CN101278829A - Portable in vivo flow cytometry - Google Patents
Portable in vivo flow cytometry Download PDFInfo
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- CN101278829A CN101278829A CNA2008100380448A CN200810038044A CN101278829A CN 101278829 A CN101278829 A CN 101278829A CN A2008100380448 A CNA2008100380448 A CN A2008100380448A CN 200810038044 A CN200810038044 A CN 200810038044A CN 101278829 A CN101278829 A CN 101278829A
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Abstract
The invention relates to a portable type flow cytometry in vivo, which comprises a light source irradiating system, an optical splitting system, a signal detecting, analyzing and processing system, a lighting system for positioning, an observation system for positioning and a sample platform. The light source irradiating system carries out laser irradiation on the marked interesting cell flow, so as to arouse and obtain the marked fluorescent transmit signal; after focalized by a microscope, the transmit signal enters into the signal detecting, analyzing and processing system by the optical splitting system to carry out detection, analysis and output of the signal. The invention adopts the semi-conductive laser as light source and has the advantages of small structure and portability, and a plurality of excitation wavelengths are selected. The detected signal provides the detailed information of the interesting cell such as cytometry. According to different detected cell types or different marking fluorescent agents, light source, light splitting and light-filtering optical components can be replaced, thus increasing the use range of the system.
Description
Technical field
The present invention relates to a kind of biomedical cell instrument that detects that is used for, especially a kind of body, real-time that can be implemented in, noinvasive ground analysis blood vessel inner cell flow behavior-at vivo flow cytometry.
Background technology
The method of circulating cells mainly is to take out in the patient body or in the animal body in current detection and the various sample blood of quantification, does to measure external after the fluorescent labeling.For example, the flow cytometer of standard is exactly like this to the analysis of various cells in the blood.The shortcoming of this detection method is, can not be at body, detect the flow behavior of various cells in the blood vessel in real time, during detection, need carry out sample making courses such as labelling to sample, and complicated operation is wasted time and energy; Because sample preparation and detection need regular hour and extra reagent, so the precision of measurement result is difficult to guarantee; Each measurement can only provide the result on the single time point, so be difficult to obtain one group of time dependent cell distribution characteristic; And the funds that such processing needs are more.
Current device at body detection tissue characteristics also has a lot, as laser confocal microscope, equipment such as optical tomography but this two kinds of equipment can only be more shallow to body surface (in 100 ~ 300um) scopes to imaging of tissue, and because the speed of its imaging, be difficult to the cell in the blood particularly the cell in the tremulous pulse follow the tracks of.
Therefore, in order to be implemented in body, real-time, portable analysis to cell in the blood vessel, be necessary to provide a kind of can be portable at the body stream type cell analyzer.
Summary of the invention
The present invention will solve the flow behavior technical problem that detects various cells in the blood vessel at body, in real time, and provides a kind of portable at vivo flow cytometry.
The present invention is achieved by the following technical solutions: of the present invention a kind of portable at vivo flow cytometry, comprise source illumination system, beam splitting system, acquisition of signal analysis process system, location illuminator, location observation system and example platform; Source illumination system carries out laser irradiation to the interested stream of cells through labelling, after exciting the microcobjective in source illumination system of transmitting of the fluorescence that obtains institute's labelling to focus on, enter into the acquisition of signal analysis process system by beam splitting system and carry out acquisition of signal and analysis and processing and output, the focused light that is sent with illuminator by the location shines the location luminous point that is shone by source illumination system on the example platform, the microcobjective imaging of location luminous point in source illumination system, enter the location through beam splitting system again and use the observation system imaging, on display screen, show.
Described source illumination system is by forming as the semiconductor laser diode LD of light source, extender lens, spatial filter, collimating lens, reflecting mirror with the microcobjective that the light beam behind the beam-expanding collimation focuses on.Source illumination system expands bundle with the oval-shaped beam of semiconductor laser diode LD emission by extender lens and enters spatial filter and leach behind the light beam high-order composition parallel beam to be entered focus on through collimating lens again and use to such an extent that microcobjective is focused into light beam the strip hot spot of 10 * 70 μ m.Such beneficial effect is the optical element that can reduce beam shaping, reduces cost and the complexity of system, improves the reliability of system.This light beam also is that flow cytometry analysis is needed simultaneously, mainly is in order to carry out the irradiation of light when individual cells flows through blood vessel, to avoid the irradiation to a plurality of cells.
Described beam splitting system comprises double-colored beam split optical filter, and between the reflecting mirror and microcobjective that are positioned over source illumination system of double-colored beam split optical filter, the light wave that makes light source and light source irradiation are separated at the interested fluorescence signal that ejects on unicellular.
Described acquisition of signal analysis process system is made up of detector, analog-to-digital conversion process circuit A/D, computer, optical filter, focusing coupled lens and spatial filter; Optical filter be one only by exciting the fluorescence signal of generation, block the bandpass filter of the optical signalling of other wave band, the purified fluorescence signal line focus coupled lens of other lightwave signal of optical filter filtering converges to spatial filter, the focus conjugation of spatial filter and microcobjective forms confocal system; Fluorescence signal enters detector D by spatial light filter and converts optical signal to the signal of telecommunication, and the signal of telecommunication is processed into through analog-to-digital conversion process circuit A/D again can data presented machine demonstration output as calculated.
Described location is made up of lighting source LED and condenser lens with illuminator; The position of the light line focus lens focus that lighting source LED sends excitation source irradiation in the sample is used to locate the observation with observation system.
Described location is made up of microcobjective, optical filter, focusing objective len, imaging CCD and display screen with observation system, optical filter is placed on the back of the dichroic filter of beam splitting system, the LED light wave of condenser lens coupling microcobjective drops on the imaging CCD plane focus of light wave, makes its imaging.
Described example platform be one can the three-dimensional platform that moves, the platform mobile accuracy is a micron dimension, example platform is provided with can control animal fixed restraint device on platform.
The invention has the beneficial effects as follows:
The present invention includes semiconductor laser (LD), energy of light source is focused on the unicellular irradiation Focused Optical system that carries out, the CCD imaging system that is used to observe the LD irradiation position, beam splitting system in the blood capillary and detection optical system that special cells in the blood vessel is detected as light source.
Adopting the beneficial effect of semiconductor laser in the source illumination system is that the general emitted light beams of this kind light source is oval-shaped beam, has reduced the required optical element of many beam shapings, has saved cost, has reduced system bulk and weight.It is that volume is little that lighting source adopts the beneficial effect of LED, and in light weight, caloric value is low, can not produce adverse influence to sample; Adopting the more useful effect of green LED irradiation is that green light is the main absorption bands of blood, so good to the blood vessel imaging in the tissue.
The present invention adopts semiconductor laser as light source, and other optical element volume is also controlled volume, so the whole system structure is small and exquisite, is easy to carry.The signal of surveying provides the details of interested cell such as cell counting etc.Can change light source and beam split, optical filtering optical element according to the difference of surveying cell type or mark fluorescent agent.The scope of application of increase system.
The present invention can be implemented in body, real-time, portable analysis to cell in the blood vessel.And it is small and exquisite to have volume, can be to the mobile unicellular detail analysis of carrying out in the blood capillary in the interested body, as interested cell (cancerous cell) counting, cell flowing velocity etc.
Description of drawings
Fig. 1 is a structured flowchart of the present invention;
Fig. 2 is a system diagram of the present invention;
Fig. 3 is a date processing block diagram of the present invention.
The specific embodiment
The present invention is further illustrated below in conjunction with accompanying drawing and embodiment.
The present invention includes: the unicellular radiating light source system of irradiation blood capillary internal labeling, the luminous point that light-source system is focused into 10 * 70 μ m with the luminous energy of semiconductor light sources is used to excite that the cell through labelling gives off fluorescence signal in the blood capillary; The CCD imaging system of observation light source irradiation position; Be used for exciting light, the beam splitting system of observing light and separating through the phosphor region that exciting radiation is come out; The detection light path and the analytical system of the fluorescence that detection radiates on unicellular through laser irradiation.
In the described light-source system, the light source of various light sources and various emission wavelengths can be applied to the cell of excitation labeling, adopt semiconductor laser for the volume that reduces system in the present invention, the hot spot form that the oval-shaped hot spot of semiconductor laser output adopts in the flow cytometer just, reduced the optical element of many beam shapings, for the miniaturization of system has been created condition.And for the control of semiconductor laser also than being easier to, its light wave is output as continuously and the pulse dual mode.The focusing system that also comprises laser beam in the light-source system adopts microcobjective that hot spot is focused into the hot spot of one 10 * 70 μ m so that shine single interested cell in the blood capillary in this system.This microcobjective also is the object lens of CCD imaging system and detection system simultaneously.Comprise two optical lenses and a microcobjective in the whole light source system, the effect of two optical lenses is with the laser beam beam-expanding collimation, is focused into the hot spot of one 10 * 70 μ m again through microcobjective.
Described CCD imaging system comprises the green light LED light source that is used to throw light on, condenser lens, optical filter and a CCD image-generating unit and display screen or computer monitor etc.To be that green light LED institute is luminous focus on the luminous point that sample shone by light-source system through laser mirror and locate after the microcobjective imaging in the light-source system concrete work process, enter into the CCD image-generating unit through beam splitting system again, the CCD image-generating unit comprises picture coupling mirror and CCD camera, shows the position of guaranteeing laser spot after the CCD imaging on display screen.
Described beam splitting system comprises two dichroic filter, and the function of these two optical filters is that a filtering excitation source light wave sees through the fluorescence light wave and will reflex to the CCD image-generating unit through the green glow that microcobjective is collected and be used for observation; Another fluorescence that is filtering laser light wave ejects cell after illuminated reflexes to detection system and is used for surveying;
Described detection light path and analytical system comprise a bandpass filter, an optical lens, and a spatial filter makes fluorescence signal focus on optical detection device and computer system; Concrete work process is that the optical lens that bandpass filter purification signal is collected usefulness through fluorescence signal incides optical detection device such as photomultiplier tube etc. through spatial filter again; After being received, this fluorescence signal also shown by software system analysis in the computer again.
The operation principle of native system is, the interested cell of object of study (animal or human body body surface position) is carried out labelling, through the interested stream of cells of labelling during through the blood capillary of laser irradiation, must obtain the transmitting of fluorescence of institute's labelling, this fluorescence signal is reflected back toward system, after microcobjective focuses on, enter into the detection light path by beam splitting system, detector detects this signal after analytical system, obtains cells of interest flowing and residence characteristics in vivo as the analysis of counting or intensity; Further can analyze transfer, the diffusion property of cancerous cell; Different according to object of study and mark mode, LASER Light Source among the present invention and beam splitting system and filter system can be changed, and can enlarge the scope of application of a whole set of instrument like this.
As shown in Figure 1, the present invention divides from function, comprises source illumination system 1; Beam splitting system 2; Acquisition of signal analysis process system 3; Location illuminator 4; Location observation system 5 and example platform 6.
By shown in Figure 2, described source illumination system 1 is by as the semiconductor laser diode LD of light source, extender lens 10, spatial filter 11, collimating lens 12, form for the reflecting mirror 13 that reduces system dimension with the microcobjective 14 that the light beam behind the beam-expanding collimation focuses on.Source illumination system 1 expands bundle with the oval-shaped beam of light source LD emission by extender lens 10 and enters spatial filter 11 and leach behind the light beam high-order composition parallel beam to be entered focus on through collimating lens 12 again and use to such an extent that microcobjective 14 is focused into the strip hot spot of 10 * 70 μ m with light beam, and the beneficial effect of such hot spot is the single interested labeled cell that can guarantee to shine in the single blood capillary.And the beneficial effect of employing semiconductor laser is that the general emitted light beams of this kind light source is oval-shaped beam in the source illumination system 1, has reduced the required optical element of many beam shapings, has saved cost, has reduced system bulk and weight.
By shown in Figure 2, described beam splitting system comprises double-colored beam split optical filter 20 and 21 two optical elements, double-colored beam split optical filter 20 be positioned over source illumination system 1 reflecting mirror 13 and microcobjective 14 between, effect is that the light wave of light source and light source irradiation are separated at the interested fluorescence signal that ejects on unicellular, promptly shield the optical signal of light source, make fluorescence signal enter into detection analysis processing system 3 and carry out acquisition of signal and analysis.
By shown in Figure 2, described acquisition of signal analysis process system 3 is made up of detector D, analog-to-digital conversion process circuit A/D, computer Computer, optical filter 31, focusing coupled lens 32 and spatial filter 33; Optical filter 31 is a bandpass filter, only by exciting the fluorescence signal of generation, blocks the optical signalling of other wave band; Purified fluorescence signal line focus coupled lens 32 through optical filter 31 other lightwave signals of filtering converges to spatial filter 33, the focus conjugation of spatial filter 33 and microcobjective 14, form confocal system, the beneficial effect of doing like this is to reduce background noise, improves the resolution of system; Fluorescence signal enters detector D by spatial light filter 33 and converts optical signal to the signal of telecommunication, and the signal of telecommunication is processed into through analog-to-digital conversion process circuit A/D again can data presented machine Computer demonstration output as calculated.
As shown in Figure 3, the fluorescence signal of surveying converts the signal of telecommunication to after enter analog-to-digital conversion process circuit A/D after the data collecting card collection through detector D, forms data type that machine data card as calculated handles display process on computers through data processing unit again.
By shown in Figure 2, described location is made up of lighting source LED and condenser lens 40 with illuminator 4; It is that volume is little that lighting source adopts the beneficial effect of LED, and in light weight, caloric value is low, can not produce adverse influence to sample; Adopting the more useful effect of green LED irradiation is that green light is the main absorption bands of blood, so good to the blood vessel imaging in the tissue.The effect of condenser lens 40 is the positions that make the excitation source irradiation in the sample of light focusing that illumination LED sends.So that the location is observed with observation system 5.
By shown in Figure 2, the location is made up of microcobjective 14, optical filter 42, focusing objective len 43, imaging CCD and display screen Screen with observation system 5, optical filter 42 is placed on the back of 2 kinds of dichroic filter 21 of beam splitting system, its effect is the light wave of only launching by LED, blocks other lightwave signal and enters CCD; The definition that helps imaging; The LED light wave of condenser lens 43 coupling microcobjectives 14 drops on the imaging CCD plane focus of light wave, makes its imaging.
By shown in Figure 2, example platform 6 be one can the three-dimensional platform that moves, mobile accuracy is controlled at micron dimension, more helps the focusing of radiation source system 1, finds suitable blood capillary to survey.Example platform 6 is provided with animal restraint device, can control animal and maintain static on platform 6.
Elements such as the light source in the above system, beam split optical filter can according to the difference of the interested cell classification of being studied the characteristics of mark fluorescent change; Make systemic-function more powerful.In addition,, be convenient to the industrialization of system, the element that all elements all adopt standard or can buy in general producer in order to reduce cost.
Claims (7)
- One kind portable at vivo flow cytometry, it is characterized in that, comprise source illumination system (1), beam splitting system (2), acquisition of signal analysis process system (3), location illuminator (4), location observation system (5) and example platform (6), described source illumination system (1) carries out laser irradiation to the interested stream of cells through labelling, excite transmitting of the fluorescence that obtains institute's labelling, after microcobjective focuses on, enter into acquisition of signal analysis process system (3) by beam splitting system (2) and carry out acquisition of signal, processing and output analysis result; The focused light that is sent with illuminator (5) by the location shines the location luminous point that example platform (6) is upward shone by source illumination system (1), the microcobjective imaging of location luminous point in source illumination system (1), enter the location through beam splitting system again and use the observation system imaging, on display screen, show.
- 2. according to claim 1 portable at vivo flow cytometry, it is characterized in that described source illumination system (1) is formed by semiconductor laser diode (LD), extender lens (10), spatial filter (11), collimating lens (12), reflecting mirror (13) with the microcobjective (14) that the light beam behind the beam-expanding collimation focuses on; The oval-shaped beam of semiconductor laser diode (LD) emission expands by extender lens (10) makes parallel beam enter the strip hot spot that the microcobjective (14) that focuses on usefulness is focused into light beam 10 * 70 μ m through collimating lens (12) after bundle enters spatial filter (11) filtering light beam high-order composition again.
- 3. according to claim 1 portable at vivo flow cytometry, it is characterized in that, described beam splitting system (2) comprises double-colored beam split optical filter (20,21), between the reflecting mirror that is positioned over source illumination system (1) (13) and microcobjective (14) of double-colored beam split optical filter (20), the light wave that makes light source and light source irradiation are separated at the interested fluorescence signal that ejects on unicellular.
- 4. according to claim 1 portable at vivo flow cytometry, it is characterized in that described acquisition of signal analysis process system (3) is made up of detector (D), analog-to-digital conversion process circuit (A/D), computer, optical filter (31), focusing coupled lens (32) and spatial filter (33); Optical filter (31) be one only by exciting the fluorescence signal of generation, block the bandpass filter of the optical signalling of other wave band, the purified fluorescence signal line focus coupled lens (32) of optical filter (31) other lightwave signal of filtering converges to spatial filter (33), the focus conjugation of spatial filter (33) and microcobjective (14) forms confocal system; Fluorescence signal enters detector (D) by spatial light filter (33) and converts optical signal to the signal of telecommunication, and the signal of telecommunication is processed into through analog-to-digital conversion process circuit (A/D) again can data presented machine demonstration output as calculated.
- 5. according to claim 1 portable at vivo flow cytometry, it is characterized in that described location is made up of lighting source LED and condenser lens (40) with illuminator (4); The light line focus lens (40) that lighting source LED sends focus on the position of excitation source irradiation in the sample, are used for the observation of location with observation system (5).
- 6. according to claim 1 portable at vivo flow cytometry, it is characterized in that, described location is made up of microcobjective (14), optical filter (42), focusing objective len (43), imaging CCD and display screen with observation system (5), optical filter (42) is placed on the back of the dichroic filter (21) of beam splitting system (2), the LED light wave of condenser lens (43) coupling microcobjective (14), the focus of light wave is dropped on the imaging CCD plane, make its imaging.
- 7. according to claim 1 portable at vivo flow cytometry, it is characterized in that, described example platform (6) be one can the three-dimensional platform that moves, the platform mobile accuracy is a micron dimension, and example platform (6) is provided with can control animal at the last fixed restraint device of platform (6).
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| CN110687034A (en) * | 2018-07-05 | 2020-01-14 | 深圳迈瑞生物医疗电子股份有限公司 | Laser irradiation system of flow cytometer and flow cytometer |
| CN110376171B (en) * | 2019-07-15 | 2021-11-19 | 上海理工大学 | Transmission type fluorescence detection imaging system applied to dPCR detector |
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