaneously turned on or off, and the light source intensity can be adjusted.
  • the fluorescence excitation light source device and the transmission bright field light source device are both located in the sample Above the stage device, a transmission bright field light source device is installed in the middle of the light source frame, and the fluorescence excitation light source device is distributed around the transmission bright field light source device centering on the transmission bright field light source.
  • the transmissive bright field light source device and the fluorescent excitation light source device are respectively located on upper and lower sides of the sample stage device, and the fluorescent excitation light source device is evenly distributed around the center of the light source frame around the periphery of the light source frame.
  • the integrated fluorescent excitation light source device further includes: a light source adjustment frame;
  • the fluorescent excitation light source device is fixedly mounted on the light source adjusting frame, and a side of the light source adjusting frame is provided with a rotating shaft, wherein the light source frame is provided with a shaft hole corresponding to the rotating shaft, and the rotating shaft is screwed through the shaft hole and an adjusting nut to realize Adjustment and fixation of the direction and position of the oblique fluorescent excitation light source device.
  • the fluorescent excitation light source device comprises:
  • the concentrating module is disposed between the monochromatic LED module and the band pass filter, and the adjustable fixing frame can adjust the monochromatic LED module and the concentrating module. The distance between them.
  • the fluorescent excitation light source device comprises:
  • the band pass filter is disposed between the monochrome LED module and the concentrating module, and the adjustable fixing frame can adjust the monochrome LED module and the concentrating module. The distance between them.
  • the concentrating module comprises a collecting lens or a lens group.
  • the single color LED module comprises at least one monochromatic LED bead.
  • the sample stage device comprises: a sample stage, a sample plate, and a first motor and a second motor;
  • the sample plate is mounted on the sample stage
  • the first motor and the second motor are coupled to the automatic control system, and the sample stage is driven to complete X-axis and Y-axis movement under the control of the automatic control system to automatically replace the sample field of view in the sample plate.
  • the sample plate is composed of a double-sided transparent slide and a spacer structure
  • the spacing structure forms a micro-slot groove of independent fixed distance in the middle of the transparent glass plate, and the sample filling hole is opened at both ends of the micro-slot groove.
  • the microscopic imaging device comprises: an objective lens, a lens barrel, a third motor, an emission light filter module and an image acquisition device; wherein:
  • the objective lens and the image acquiring device are respectively fixed at both ends of the lens barrel;
  • the emission filter module is fixed on the lens barrel and located between the objective lens and the image acquisition device;
  • the image acquisition device is connected to the analysis processing system
  • the third motor is respectively connected to the lens barrel and the automatic control system, and under the control of the automatic control system, the lens barrel and the objective lens are moved up and down to perform automatic adjustment of the measurement focal length;
  • the objective lens is configured to enlarge a preset multiple of a preset detection area value
  • the emission filter module is configured to filter the fluorescence of the sample particles in the predetermined detection area after being excited by the fluorescent excitation light;
  • the image obtaining device is configured to acquire an image of the preset detection area after being enlarged to a preset multiple, and send the image to the analysis processing system for analysis processing.
  • the emission light filter module comprises: a sealing upper disk, a sealing lower disk, a rotating disk, a filter, a filter snap ring, a turntable shaft and a fourth motor; wherein:
  • the sealed upper disc and the sealed lower disc are fixed at both ends of the turntable shaft at the center of the turntable;
  • the filter is fixed on the turntable by a filter snap ring;
  • the fourth motor is respectively connected to the turntable and the automatic control system, and the turntable is rotated under the control of the automatic control system to switch the filter.
  • the technical solution provided by the present invention has at least the following advantages:
  • the invention provides a fluorescence excitation light source device and system and a fluorescence microscope, comprising: an adjustable fixing frame; and a monochrome LED module, a concentrating module and a band pass filter fixed in the adjustable fixing frame
  • the concentrating module is disposed between the monochromatic LED module and the band pass filter, or the band pass filter is disposed on the monochromatic LED module and the concentrating mode
  • the adjustable fixture can adjust the distance between the monochrome LED module and the concentrating module.
  • the present invention provides an automatic multi-channel flow image fluorescence analysis system.
  • the sample stage device can be controlled by the automatic control system, and the sample stage device can be realized.
  • the sample in the sample is measured at different positions;
  • the microscopic imaging device is controlled by the automatic control system, and the automatic adjustment of the measurement focal length and the automatic switching of the filter can be realized;
  • the microscopic imaging device can be realized by the analysis processing system.
  • the image is analyzed and processed; in summary, the invention can rapidly acquire and analyze imaging information of a large number of samples based on microscopic imaging technology, and analyzes by using a density function algorithm to simultaneously obtain morphological imaging analysis results of each cell. And stream-like data analysis results based on a large sample size density function.
  • FIG. 1 is a schematic structural view of an automatic multi-channel flow image fluorescence analysis system according to the present disclosure
  • FIG. 2 is a schematic structural view of an integrated fluorescent excitation light source device according to the present invention.
  • FIG. 3 is a schematic structural view of another integrated fluorescent excitation light source device according to the present disclosure.
  • FIG. 4 is a schematic view showing a cut surface structure of a fluorescence excitation light source device according to the present invention.
  • FIG. 5 is a schematic view showing a sectional structure of another fluorescent excitation light source device according to the present invention.
  • FIG. 6 is a schematic structural view of a mechanical part of an automatic multi-channel flow image fluorescence analysis system according to the present disclosure
  • Figure 7 is a schematic structural view of a sample plate disclosed in the present invention.
  • FIG. 8 is a schematic structural diagram of a light emitting filter module according to the present disclosure.
  • an automatic multi-channel flow image fluorescence analysis system disclosed in an embodiment of the invention includes: an integrated fluorescence excitation light source device 11 , a sample stage device 12 , a microscopic imaging device 13 , and an automatic control system 14 and an analytical processing system 15; wherein:
  • the automatic control system 14 is respectively connected to the integrated fluorescent excitation light source device 11, the sample stage device 12 and the microscopic imaging device 13, and controls the integrated fluorescent excitation light source device 11, the sample stage device 12 and the microscopic imaging device 13, respectively;
  • the integrated fluorescent excitation light source device 11, the sample stage device 12 and the microscopic imaging device 13 constitute a microscopic optical system
  • the analytical processing system 15 is coupled to the microscopic imaging device 14 for performing an analytical process on the images acquired by the microscopic imaging device 13.
  • the sample stage device is controlled by the automatic control system 14. 12, enabling measurement of different positions of the sample in the sample stage device 12; controlling the microscopic imaging device 13 by the automatic control system 14, enabling automatic adjustment of the measurement focal length, and automatic switching of the filter; by analyzing the processing system 15 can realize the analysis and processing of the microscopic image collected by the microscopic imaging device 13; in summary, the invention can quickly acquire and analyze the imaging information of a large number of samples based on the microscopic imaging technology, and analyze the image using a density function algorithm. To obtain both morphological imaging results of each cell and stream-like data analysis results based on a large sample size density function.
  • one of the structures of the integrated fluorescent excitation light source device is as shown in FIG. 2, and includes: a light source frame 111, and a transmission bright field light source device 112 and 4 vertically disposed at the center of the light source frame 111. More than one center of the surrounding light source frame 111 is obliquely disposed on the light source frame 111 on the fluorescent excitation light source device 113;
  • the fluorescence excitation light source device 113 and the transmission bright field light source device 112 are both located above the sample plate (not shown), the transmission bright field light source device 112 is mounted in the middle of the light source frame 111, and the four fluorescent excitation light source devices 113 are transparent.
  • the field light source device 112 is centrally distributed around the transmission bright field light source device Around 112.
  • the fluorescent excitation light source device 113 is fixedly mounted on the light source adjusting frame 114.
  • the light source adjusting frame 114 is connected to the light source frame 111 through the rotating shaft 115 and the adjusting nut 116, and the relative position between the light source adjusting frame 114 and the light source frame 111 is adjusted, which can be conveniently adjusted.
  • the incident angle of the oblique fluorescent excitation light source device 113 is adjusted.
  • the adjusting nut 116 When using, first loosen the adjusting nut 116, adjust the incident angle of the oblique light source, adjust the appropriate angle according to the requirements of clear imaging and uniform background of the field of view, and then tighten the adjusting nut 116 to fix the position of the oblique light source.
  • the transmission bright field source device 112 is in the center position and the incident light is perpendicular to the sample plate (not shown). During the experiment, only the fluorescent excitation filter needs to be replaced to complete the fluorescence signal collection of the multi-color channel, and the switching of the oblique light source is automatically completed by the circuit control system.
  • FIG. 3 is different from the above-mentioned integrated fluorescent excitation light source device in that the fluorescent excitation light source device 113 and the transmission bright field light source are different.
  • the devices 112 are respectively located on the upper and lower sides of the sample plate 117, and the four fluorescent excitation light source devices 113 are evenly distributed around the center of the light source frame 111 at the peripheral edges of the transmission bright field light source device 112.
  • the number of the fluorescent excitation light source devices 113 is not limited to four, and may be increased according to actual conditions.
  • the angle of cooperation between the oblique light source (the light source emitted from the fluorescent excitation light source device 113) and the direct light source (the light source transmitted through the bright field light source device 112) can be arbitrarily changed as needed.
  • the fluorescent excitation lamp can be an LED lamp bead or a laser lamp.
  • a sectional structure of a fluorescent excitation light source device is as shown in FIG. 4, including: an adjustable fixing frame 100;
  • the concentrating module 300 is disposed between the monochromatic LED module 200 and the band pass filter 400, and the adjustable fixing frame 100 can adjust the distance between the monochromatic LED module 200 and the concentrating module 300.
  • the technical solution provided by the embodiment of the present application adopts the LED module as a fluorescent excitation light source, and the LED has the characteristics of short response time, low cost, long service life, small volume, etc., so that the fluorescent excitation light source device has Short response time, low cost, long service life and volume Small and other advantages. And, because the attenuation of LED light is fast and accurate, the phototoxicity can be greatly reduced under long-term living cell test, so that the fluorescence excitation light source device is beneficial to long-term living cell experiments.
  • the LED since the LED only needs 3W of power to realize the same wavelength of fluorescence excitation light provided by the mercury lamp, the xenon lamp, etc., compared with the mercury lamp requires 50W to 100W, and the xenon lamp requires 100W of power, the LED has The low energy consumption makes the fluorescence excitation light source device greatly reduce the power consumption.
  • the concentrating module is disposed at the front end of the monochromatic LED module, which not only makes the fluorescent excitation light source device have high brightness, but also can adjust the concentrating module and the monochromatic LED module through the adjustable fixing frame. The distance is adjusted to adjust the size of the light spot of the fluorescent excitation light source device, thereby adjusting the concentration of the excitation light source energy of the light emitted by the fluorescent excitation light source device.
  • a band pass filter is disposed at the front end of the monochromatic LED module to constrain the light emitting wavelength range of the fluorescent excitation light source device, thereby further improving the singularity of the fluorescent excitation light source emitted by the fluorescent excitation light source device.
  • the blue LED module has a peak wavelength range of 470 ⁇ 15 nm (including the endpoint value), and its corresponding bandpass filter can pass light with a wavelength range of 470 ⁇ 15 nm (including the endpoint value), so The pass filter can concentrate the wavelength of the fluorescent excitation source emitted by the fluorescence excitation source device between 465 nm and 485 nm (including the endpoint value), further improving the unity of the fluorescent excitation source emitted by the fluorescent excitation source device.
  • the concentrating module provided by the embodiment of the present invention includes, but is not limited to, a concentrating lens or a lens group. That is, the concentrating module provided by the embodiment of the present application may be a single concentrating lens, or may be more A lens group having a condensing function, which is composed of a lens, is not specifically limited in this application, and needs to be specifically designed according to actual needs. Moreover, the material of the lens in the condensing lens and the lens group is also not limited in the present application.
  • the monochrome LED module provided by the embodiment of the present application includes at least one single-color LED lamp bead.
  • the number of the single-color LED lamp bead in the LED module specific design is required according to the actual application, and the present application does not specifically limit the application.
  • the adjustable fixing frame provided by the embodiment of the present application is a sealed adjustable fixing frame, and the monochrome LED module and the concentrating module and The band pass filters are sealed and fixed in the adjustable holder.
  • the monochromatic LED module, the concentrating module and the band pass filter are both sealed and fixed in the adjustable fixing frame, so that all the fluorescent excitation light sources emitted by the monochromatic LED module are incident on the concentrating module, and then The fluorescent excitation light source of the concentrating module is all incident on the band pass filter, and then filtered and then emitted, thereby avoiding waste before the light of the fluorescent excitation light source device, and improving the light intensity of the fluorescent excitation light source device.
  • the present application does not specifically limit the shape of the adjustable fixing frame.
  • the method for adjusting the distance between the monochrome LED module and the collecting lens of the adjustable fixing frame is not specifically limited, and The actual application is designed specifically.
  • the concentrating module is disposed between the monochromatic LED module and the band pass filter.
  • the concentrating module can also Set on the side of the bandpass filter that faces away from the monochrome LED module.
  • FIG. 5 a schematic diagram of a cross-sectional structure of a fluorescent excitation light source device according to another embodiment of the present application, wherein the fluorescent excitation light source device includes:
  • Adjustable fixing frame 100
  • the band pass filter 400 is disposed between the monochromatic LED module 200 and the concentrating module 300, and the adjustable mount 100 can adjust the distance between the monochromatic LED module 200 and the concentrating module 300.
  • the technical solution provided by the embodiment of the present application adopts the LED module as a fluorescent excitation light source, and the LED has the characteristics of short response time, low cost, long service life, small volume, etc., so that the fluorescent excitation light source device has Short response time, low cost, long service life and small size. And, because the attenuation of LED light is fast and accurate, the phototoxicity can be greatly reduced under long-term living cell test, so that the fluorescence excitation light source device is beneficial to long-term living cell experiments.
  • the LED since the LED only needs 3W of power to realize the same wavelength of fluorescence excitation light provided by the mercury lamp, the xenon lamp, etc., compared with the mercury lamp requires 50W to 100W, and the xenon lamp requires 100W of power, the LED has The low energy consumption makes the fluorescence excitation light source device greatly reduce the power consumption.
  • the concentrating module is disposed at the front end of the monochromatic LED module, which not only makes the fluorescent excitation light source device have high brightness, but also can adjust the concentrating module and the monochromatic LED module through the adjustable fixing frame. The distance is adjusted to adjust the size of the light spot of the fluorescent excitation light source device, thereby adjusting the concentration of the excitation light source energy of the light emitted by the fluorescent excitation light source device.
  • a band pass filter is disposed at the front end of the monochromatic LED module to constrain the light emitting wavelength range of the fluorescent excitation light source device, thereby further improving the singularity of the fluorescent excitation light source emitted by the fluorescent excitation light source device.
  • the blue LED module has a peak wavelength range of 470 ⁇ 15 nm (including the endpoint value), and its corresponding bandpass filter can pass light with a wavelength range of 470 ⁇ 15 nm (including the endpoint value), so The pass filter can concentrate the wavelength of the fluorescent excitation source emitted by the fluorescence excitation source device between 465 nm and 485 nm (including the endpoint value), further improving the unity of the fluorescent excitation source emitted by the fluorescent excitation source device.
  • the concentrating module provided by the embodiment of the present invention includes, but is not limited to, a concentrating lens or a lens group. That is, the concentrating module provided by the embodiment of the present application may be a single concentrating lens, or may be more A lens group having a condensing function, which is composed of a lens, is not specifically limited in this application, and needs to be specifically designed according to actual needs. Moreover, the material of the lens in the condensing lens and the lens group is also not limited in the present application.
  • the monochrome LED module provided by the embodiment of the present application includes at least one single-color LED lamp bead.
  • the number of the single-color LED lamp bead in the LED module specific design is required according to the actual application, and the present application does not specifically limit the application.
  • FIG. 6 is a schematic structural diagram of a mechanical part of an automatic multi-channel flow image fluorescence analysis system according to the present invention, comprising: an integrated fluorescence excitation light source device 601, a sample stage 602, a sample plate 603, a first motor 604, a second motor 605, an objective lens 606, a lens barrel 607, a third motor 608, an emission light filter module 609, a fourth motor 610, and an image acquisition device 611;
  • the integrated fluorescent excitation light source device 601 is controlled by an automatic control system to realize a combination of different light sources in which a single light source is sequentially opened and closed, a plurality of light sources are simultaneously turned on or off, and the light source intensity can be adjusted;
  • the sample plate 603 is mounted on the sample stage 602;
  • the first motor 604 and the second motor 605 are connected to the automatic control system, and the sample stage 602 is driven to complete the X-axis and the Y-axis movement under the control of the automatic control system to perform automatic replacement of the sample field of view in the sample plate 603;
  • the objective lens 606 and the image acquiring device 611 are respectively fixed to both ends of the lens barrel 607;
  • the emission filter module 609 is fixed on the lens barrel 607, and is located at the objective lens 606 and the image acquisition device 611. between;
  • the image acquisition device 611 is connected to the analysis processing system
  • the third motor 608 is respectively connected to the lens barrel 607 and the automatic control system, and under the control of the automatic control system, the lens barrel 607 and the objective lens 606 are moved up and down to perform automatic adjustment of the measurement focal length;
  • the objective lens 606 is configured to enlarge a preset multiple of the preset detection area value
  • the emission filter module 609 is configured to filter the fluorescence of the sample particles in the preset detection area after being excited by the fluorescent excitation light;
  • the fourth motor 610 of the emission filter module 609 switches the filter in the emission filter module 609 under the control of the automatic control system
  • the image obtaining device 611 is configured to acquire an image of the preset detection area after being enlarged to a preset multiple, and send the image to the analysis processing system for analysis processing.
  • the sample plate is composed of a double-sided transparent slide 711 and a spacing structure 712 ;
  • the spacer structure 712 forms a micro slit slot 713 of a fixed fixed distance in the middle of the transparent slide 711, and sample filling holes 714 are formed at both ends of the micro slit groove 713.
  • the sample plate is composed of two transparent glass materials and a plurality of spacer structures, and a micro slit groove (50 ⁇ m to 300 ⁇ m) is formed between the two transparent materials at a fixed distance, and sample filling holes are formed at both ends of the micro slit groove. After the sample is injected through the sample filling hole, it is evenly distributed in the micro slit groove to form a predetermined thin layer of liquid of a certain thickness. Since the height of the metering microslits is fixed, the volume of the observation or imaging field of view can be obtained by measuring the observed area of the observation or photographing field of view* the height of the microslot, and then the following formula can be used to convert the volume. Sample concentration:
  • Sample concentration number of fields of view (cells) * 1 mL / field of view * microslot height.
  • the sample does not flow after the filling and the detection process, and even the original adherence state of the cell, such a state has no influence of the flow rate and the pressure, and the cell state and the detection result are more in line with the original state of the sample.
  • FIG. 8 it is a schematic structural diagram of the light emitting filter module, including: a sealing upper disk 801 , a sealing lower disk 802 , a rotating disk 803 , a filter 804 , a filter retaining ring 805 , and a rotating shaft 806 . And a fourth motor (not shown); wherein:
  • the sealing upper disc 801 and the sealing lower disc 802 are fixed at both ends of the rotating shaft 806 at the center of the turntable 803;
  • the filter 804 is fixed on the turntable 803 through the filter retaining ring 805;
  • the fourth motor is respectively connected to the turntable 803 and the automatic control system, and under the control of the automatic control system, the turntable 803 is rotated to switch the filter.
  • the computer is connected to the automatic control system for communication, and the computer generates an automatic control flow program according to the manual instruction or the automatic operation programming instruction, and automatically controls the computer through interaction with the instruction information of the automatic control system and the image acquisition device.
  • the automatic control system is connected to each light source lamp of the integrated fluorescent excitation light source device, supplies power thereto, and issues commands such as switching and adjusting voltage and current to control the opening and closing of each light source lamp.
  • the sample stage device is driven by a first motor and a second motor disposed on the X and Y axes, and the electric first motor and the second motor are connected to the automatic control system, and receive an instruction of the automatic control system to complete the X axis of the sample stage.
  • the lens barrel of the system is fixed on the vertical motor-moving sliding table driven by the third motor, and the third motor is connected with the automatic control system, and receives the instruction of the automatic control system to complete the axial reciprocating movement, and drives the lens barrel and the objective lens. Move up and down, and finally realize the function of automatically adjusting the measuring focal length according to the instruction.
  • the movable switching light (cut-off light) filter group of the system and the bracket thereof are driven by the fourth motor, the fourth motor and the automatic
  • the control system is connected and receives an instruction from the automatic control system to complete the switching of the emitted optical filter.
  • the image acquisition device of the system is connected with a computer, and is acquired by the instruction of the computer and transmitted to the computer to realize the function of acquiring the microscopic image.
  • the data analysis system of the system is an analysis processing system placed in a computer.
  • the analysis processing system mainly includes the following software function modules:
  • Electronic image generation and optimization function Through this function, the electronic signals acquired by the image acquisition device can be generated into electronic images of various formats (PNG, jpg, tif, etc.). Image optimization such as noise removal and fluorescence background removal are performed on the electronic image.
  • Multi-image fusion function On the basis of the above a, a plurality of pictures taken at the same position of the same sample can be fused into one picture by arbitrarily combining two or more multiple pictures.
  • Intelligent image recognition function Through the automatic image recognition program, it is possible to automatically analyze various shapes of particles (cells) in the image, and calculate the position, shape, size, agglomeration, fluorescence, and whether each particle (cell) is excited at the same time.
  • the intensity of various fluorescences accumulated fluorescence intensity, average fluorescence intensity), fluorescence position, area of fluorescence, fluorescence diameter, shape of fluorescence, maximum fluorescence value, minimum fluorescence value, fluorescence drop value, fluorescence edge Smoothness and other information, based on the image identification information, automatically classify the samples according to the screening rules, and classify and calculate the number, average area, average diameter, average fluorescence intensity, average fluorescence area, average fluorescence diameter, etc. index.
  • Asynchronous multi-channel fluorescence image analysis function Multi-channel fluorescence image analysis of asynchronous single channel, dual channel, three channel, four channel... can be completed.
  • Synchronous multi-channel fluorescence image analysis function Synchronous multi-channel fluorescence image analysis such as simultaneous dual channel, three channel and four channel can be completed.
  • f automatic statistical analysis function: based on c, d, e, according to the logical structure of various experiments, automatically calculate the sample concentration, viability (mortality), transfection rate, apoptotic rate, the proportion of each cell cycle Wait for the final result.
  • the invention can preset a large sample size collection limit value (for example, 10,000 cell samples) like a flow cytometer, and automatically acquire multiple visual field sample images through an automatic control system.
  • the software system automatically analyzes the images of the multiple fields of view, and uses multi-threaded computer technology to analyze and calculate while acquiring image signals, so as to realize the collection and analysis of a large number of quantitative samples quickly (within 3 minutes). It solves the problem that it is difficult to quickly analyze a large number of samples under the microscopic observation method.
  • Custom editing function The software of the invention comprises a custom editing module, which can customize the editing experiment process, the device running process, the image analysis method, and each according to different fluorescence experiment requirements (such as sampling amount, fluorescence channel, and analysis method). Parameters and thresholds, data calculation formulas, result presentation names and formats, interface formats, and automatic generation and saving of new standard experiment modules. This function breaks through the traditional analytical instrument curing function and process ideas, allowing users to innovate experimental processes and methods, and boldly explore new methods and attempts.

Abstract

一种自动多通道类流式图像荧光分析系统，包括：集成式荧光激发光源装置(11)、样品台装置(12)、显微成像装置(13)、自动控制系统(14)和分析处理系统(15)；其中：自动控制系统(14)分别与集成式荧光激发光源装置(11)、样品台装置(12)和显微成像装置(13)相连，分别对集成式荧光激发光源装置(11)、样品台装置(12)和显微成像装置(13)进行控制；集成式荧光激发光源装置(11)、样品台装置(12)和显微成像装置(13)构成显微光学系统；分析处理系统(15)与显微成像装置(13)相连，对显微成像装置(13)采集的图像进行分析处理。能够以显微成像技术为基础、快速采集和分析大量样品的成像信息，并采用密度函数的算法进行分析，以同时获得每个细胞的形态影像学分析结果和基于大样本量的密度函数的类流式数据分析结果。

Description

自动多通道类流式图像荧光分析系统
本申请要求于2015年07月01日提交中国专利局、申请号为201510376530.0、发明名称为“集成式荧光激发光源装置”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。本申请要求于2015年07月28日提交中国专利局、申请号为201510452591.0、发明名称为“自动多通道类流式图像荧光分析系统”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明涉及荧光分析技术领域，更为具体的说，涉及一种自动多通道类流式图像荧光分析系统。
背景技术
细胞分析在生物学基础研究和临床诊断应用中都具有非常重要的意义，细胞的分析和研究又细分为很多的方向和应用，从数量和性质的角度，可分为定性研究与定量研究。
现有技术中，对于细胞的定性研究，是从传统用显微镜观察和测量开始的，通过对细胞的大小、死活、形状、内部结构等通过光学可见的特征来进行细胞性质的判断。随着现代荧光标定和检测技术的发展，用荧光试剂对细胞或细胞内部特定组成目标进行染色，然后，采用荧光显微镜进行观察，这给细胞观察和分析增加了很多荧光属性指针，可以更深入和精确的对细胞性质进行分辨和判断。显微镜的放大倍数越大，定性研究可以越准确和深入。但同时的弊端是，单一视野能够观察到的数量就会越少，因此，为了比较准确的进行数量统计，需要借助血球计数板，在该计数板上预先用线条标刻了固定面积的区域，然后，通过不断移动显微镜的样品台，以实现多个视野对需要统计区域的全覆盖，然后，用人工计数统计的方法，统计出一定面积下的各种类型细胞的数量，再通 过公式计算出浓度、比例等数量指标。这样的计数方法，操作复杂，耗时长、人工误差大，并且，无法实现对每个细胞的大小、荧光强度等特征进行精确分析。总体来说，荧光显微观察方法的长处是能够让细胞特征以可见的方式直接呈现，是对单个细胞及细胞内部特征进行定性观察和研究最基础和最重要的方法。但是，这一方法难以实现对大量样本的精确和快速分析，开展定量研究是这一方法的弱点和难点。
为解决快速细胞定量分析的难题，自20世纪30年代开始，科学家对细胞计数的方法进行了不懈的研究和改良，现在比较广泛应用的技术方案，是让细胞以单个的方式快速通过特定检测区域，以实现快速计数和分析。为了得到单个排列的细胞流，一种方法是采用细胞流通过比细胞直径略大的毛细管的方式来实现，这一方式的缺点是对不同大小的细胞需要更换相应的毛细管，对于大小不一和成团的细胞分析精度降低，目前这一方法在库尔特计数方法中仍然被广泛运用。另一种方法，是运用分层鞘流原理，用鞘液流来形成单个排列并快速通过的细胞流，流式细胞分析技术就是基于这一原理，并以此命名。这种形成快速流动的单细胞流的方法优势是可以确保在获得单个细胞信息的基础上，快速采集大量单细胞样本信息，并在此基础开展定量分析。但缺点也很明显，由于细胞的快速流动通过，这让采用显微镜方法进行可视观察分析难以实现，因此，不管是库尔特原理，还是流式细胞术，均是采集电子检测信号来进行分析和统计，这种方式下得出的细胞直径等指标，是通过电信号强弱、通过时间来进行模拟计算的，这种计算是否准确，最终还是要与显微镜检测的一致来进行评判。因此，这种模拟信号并不直观，也因为此，排除杂质干扰的能力也就有限。同时，用这种方法只能检测悬浮细胞，贴壁细胞无法采用这种方法进行检测。
综上所述，显微荧光观察和分析方法擅长细胞观察和定性研究，但对于大样本的分类、计数等定量分析是其弱点和难点。流式荧光分析方法的优势是快速实现对大量细胞样品、多种荧光特征信号的采集，基于密度函数概率分类分析原理，最终达到对样本的定性研究，这种方法的缺点是无法获得如显微镜观察下的单个细胞的成像，无法直接对单个细胞进行深入的观察研究和判断，其对细胞性质的判断是一种概率判断，而无法做到绝对判断。
因此，在细胞的分析过程中，如何解决显微镜方法难以大量采集样本并开展基于密度函数的类流式分析，是一项亟待解决的问题。
发明内容
有鉴于此，本发明提供了一种自动多通道类流式图像荧光分析系统，能够以显微成像技术为基础、快速采集和分析大量样品的成像信息，并采用密度函数的算法进行分析，以同时获得每个细胞的形态影像学分析结果和基于大样本量的密度函数的类流式数据分析结果。
为实现上述目的，本发明提供的技术方案如下：
一种自动多通道类流式图像荧光分析系统，包括：集成式荧光激发光源装置、样品台装置、显微成像装置、自动控制系统和分析处理系统；其中：
所述自动控制系统分别与所述集成式荧光激发光源装置、样品台装置和显微成像装置相连，分别对所述集成式荧光激发光源装置、样品台装置和显微成像装置进行控制；
所述集成式荧光激发光源装置、样品台装置和显微成像装置构成显微光学系统；
所述分析处理系统与所述显微成像装置相连，对所显微成像装置采集的图像进行分析处理。
优选地，所述集成式荧光激发光源装置包括：光源架、1个垂直设置于光源架中心的透射明场光源装置及2个以上环绕光源架的中心倾斜设置于光源架上的荧光激发光源装置；
所述荧光激发光源装置所发光束与透射明场光源装置所发光束在某一位置形成交汇，所述荧光激发光源装置、所述透射明场光源装置均与所述自动控制系统相连接，透射明场光源装置与荧光激发光源装置通过所述自动控制系统控制，以实现单一光源顺序间隔开启和关闭、多种光源同时开启或关闭的不同光源组合，及实现光源光强可调整。
优选地，所述荧光激发光源装置与所述透射明场光源装置均位于所述样品 台装置的上方，透射明场光源装置安装于所述光源架的中部，荧光激发光源装置以透射明场光源为中心环绕分布于透射明场光源装置的周围。
优选地，所述透射明场光源装置与所述荧光激发光源装置分别位于所述样品台装置的上下两侧，荧光激发光源装置环绕光源架的中心均匀分布于光源架的四周边缘。
优选地，所述集成式荧光激发光源装置还包括：光源调整架；
所述荧光激发光源装置固定安装于该光源调整架上，光源调整架的侧面设置有转轴，所述光源架对应于转轴设置有轴孔，转轴穿过轴孔与一调节螺母螺接，以实现对斜射的荧光激发光源装置的方向与位置的调节及固定。
优选地，所述荧光激发光源装置包括：
可调整固定架；
以及，固定于所述可调整固定架中的单色LED模组、聚光模组和带通滤光片；
其中，所述聚光模组设置于所述单色LED模组和带通滤光片之间，且所述可调整固定架能够调整所述单色LED模组和所述聚光模组之间的距离。
优选地，所述荧光激发光源装置包括：
可调整固定架；
以及，固定于所述可调整固定架中的单色LED模组、聚光模组和带通滤光片；
其中，所述带通滤光片设置于所述单色LED模组和聚光模组之间，且所述可调整固定架能够调整所述单色LED模组和所述聚光模组之间的距离。
优选地，所述聚光模组包括聚光透镜或透镜组。
优选地，所述单色LED模组包括至少一个单色LED灯珠。
优选地，所述样品台装置包括：样品台、样品板和第一电机和第二电机；
所述样品板安装在所述样品台上；
所述第一电机和第二电机与所述自动控制系统连接，在所述自动控制系统的控制下驱动所述样品台完成X轴和Y轴移动，进行样品板中样品视野的自动更换。
优选地，所述样品板由两面透明玻片和间隔结构构成；
所述间隔结构在所述透明玻片中间形成独立固定距离的微缝槽，所述微缝槽的两端开有样品加注孔。
优选地，所述显微成像装置包括：物镜、镜筒、第三电机、发射光滤光模组和图像获取装置；其中：
所述物镜和图像获取装置分别固定于所述镜筒的两端；
所述发射滤光模组固定在所述镜筒上，位于所述物镜和所述图像获取装置之间；
所述图像获取装置与所述分析处理系统相连；
所述第三电机分别与所述镜筒和自动控制系统连接，在所述自动控制系统的控制下，带动所述镜筒和物镜上下移动，进行测量焦距的自动调节；
所述物镜用于放大预设检测区域值预设倍数；
所述发射滤光模组用于对所述预设检测区域内样品颗粒受所述荧光激发光激发后的荧光进行滤光处理；
所述图像获取装置用于获取放大至预设倍数后所述预设检测区域的图像，并将所述图像发送至所述分析处理系统进行分析处理。
优选地，所述发射光滤光模组包括：密封上盘、密封下盘、转盘、滤光片、滤光片卡环、转盘轴和第四电机；其中：
所述密封上盘和密封下盘固定于所述转盘中心的转盘轴两端；
所述滤光片通过滤光片卡环固定在所述转盘上；
所述第四电机分别与所述转盘和自动控制系统相连，在所述自动控制系统的控制下带动所述转盘转动，切换所述滤光片。
相较于现有技术，本发明提供的技术方案至少具有以下优点：
本发明提供了一种荧光激发光源装置及系统和荧光显微镜，包括：可调整固定架；以及，固定于所述可调整固定架中的单色LED模组、聚光模组和带通滤光片；其中，所述聚光模组设置于所述单色LED模组和带通滤光片之间，或者，所述带通滤光片设置于所述单色LED模组和聚光模组之间，且所述可调整固定架能够调整所述单色LED模组和所述聚光模组之间的距离。
由上述方案可知，本发明提供的一种自动多通道类流式图像荧光分析系 统，通过自动控制系统控制集成式荧光激发装置，能够实现单一光源顺序间隔开启和关闭、多种光源同时开启或关闭的不同光组合；通过自动控制系统控制样品台装置，能够实现对样品台装置中的样品进行不同位置的测量；通过自动控制系统控制显微成像装置，能够实现测量焦距的自动调节，以及滤光片的自动切换；通过分析处理系统能够实现对显微成像装置采集的显微图像进行分析处理；综上，本发明能够以显微成像技术为基础、快速采集和分析大量样品的成像信息，并采用密度函数的算法进行分析，以同时获得每个细胞的形态影像学分析结果和基于大样本量的密度函数的类流式数据分析结果。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据提供的附图获得其他的附图。
图1为本发明公开的一种自动多通道类流式图像荧光分析系统的结构示意图；
图2为本发明公开的一种集成式荧光激发光源装置的结构示意图；
图3为本发明公开的另一种集成式荧光激发光源装置的结构示意图；
图4为本发明公开的一种荧光激发光源装置的切面结构的示意图；
图5为本发明公开的另一种荧光激发光源装置的切面结构的示意图；
图6为本发明公开的自动多通道类流式图像荧光分析系统的机械部分结构示意图；
图7为本发明公开的样品板的结构示意图；
图8为本发明公开的发射光滤光模组的结构示意图。
具体实施方式
下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是 全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
如图1所示，为本发明实施例公开的一种自动多通道类流式图像荧光分析系统，包括：集成式荧光激发光源装置11、样品台装置12、显微成像装置13、自动控制系统14和分析处理系统15；其中：
自动控制系统14分别与集成式荧光激发光源装置11、样品台装置12和显微成像装置13相连，分别对集成式荧光激发光源装置11、样品台装置12和显微成像装置13进行控制；
集成式荧光激发光源装置11、样品台装置12和显微成像装置13构成显微光学系统；
分析处理系统15与显微成像装置14相连，对显微成像装置13采集的图像进行分析处理。
在上述实施例中，通过自动控制系统14控制集成式荧光激发装置11，能够实现单一光源顺序间隔开启和关闭、多种光源同时开启或关闭的不同光组合；通过自动控制系统14控制样品台装置12，能够实现对样品台装置12中的样品进行不同位置的测量；通过自动控制系统14控制显微成像装置13，能够实现测量焦距的自动调节，以及滤光片的自动切换；通过分析处理系统15能够实现对显微成像装置13采集的显微图像进行分析处理；综上，本发明能够以显微成像技术为基础、快速采集和分析大量样品的成像信息，并采用密度函数的算法进行分析，以同时获得每个细胞的形态影像学分析结果和基于大样本量的密度函数的类流式数据分析结果。
具体的，在上述实施例中，集成式荧光激发光源装置的其中一种结构如图2所示，包括：光源架111、1个垂直设置于光源架111中心的透射明场光源装置112及4个以上环绕光源架111的中心倾斜设置于光源架111上的荧光激发光源装置113；
荧光激发光源装置113与透射明场光源装置112均位于样品板(图中未画出)的上方，透射明场光源装置112安装于光源架111的中部，4个荧光激发光源装置113以透射明场光源装置112为中心环绕分布于透射明场光源装置 112的周围。
荧光激发光源装置113固定安装于光源调整架114上，光源调整架114通过转轴115及调节螺母116与光源架111连接，调整光源调整架114与光源架111之间的相对位置，可以方便的调节斜射的荧光激发光源装置113的入射角度。
使用时，先拧松调节螺母116，调节斜射光源的入射角度，根据成像清晰、视野背景均匀等要求，调节出合适的角度后，拧紧调节螺母116，固定斜射光源的位置。透射明场光源装置112位于正中位置，入射光线与样品板(图中未画出)保持垂直。实验过程中，只需要更换荧光激发滤光片即可完成多颜色通道的荧光信号采集，而斜射光源的切换由电路控制系统自动完成。
具体的，在上述实施例中，集成式荧光激发光源装置的另一种结构如图3所示，与上一种集成式荧光激发光源装置的区别在于：荧光激发光源装置113与透射明场光源装置112分别位于样品板117的上下两侧，4个荧光激发光源装置113环绕光源架111的中心均匀分布于透射明场光源装置112的四周边缘。
当然，本发明的保护范围不局限于以上实施例。其中，荧光激发光源装置113的数量不单单只有4个一种情况，可以根据实际情况增加。斜射光源(荧光激发光源装置113发出的光源)与直射光源(透射明场光源装置112发出的光源)的配合角度可以根据需要任意变化。荧光激发灯可以为LED灯珠或激光灯。
具体的，在上述实施例中，一种荧光激发光源装置的切面结构如图4所示，包括：可调整固定架100；
以及，固定于可调整固定架100中的单色LED模组200、聚光模组300和带通滤光片400；
其中，聚光模组300设置于单色LED模组200和带通滤光片400之间，且可调整固定架100能够调整单色LED模组200和聚光模组300之间的距离。
由上述内容可知，本申请实施例提供的技术方案，采用但是LED模组作为荧光激发光源，并且由于LED具有响应时间短、成本低、使用寿命长、体积小等特点，使得荧光激发光源装置具有响应时间短、成本低、使用寿命长、体积 小等优点。以及，由于LED光的衰减即快又精确，长期活细胞试验下可大大减少光毒性，使得荧光激发光源装置利于长期活细胞实验。并且，由于LED仅仅需要3W的功率即可实现与汞灯、氙灯等提供的相同波段的荧光激发光，相较于汞灯需要50W至100W的功率，而氙灯需要100W的功率的情况，LED具有耗能低的特点，使得荧光激发光源装置大大降低了功耗。
另外，将聚光模组设置于单色LED模组的前端，不仅使荧光激发光源装置的出光亮度高，而且还能可以通过可调固定架调整聚光模组和单色LED模组之间的距离，以调节荧光激发光源装置的出光光斑大小，进而调节荧光激发光源装置的出光的激发光源能量的聚集程度。
此外，将带通滤光片设置于单色LED模组的前端，以约束荧光激发光源装置的出光波长范围，进一步提高荧光激发光源装置的发出的荧光激发光源的单一性。例如，蓝色LED模组的峰值波长范围为470±15nm(包括端点值)，而与其对应的带通滤光片能够通过波长范围为470±15nm(包括端点值)的光，因此，通过带通滤光片能够使得荧光激发光源装置发出的荧光激发光源的波长集中在465nm～485nm(包括端点值)之间，进一步提高荧光激发光源装置的发出的荧光激发光源的单一性。
其中，本申请实施例提供的聚光模组包括但不限于聚光透镜或透镜组，即，本申请实施例提供的聚光模组可以为单独的一个聚光透镜，或者，还可以为多个透镜组成的具有聚光功能的透镜组，对此本申请不作具体限制，需要根据实际需要进行具体设计。而且，本申请对于聚光透镜和透镜组中透镜的材质同样不作限制。
此外，本申请实施例提供的单色LED模组包括至少一个单色LED灯珠，对于LED模组中单色LED灯珠的数量，需要根据实际应用进行具体设计，本申请不作具体限制。
进一步的，为了避免荧光激发光源装置中LED模组提供的荧光激发光源的浪费，本申请实施例提供的可调整固定架为密封可调整固定架，且单色LED模组、聚光模组和带通滤光片均密封固定于可调整固定架内。
即，将单色LED模组、聚光模组和带通滤光片均密封固定于可调整固定架内，保证单色LED模组发出的荧光激发光源全部入射至聚光模组，而后经 过聚光模组的荧光激发光源全部入射至带通滤光片，而后通过经过滤波后出射，避免了荧光激发光源装置的出光之前的浪费，提高了荧光激发光源装置的出光强度。
需要说明的是，本申请对于可调整固定架的形状不作具体限制，另外，本申请对于可调整固定架调整单色LED模组和聚光透镜之间的距离的方式不作具体限定，均需要根据实际应用进行具体设计。
本申请上述实施例提供的荧光激发光源装置，其聚光模组设置于单色LED模组和带通滤光片之间；此外，在本申请另一实施例中，聚光模组还可以设置于带通滤光片背离单色LED模组的一侧。具体的，参考图5所示，为本申请另一实施例提供的一种荧光激发光源装置的切面结构示意图，其中，荧光激发光源装置，包括：
可调整固定架100；
以及，固定于可调整固定架100中的单色LED模组200、聚光模组300和带通滤光片400；
其中，带通滤光片400设置于单色LED模组200和聚光模组300之间，且可调整固定架100能够调整单色LED模组200和聚光模组300之间的距离。
由上述内容可知，本申请实施例提供的技术方案，采用但是LED模组作为荧光激发光源，并且由于LED具有响应时间短、成本低、使用寿命长、体积小等特点，使得荧光激发光源装置具有响应时间短、成本低、使用寿命长、体积小等优点。以及，由于LED光的衰减即快又精确，长期活细胞试验下可大大减少光毒性，使得荧光激发光源装置利于长期活细胞实验。并且，由于LED仅仅需要3W的功率即可实现与汞灯、氙灯等提供的相同波段的荧光激发光，相较于汞灯需要50W至100W的功率，而氙灯需要100W的功率的情况，LED具有耗能低的特点，使得荧光激发光源装置大大降低了功耗。
另外，将聚光模组设置于单色LED模组的前端，不仅使荧光激发光源装置的出光亮度高，而且还能可以通过可调固定架调整聚光模组和单色LED模组之间的距离，以调节荧光激发光源装置的出光光斑大小，进而调节荧光激发光源装置的出光的激发光源能量的聚集程度。
此外，将带通滤光片设置于单色LED模组的前端，以约束荧光激发光源装置的出光波长范围，进一步提高荧光激发光源装置的发出的荧光激发光源的单一性。例如，蓝色LED模组的峰值波长范围为470±15nm(包括端点值)，而与其对应的带通滤光片能够通过波长范围为470±15nm(包括端点值)的光，因此，通过带通滤光片能够使得荧光激发光源装置发出的荧光激发光源的波长集中在465nm～485nm(包括端点值)之间，进一步提高荧光激发光源装置的发出的荧光激发光源的单一性。
其中，本申请实施例提供的聚光模组包括但不限于聚光透镜或透镜组，即，本申请实施例提供的聚光模组可以为单独的一个聚光透镜，或者，还可以为多个透镜组成的具有聚光功能的透镜组，对此本申请不作具体限制，需要根据实际需要进行具体设计。而且，本申请对于聚光透镜和透镜组中透镜的材质同样不作限制。
此外，本申请实施例提供的单色LED模组包括至少一个单色LED灯珠，对于LED模组中单色LED灯珠的数量，需要根据实际应用进行具体设计，本申请不作具体限制。
如图6所示，为本发明公开的自动多通道类流式图像荧光分析系统的机械部分结构示意图，包括：集成式荧光激发光源装置601、样品台602、样品板603、第一电机604、第二电机605、物镜606、镜筒607、第三电机608、发射光滤光模组609、第四电机610和图像获取装置611；
集成式荧光激发光源装置601通过自动控制系统的控制，以实现单一光源顺序间隔开启和关闭、多种光源同时开启或关闭的不同光源组合，及实现光源光强可调整；
样品板603安装在样品台602上；
第一电机604和第二电机605与自动控制系统连接，在自动控制系统的控制下驱动样品台602完成X轴和Y轴移动，进行样品板603中样品视野的自动更换；
物镜606和图像获取装置611分别固定于镜筒607的两端；
发射滤光模组609固定在镜筒607上，位于物镜606和图像获取装置611 之间；
图像获取装置611与分析处理系统相连；
第三电机608分别与镜筒607和自动控制系统连接，在自动控制系统的控制下，带动镜筒607和物镜606上下移动，进行测量焦距的自动调节；
物镜606用于放大预设检测区域值预设倍数；
发射滤光模组609用于对预设检测区域内样品颗粒受所述荧光激发光激发后的荧光进行滤光处理；
发射滤光模组609中的第四电机610，在自动控制系统的控制下对发射滤光模组609中的滤光片进行切换；
图像获取装置611用于获取放大至预设倍数后预设检测区域的图像，并将图像发送至分析处理系统进行分析处理。
具体的，如图7所示，为样品板的结构示意图，样品板由两面透明玻片711和间隔结构712构成；
间隔结构712在透明玻片711中间形成独立固定距离的微缝槽713，微缝槽713的两端开有样品加注孔714。
样品板由两面透明玻片材料和一些间隔结构组合，在两片透明材料中间形成独立固定距离的微缝槽(50μm～300μm)，该微缝槽的两端开有样品加注孔。样品通过样品加注孔注入后，在微缝槽中均布，形成预设的一定厚度的液体薄层。由于该等计量微缝的高度固定，因此，通过测定的一个观察或拍照视野的观察面积*该微缝槽的高度，可以得到该观察或摄像视野的体积，然后，通过以下公式可以换算出该样品浓度：
样品浓度＝视野颗粒(细胞)个数*1mL/视野面积*微缝槽高度。
视野数量越多，可观察和分析的样品量越多，测量的浓度偏差就会减小。
本发明采用的样品板，具有以下优势：
1)相对于普通的载玻片无法形成固定的液体厚度，而只能观察数数，却无法转换为最终需要的浓度信息，采用样品板解决了这一问题。
2)相对于普通雪球计数板在计数板上进行刻画标准的框格，然后识别计数该框格中细胞数量然后转换为浓度的方法，采用样品板直接测定并采用视野面积而进行计数于换换，更为简单，易操作，误差小。
3)相对于流式通过在微管或鞘液中流动的方式，由于细胞处于一种不断的运动状态中，其必然受到流速和压力等因素的影响，这种方法本身会造成细胞状态的失真。例如：处于凋亡后期的细胞，在这种流速的作用下很容易造成细胞核与细胞膜的脱落和分离，从而造成测量的结果与样品本来的状态有偏差。基于同样的原因，使用流式后的样品是无法再重复使用或测量的。采用样品板的方法，样品加注后及检测过程中不流动的，甚至可以是细胞本来的贴壁状态，这样的状态没有流速和压力的影响，细胞状态和检测结果更符合样品本来的状态。
具体的，如图8所示，为发射光滤光模组的结构示意图，包括：密封上盘801、密封下盘802、转盘803、滤光片804、滤光片卡环805、转盘轴806和第四电机(图中未画出)；其中：
密封上盘801和密封下盘802固定于转盘803中心的转盘轴806两端；
滤光片804通过滤光片卡环805固定在转盘803上；
第四电机分别与转盘803和自动控制系统相连，在自动控制系统的控制下带动转盘803转动，切换所述滤光片。
综上所述，通过计算机与自动控制系统连接通信，计算机根据人工指令或自动运算编程指令生成自动控制流程程序，并通过计算机与自动控制系统和图像获取装置的指令信息交互来进行自动控制。
自动控制系统与集成式荧光激发光源装置的各个光源灯连接，为其提供电源并发出开关和调节电压电流等指令，以控制各光源灯的开启和关闭。
样品台装置通过X、Y轴向上设置的第一电机和第二电机进行传动，该电第一电机和第二电机与自动控制系统连接，并接收自动控制系统的指令完成样品台的X轴和Y轴的移动，最终实现可对平置于样品台上的样品板的不同位置进行测量的功能。
本系统的镜筒固定在第三电机驱动的可垂直轴向移动的滑台上，第三电机与自动控制系统连接，并接收自动控制系统的指令完成轴向往复移动，带动镜筒和物镜的上下移动，最终实现依照指令自动调节测量焦距的功能。
本系统的可移动切换发射光(截止光)滤光片组及其支架(不限于只圆盘转动方式，也可以是线性往复运动方式)，是通过第四电机进行驱动，第四电机与自动控制系统连接，并接收自动控制系统的指令完成发射光滤光片的切换。
本系统的图像获取装置与计算机相连接，受计算机的指令获取信息并传输给计算机，实现获取显微图像的功能。
本系统的数据分析系统是置于计算机中的分析处理系统。该分析处理系统主要包含以下软件功能模块：
a、电子图像生成和优化功能：通过该功能可以将图像获取装置获取的电子信号，生成为多种格式(PNG、jpg、tif等)的电子图像。并对电子图像进行噪点去除、荧光背景去除等图像优化。
b、多图像融合功能：在上述a的基础上，对同一样品位于同一位置所拍摄的多张图片，可以任意组合将两张或两张以上的多图融合为一张图。
c、智能图像识别功能：通过自动图像识别程序，可以自动分析图像中各种形态的颗粒(细胞)，计算出每个颗粒(细胞)的位置、形状、大小、团聚、是否激发荧光、是否同时激发多种荧光、激发的各种荧光的强度(累计荧光强度、平均荧光强度)、荧光位置、荧光的面积、荧光直径、荧光的形状、最大荧光值、最小荧光值、荧光落差值、荧光边缘平滑度等信息，并基于该等图像识别信息，根据筛选规则自动对样品进行归类、并分类计算各类的个数、平均面积、平均直径、平均荧光强度、平均荧光面积、平均荧光直径等指标。
d、异步多通道荧光图像分析功能：可完成异步单通道、双通道、三通道、四通道……的多通道荧光图像分析。
e、同步多通道荧光图像分析功能：可完成同步双通道、三通道、四通道等同步多通道荧光图像分析。
f、自动统计分析功能：在c、d、e的基础上，根据各种实验的逻辑结构，自动计算出样品浓度、活率(死亡率)、转染率、凋亡率、各细胞周期比例等最终结果。
g、自动类流式分析功能：基于密度函数原理，以上述c、d、e点所获得的图像荧光数量、大小、强度等信息指标替换流式细胞仪系统下通过pmt采集换算的颗粒数量、荧光大小、荧光强度等信息，构件起基于电子图像信息而非PMT电子信号信息的生成散点图、设门和密度函数分析算法。通过本功能，在样品浓度、活率、转染率、凋亡率、细胞周期比率检测等试验中，可获得类流式分析的结果。这一结果由于分析方法的基本原理一致，相对于以上f的直接统计结果，与流式细胞分析的数据更具有一致性和可对比性。
h、快速自动大样品量采集分析功能：本发明可像流式细胞仪一样预设大样本量采集限量值(例如：1万个细胞样品)，通过自动控制系统自动获取多个视野样品图像，软件系统自动分析该多个视野的图像，并采用多线程计算机技术，边采集图像信号边进行分析计算，以快速(3分钟内)实现大量定量样本的采集和分析。解决了显微观察方法下难以快速分析大量样本的难题。
i、自定义编辑功能：本发明软件包含自定义编辑模块，可以根据不同的荧光实验需求(如采样量、荧光通道、分析方法)，自定义编辑实验流程、设备运行流程、图像分析方法、各种参数和阀值、数据计算公式、结果呈现名称和格式、界面格式，并自动生成和保存新的标准实验模块。本功能突破的传统分析仪器固化功能和流程的思路，让用户可以创新实验流程和方法，大胆展开新方法的摸索和尝试。
对所公开的实施例的上述说明，使本领域专业技术人员能够实现或使用本发明。对这些实施例的多种修改对本领域的专业技术人员来说将是显而易见的，本文中所定义的一般原理可以在不脱离本发明的精神或范围的情况下，在其它实施例中实现。因此，本发明将不会被限制于本文所示的这些实施例，而是要符合与本文所公开的原理和新颖特点相一致的最宽的范围。

Claims (13)

  1. 一种自动多通道类流式图像荧光分析系统，其特征在于，包括：集成式荧光激发光源装置、样品台装置、显微成像装置、自动控制系统和分析处理系统；其中：
    所述自动控制系统分别与所述集成式荧光激发光源装置、样品台装置和显微成像装置相连，分别对所述集成式荧光激发光源装置、样品台装置和显微成像装置进行控制；
    所述集成式荧光激发光源装置、样品台装置和显微成像装置构成显微光学系统；
    所述分析处理系统与所述显微成像装置相连，对所述显微成像装置采集的图像进行分析处理。
  2. 根据权利要求1所述的系统，其特征在于，所述集成式荧光激发光源装置包括：光源架、1个垂直设置于光源架中心的透射明场光源装置及2个以上环绕光源架的中心倾斜设置于光源架上的荧光激发光源装置；
    所述荧光激发光源装置所发光束与透射明场光源装置所发光束在某一位置形成交汇，所述荧光激发光源装置、所述透射明场光源装置均与所述自动控制系统相连接，透射明场光源装置与荧光激发光源装置通过所述自动控制系统控制，以实现单一光源顺序间隔开启和关闭、多种光源同时开启或关闭的不同光源组合，及实现光源光强可调整。
  3. 根据权利要求2所述的系统，其特征在于，所述荧光激发光源装置与所述透射明场光源装置均位于所述样品台装置的上方，透射明场光源装置安装于所述光源架的中部，荧光激发光源装置以透射明场光源为中心环绕分布于透射明场光源装置的周围。
  4. 根据权利要求2所述的系统，其特征在于，所述透射明场光源装置与所述荧光激发光源装置分别位于所述样品台装置的上下两侧，荧光激发光源装置环绕光源架的中心均匀分布于光源架的四周边缘。
  5. 根据权利要求3或4所述的系统，其特征在于，所述集成式荧光激发光源装置还包括：光源调整架；
    所述荧光激发光源装置固定安装于该光源调整架上，光源调整架的侧面设置有转轴，所述光源架对应于转轴设置有轴孔，转轴穿过轴孔与一调节螺母螺接，以实现对斜射的荧光激发光源装置的方向与位置的调节及固定。
  6. 根据权利要求5所述的系统，其特征在于，所述荧光激发光源装置包括：
    可调整固定架；
    以及，固定于所述可调整固定架中的单色LED模组、聚光模组和带通滤光片；
    其中，所述聚光模组设置于所述单色LED模组和带通滤光片之间，且所述可调整固定架能够调整所述单色LED模组和所述聚光模组之间的距离。
  7. 根据权利要求5所述的系统，其特征在于，所述荧光激发光源装置包括：
    可调整固定架；
    以及，固定于所述可调整固定架中的单色LED模组、聚光模组和带通滤光片；
    其中，所述带通滤光片设置于所述单色LED模组和聚光模组之间，且所述可调整固定架能够调整所述单色LED模组和所述聚光模组之间的距离。
  8. 根据权利要求6或7所述的系统，其特征在于，所述聚光模组包括聚光透镜或透镜组。
  9. 根据权利要求8所述的系统，其特征在于，所述单色LED模组包括至少一个单色LED灯珠。
  10. 根据权利要求9所述的系统，其特征在于，所述样品台装置包括：样品台、样品板和第一电机和第二电机；
    所述样品板安装在所述样品台上；
    所述第一电机和第二电机与所述自动控制系统连接，在所述自动控制系统的控制下驱动所述样品台完成X轴和Y轴移动，进行样品板中样品视野的自动更换。
  11. 根据权利要求10所述的系统，其特征在于，所述样品板由两面透明玻片和间隔结构构成；
    所述间隔结构在所述透明玻片中间形成独立固定距离的微缝槽，所述微缝槽的两端开有样品加注孔。
  12. 根据权利要求11所述的系统，其特征在于，所述显微成像装置包括：物镜、镜筒、第三电机、发射光滤光模组和图像获取装置；其中：
    所述物镜和图像获取装置分别固定于所述镜筒的两端；
    所述发射滤光模组固定在所述镜筒上，位于所述物镜和所述图像获取装置之间；
    所述图像获取装置与所述分析处理系统相连；
    所述第三电机分别与所述镜筒和自动控制系统连接，在所述自动控制系统的控制下，带动所述镜筒和物镜上下移动，进行测量焦距的自动调节；
    所述物镜用于放大预设检测区域值预设倍数；
    所述发射滤光模组用于对所述预设检测区域内样品颗粒受所述荧光激发光激发后的荧光进行滤光处理；
    所述图像获取装置用于获取放大至预设倍数后所述预设检测区域的图像，并将所述图像发送至所述分析处理系统进行分析处理。
  13. 根据权利要求12所述的系统，其特征在于，所述发射光滤光模组包括：密封上盘、密封下盘、转盘、滤光片、滤光片卡环、转盘轴和第四电机；其中：
    所述密封上盘和密封下盘固定于所述转盘中心的转盘轴两端；
    所述滤光片通过滤光片卡环固定在所述转盘上；
    所述第四电机分别与所述转盘和自动控制系统相连，在所述自动控制系统的控制下带动所述转盘转动，切换所述滤光片。
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Families Citing this family (30)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105486667A (zh) * 2015-07-01 2016-04-13 上海睿钰生物科技有限公司 集成式荧光激发光源装置
CN105465745B (zh) * 2015-12-31 2019-04-05 中国科学院苏州生物医学工程技术研究所 多波段荧光照明装置
EP3405898A1 (en) 2016-01-21 2018-11-28 Protein Dynamic Solutions LLC Method and system for spectral data analysis
LU93022B1 (de) * 2016-04-08 2017-11-08 Leica Microsystems Verfahren und Mikroskop zum Untersuchen einer Probe
US20190388889A1 (en) * 2016-10-07 2019-12-26 Boehringer Ingelheim Vetmedica Gmbh Method for controlling an analysis device and analysis system
CN107037576B (zh) * 2017-05-03 2023-08-15 中国科学院苏州生物医学工程技术研究所 光学显微成像设备及其成像方法
CN107367456B (zh) * 2017-07-20 2020-04-03 上海睿钰生物科技有限公司 一种免洗涤图像类流式荧光检测方法和系统
CN107356515B (zh) * 2017-07-20 2020-04-03 上海睿钰生物科技有限公司 一种荧光图像的荧光强度确定方法和系统
CN107272196A (zh) * 2017-08-03 2017-10-20 苏州天准科技股份有限公司 一种削弱远心光学系统环形光照明鬼影的方法
CN109932316A (zh) * 2017-12-18 2019-06-25 长光华大基因测序设备（长春）有限公司 基因测序光学装置
CN108318468B (zh) * 2018-05-05 2024-01-16 哈尔滨索飞永诚科技有限公司 一种用于对液体样品中荧光染料染色微粒进行快速计数的计数系统
CN109001900A (zh) * 2018-09-05 2018-12-14 南京大学 一种明场和荧光双模态的显微成像系统及方法
CN111041076B (zh) * 2018-10-11 2023-09-26 深圳华大生命科学研究院 气泡检测方法及系统、基因测序仪、计算机可读存储介质
CN111427142A (zh) 2019-01-09 2020-07-17 卡尔蔡司显微镜有限责任公司 用于显微镜设备的照明模块及相关控制方法和显微镜设备
CN111435192B (zh) * 2019-01-15 2021-11-23 卡尔蔡司显微镜有限责任公司 利用荧光显微镜生成荧光照片的方法
WO2020183433A1 (en) * 2019-03-14 2020-09-17 3M Innovative Properties Company Sterilization indicator reading apparatus with a color sensor
CN111239086B (zh) * 2019-08-30 2024-04-05 北京临近空间飞行器系统工程研究所 视觉背景装置，荧光显微光学系统，扫描分析系统
CN110866918B (zh) * 2019-12-11 2022-04-05 上海睿钰生物科技有限公司 酵母分析方法
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CN110991379B (zh) * 2019-12-11 2022-02-22 上海睿钰生物科技有限公司 藻类计数方法
CN110987768B (zh) * 2019-12-11 2022-02-15 上海睿钰生物科技有限公司 酵母计数方法
CN111551531B (zh) * 2020-05-19 2023-04-18 北京金诺美科技股份有限公司 一种荧光激发系统及实时荧光定量pcr仪
CN113946043A (zh) * 2020-07-16 2022-01-18 香港科技大学 一种激发光源和包括该激发光源的荧光显微镜
CN112946673B (zh) * 2021-01-29 2023-01-06 上海睿钰生物科技有限公司 一种激光测距方法、对焦方法、激光测距系统及对焦系统
CN113092428B (zh) * 2021-04-02 2023-07-25 安图实验仪器(郑州)有限公司 一种多重荧光检测方法、装置、设备和系统
CN113109314A (zh) * 2021-05-28 2021-07-13 上海睿钰生物科技有限公司 多荧光信号检测系统和方法
CN114199849A (zh) * 2021-12-17 2022-03-18 广州博鹭腾生物科技有限公司 一种应用于荧光成像的荧光光路系统
CN114441496B (zh) * 2022-02-14 2024-03-01 江苏禹视科技有限公司 一种多光源荧光扫描分析系统和方法
CN115061270B (zh) * 2022-05-30 2024-01-16 中国人民解放军国防科技大学 一种倾斜模式望远显微组合成像方法
CN114813522B (zh) * 2022-06-29 2022-10-21 深圳安侣医学科技有限公司 基于显微放大数字图像的血液细胞分析方法及系统

Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2004325174A (ja) * 2003-04-23 2004-11-18 Olympus Corp 蛍光検出装置
CN101149327A (zh) * 2007-11-06 2008-03-26 浙江大学 基于细胞显微图像信息的抗肿瘤药物评价和筛选方法
CN102151122A (zh) * 2011-03-17 2011-08-17 中国科学院自动化研究所 激发荧光分子成像系统及一次荧光成像方法
US20130015362A1 (en) * 2011-07-12 2013-01-17 Sharp Kabushiki Kaisha Fluid purification and sensor system
CN104198458A (zh) * 2014-09-26 2014-12-10 哈尔滨工业大学 一种飞秒激光双光子荧光生物显微成像系统及其成像方法
CN104641222A (zh) * 2012-05-29 2015-05-20 麦考瑞大学 用于发光显微成像的双向扫描
CN104990907A (zh) * 2015-07-01 2015-10-21 上海睿钰生物科技有限公司 自动多通道类流式图像荧光分析系统
CN204945042U (zh) * 2015-07-28 2016-01-06 上海睿钰生物科技有限公司 自动多通道类流式图像荧光分析系统

Family Cites Families (59)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP3164609B2 (ja) 1990-10-31 2001-05-08 オリンパス光学工業株式会社 内視鏡装置
JP2000235002A (ja) * 1999-02-15 2000-08-29 Fuji Photo Film Co Ltd 撮影方法および撮影装置
JP3783826B2 (ja) * 2000-01-17 2006-06-07 横河電機株式会社 バイオチップ読み取り装置
GB0200938D0 (en) * 2002-01-16 2002-03-06 Solexa Ltd Prism design for scanning applications
CN2522855Y (zh) * 2002-01-30 2002-11-27 无锡市朗珈生物医学工程有限公司 带滤光片转盘的生物芯片荧光检测扫描装置
CN101072997A (zh) * 2003-08-14 2007-11-14 塞通诺米公司 用于粒子分类系统的光学检测器
JP2005062023A (ja) * 2003-08-14 2005-03-10 Japan Science & Technology Agency 落射型蛍光測定装置
JP4825426B2 (ja) * 2005-01-31 2011-11-30 財団法人 東京都医学総合研究所 生物顕微鏡に用いる暗視野照明装置
US7355696B2 (en) * 2005-02-01 2008-04-08 Arryx, Inc Method and apparatus for sorting cells
FR2889404B1 (fr) 2005-08-01 2009-03-27 Commissariat Energie Atomique Source lumineuse a deux longueurs d'onde et de puissance d'eclairement variable et utilisation d'une telle source lumineuse
GB0614297D0 (en) * 2006-07-19 2006-08-30 Shaw Water Engineering Ltd Apparatus, system and method for detecting particles
CN101191770B (zh) * 2006-11-21 2011-06-15 杭州远方光电信息股份有限公司 发光二极管荧光粉发射光谱测量方法
CN200968935Y (zh) * 2006-11-21 2007-10-31 杭州远方光电信息有限公司 用于发光二极管荧光粉测试的激发与接收装置
CN101363839B (zh) * 2008-09-12 2012-02-08 首都师范大学 可观测带荧光生物样品的非损伤微测系统
EP2331941B1 (en) * 2008-09-25 2014-11-12 Koninklijke Philips N.V. Detection system and method
JP2010134382A (ja) * 2008-12-08 2010-06-17 Olympus Corp 観察装置
JP5775004B2 (ja) 2009-03-03 2015-09-09 アクセス メディカル システムズ，リミティド 高感度蛍光分析のための検出システム及び方法
JP5499732B2 (ja) * 2009-06-23 2014-05-21 ソニー株式会社 生体サンプル像取得装置、生体サンプル像取得方法及び生体サンプル像取得プログラム
JP2011028187A (ja) * 2009-07-21 2011-02-10 Kenichi Tanehashi 実体顕微鏡のステージ上に置いて透過蛍光観察をする装置
CN102834718A (zh) * 2010-01-12 2012-12-19 耐克思乐生物科学有限责任公司 计数细胞和生物分子的系统和方法
CN102754011B (zh) * 2010-02-03 2015-08-05 株式会社尼康 观察装置及观察方法
JP2011186182A (ja) * 2010-03-09 2011-09-22 Claro Inc 顕微鏡装置
JP2011237549A (ja) * 2010-05-10 2011-11-24 Iponacology Ltd 顕微鏡照明装置における光源ユニット、およびそれを備える顕微鏡
DE102010023486A1 (de) * 2010-06-11 2011-12-15 B. Braun Avitum Ag Nachweisvorrichtung und -verfahren
CN102329725A (zh) * 2010-06-16 2012-01-25 三星泰科威株式会社 光透射温度控制装置、生物诊断设备和方法
JP5707758B2 (ja) * 2010-07-13 2015-04-30 ソニー株式会社 撮像装置、撮像システム、手術用ナビゲーションシステム、及び撮像方法
WO2012023816A2 (ko) 2010-08-18 2012-02-23 주식회사 나노엔텍 멀티 형광영상 관측용 형광현미경, 이를 이용한 형광영상의 관찰방법 및 멀티 형광영상 관측 시스템
JP2012168088A (ja) * 2011-02-16 2012-09-06 Ncd:Kk 蛍光反応検出装置
DE202011000688U1 (de) * 2011-03-25 2011-06-09 Leica Microsystems CMS GmbH, 35578 Vorrichtung zur Aufnahme von Filtern für Mikroskope
CN102305778B (zh) * 2011-05-17 2013-10-30 易定容 微多光谱荧光接收和处理系统
CN202057599U (zh) * 2011-05-17 2011-11-30 易定容 微多光谱荧光接收和处理系统
DE102011105181A1 (de) * 2011-06-17 2012-12-20 Leica Microsystems Cms Gmbh Mikroskop und Verfahren zur bildgebenden Fluoreszenzmikroskopie
DE102011051278A1 (de) * 2011-06-22 2012-12-27 Leica Microsystems Cms Gmbh Verfahren und lichtmikroskopische Einrichtung zur bildlichen Darstellung einer Probe
DE102011079942B4 (de) * 2011-07-27 2016-12-15 Leica Microsystems Cms Gmbh Mikroskopbeleuchtungsverfahren und Mikroskop
DE102011083215A1 (de) * 2011-09-22 2013-03-28 Siemens Aktiengesellschaft Probenträger und Verfahren zur mikroskopischen Untersuchung biologischer Proben
CN102507518B (zh) * 2011-10-25 2013-05-08 天津港东科技发展股份有限公司 12灯位多通道原子荧光光度计
CN103134780B (zh) * 2011-12-01 2015-12-09 中国科学院大连化学物理研究所 一种紧贴式激发光路的发光二极管诱导荧光检测器
JP5947553B2 (ja) * 2012-01-27 2016-07-06 浜松ホトニクス株式会社 撮像装置及び撮像装置の製造方法
DE102012201286A1 (de) * 2012-01-30 2013-08-01 Carl Zeiss Microscopy Gmbh Mikroskop und Verfahren für die wellenlängenselektive und örtlich hochauflösende Mikroskopie
WO2013137247A1 (ja) * 2012-03-12 2013-09-19 三菱レイヨン株式会社 蛍光検出装置及び蛍光検出方法
US8901514B2 (en) * 2012-06-28 2014-12-02 Molecular Devices, Llc Sample analysis system with spotlight illumination
CN103777336B (zh) * 2012-10-22 2017-09-05 承奕科技股份有限公司 显微光学撷取装置用荧光辅具模组、基架及该装置
US8785885B1 (en) 2013-01-30 2014-07-22 Omnivision Technologies, Inc. Fluorescence imaging module
CN103134784B (zh) * 2013-02-05 2015-04-29 华中科技大学 光纤化活体荧光激发光谱成像装置
CN105579828B (zh) * 2013-05-28 2020-02-14 克莫麦特公司 图像式细胞仪
CN203337549U (zh) * 2013-06-28 2013-12-11 陕西师范大学 一种适用于微痕量爆炸物荧光检测的光学组件
CN203811540U (zh) * 2013-10-22 2014-09-03 闽浪仪器科技（厦门）有限公司 一种高度集成的多道数字荧光成像系统
CN104568859B (zh) * 2013-10-22 2017-10-13 承奕科技股份有限公司 具多组异角度光源的荧光观测装置、基架及荧光显微镜
CN103645167A (zh) * 2013-12-13 2014-03-19 苏州东胜兴业科学仪器有限公司 一种聚合酶链反应检测仪
CN103995346A (zh) * 2014-06-09 2014-08-20 武汉理工大学 一种显微镜物镜轴向扫描装置
CN204347334U (zh) * 2014-07-09 2015-05-20 闽浪仪器科技（厦门）有限公司 一种阵列式微滤片多道荧光显微镜
CN204330595U (zh) * 2014-07-15 2015-05-13 闽浪仪器科技（厦门）有限公司 一种实时多通道荧光检测系统
CN104296687A (zh) * 2014-11-05 2015-01-21 哈尔滨工业大学 基于荧光共焦显微技术的光滑大曲率样品测量装置与方法
CN204389397U (zh) * 2015-01-21 2015-06-10 南京中科神光科技有限公司 一种快速、多通道实时荧光定量检测装置
CN104677871A (zh) * 2015-02-27 2015-06-03 中国科学院自动化研究所 多光子激发光片照明显微成像系统
CN104730054B (zh) * 2015-04-10 2018-08-21 中国科学院烟台海岸带研究所 一种一体化探头式光电水质多参数在线测量系统
CN204789341U (zh) * 2015-07-01 2015-11-18 上海睿钰生物科技有限公司 集成式荧光激发光源装置
CN204945041U (zh) * 2015-07-28 2016-01-06 上海睿钰生物科技有限公司 一种多荧光通道同步显微成像装置
CN204945040U (zh) * 2015-07-28 2016-01-06 上海睿钰生物科技有限公司 荧光激发光源装置及系统和荧光显微成像系统

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2004325174A (ja) * 2003-04-23 2004-11-18 Olympus Corp 蛍光検出装置
CN101149327A (zh) * 2007-11-06 2008-03-26 浙江大学 基于细胞显微图像信息的抗肿瘤药物评价和筛选方法
CN102151122A (zh) * 2011-03-17 2011-08-17 中国科学院自动化研究所 激发荧光分子成像系统及一次荧光成像方法
US20130015362A1 (en) * 2011-07-12 2013-01-17 Sharp Kabushiki Kaisha Fluid purification and sensor system
CN104641222A (zh) * 2012-05-29 2015-05-20 麦考瑞大学 用于发光显微成像的双向扫描
CN104198458A (zh) * 2014-09-26 2014-12-10 哈尔滨工业大学 一种飞秒激光双光子荧光生物显微成像系统及其成像方法
CN104990907A (zh) * 2015-07-01 2015-10-21 上海睿钰生物科技有限公司 自动多通道类流式图像荧光分析系统
CN204945042U (zh) * 2015-07-28 2016-01-06 上海睿钰生物科技有限公司 自动多通道类流式图像荧光分析系统

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