CN104764727A - Fluorescence imaging analysis system and fluorescence imaging analysis method thereof - Google Patents
Fluorescence imaging analysis system and fluorescence imaging analysis method thereof Download PDFInfo
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- CN104764727A CN104764727A CN201510186113.XA CN201510186113A CN104764727A CN 104764727 A CN104764727 A CN 104764727A CN 201510186113 A CN201510186113 A CN 201510186113A CN 104764727 A CN104764727 A CN 104764727A
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Abstract
The invention discloses a fluorescence imaging analysis system and a fluorescence imaging analysis method. The fluorescence imaging analysis system comprises a shell, and at least one excitation device and an imaging device, which are arranged inside the shell; the excitation device comprises an excitation light source and an excitation filter; light rays emitted by the excitation light source pass through the excitation filter to form monochromatic excitation light and the monochromatic excitation light is irradiated to a sample stage, and a detected substance emits fluorescent light after being excited; and the imaging device is used for capturing fluorescent light passing through a piece of filter glass and analyzing and determining the concentration of the fluorescent substance. Compared with the prior art, the system can acquire the reliable detection result without arranging an expensive optical grating, an interference color filter and a photomultiplier and determining an electronic circuit, the structure is skillful and simple, the preparation cost is low and the system and the method can be widely applied.
Description
Technical field
The invention belongs to field of biology, be specifically related to a kind of fluorescence imaging analysis system and fluorescence imaging analysis method thereof.
Background technology
The material that another wavelength is greater than light absorbing light wave can be launched after fluorescent dye general reference absorbs the light wave of a certain wavelength.Due to highly sensitive, easy to operate, fluorescent dye instead of radioactive isotope gradually as scientific research certification mark, and it is widely used in fluorescence immunoassay, fluorescence probe, cell dyeing etc.Comprise specific DNA to dye, for correlative studys such as chromosome analysis, cell cycle, Apoptosis.Separately have a lot of nucleic acid dye to be very useful counterstain in polychromatic dyeing system, can be used as ground control, labeled cell core makes the spatial relationship of intracellular structure very clear.But existing detection of fluorescent dyes device tends to high sensitivity, pinpoint accuracy, high automation more, cause detecting instrument complex structure, with high costs, the working service of instrument requires higher simultaneously, limits the application of fluorescent dye on detecting.Such as, but the application achievements of fluorescent dye in biological study is being generalized to wider daily use field, medical test, food inspection etc.Therefore, need exploitation badly a kind of general, cheap, possess the pick-up unit of enough accuracy of detection and sensitivity simultaneously.
Summary of the invention
The present invention proposes a kind of fluorescence imaging analysis system, it at least one excitation apparatus and imaging device of comprising shell and being arranged on enclosure; Wherein,
Described shell is closed hollow housing, its inner formation darkroom, and the inside of described shell is provided with sample stage, for placing the detected material carrying fluorescent material; Described shell is provided with light hole, and described light hole is just corresponding to the position of described sample stage, by sample stage described in light hole Observable;
Described excitation apparatus is arranged on the inside of described shell, and it comprises excitation source and exciter filter; The light that described excitation source sends forms monochromatic exciting light through described exciter filter and is irradiated to described sample stage; Fluorescence is sent after the described detected material be positioned on described sample stage is stimulated;
Described imaging device comprises filter glass and image unit; Described filter glass is right against described light hole and arranges, and for the veiling glare except fluorescence except of filtering through described light hole, described image unit is right against described filter glass and arranges, for catching the fluorescence through described filter glass.Described imaging device can arrange light hole position on the housing, is right against described sample stage.
In the present invention, described fluorescent material include but not limited to fluorescent dye, fluorescin or other any be stimulated after can there is the material of fluorescence.
In fluorescence imaging analysis system of the present invention, the optical property of the fluorescent material that can contain according to detected material, determines to select to arrange described excitation source, described exciter filter or described filter glass accordingly.
Excite according to difference and need to carry out selecting different excitation sources, the cutoff wavelength of exciter filter will be taken into account simultaneously.In fluorescence imaging analysis system of the present invention, the cutoff wavelength of described exciter filter is the wavelength values scope between the excitation wavelength corresponding with described fluorescent material and emission wavelength, thus guarantee only to allow exciting light through described exciter filter, and the utilizing emitted light generated by fluorescent material can not through this exciter filter.Such as, the detected nothing of the fluorescent material containing SYBR Green I+DNA solution, the excitation wavelength of SYBR Green I is 497nm, and the emission wavelength of the exciting light generated is 520nm.In fluorescence imaging analysis system, excitation source is blue led lamp, and the wavelength of blue light is 476-495nm; Exciter filter is ZB1 model, and its transmittance spectrum scope is 300 ~ 500nm.The cutoff wavelength of this exciter filter is between excitation wavelength and emission wavelength.
In fluorescence imaging analysis system of the present invention, described excitation source includes but not limited to LED light source and any suitable light source etc.
In a specific embodiment, when fluorescent material is green fluorescence material, the spectral range of described excitation source is 445 ~ 475nm; The transmittance spectrum scope of described exciter filter is 325nm ~ 500nm; The transmittance spectrum scope of described filter glass is 500nm ~ 2500nm; Or,
In another embodiment, when fluorescent material is red fluorescent material, the spectral range of described excitation source is 585nm ± 29nm; The transmittance spectrum of described exciter filter is 500nm ~ 620nm; The transmittance spectrum scope of described filter glass is 620nm ~ 2500nm; Or,
In another embodiment, when fluorescent material is yellow fluorescent substance, the spectral range of described excitation source is 531nm ± 40nm; The transmittance spectrum of described exciter filter is 350nm ~ 580nm; The transmittance spectrum scope of described filter glass is 580nm ~ 2500nm; Or,
In another embodiment, when fluorescent material is blue fluorescent substance, the spectral range of described excitation source is 357nm ± 44nm; The transmittance spectrum of described exciter filter is 280nm ~ 410nm; The transmittance spectrum scope of described filter glass is 410nm ~ 2500nm; Or,
In other embodiments, according to the optical property of any fluorescent dye/fluorescin/or other fluorescent material, the excitation source of suitable spectral range, exciter filter or filter glass can be selected accordingly.
Select crest in one embodiment in the LED of 453nm as excitation source.
In fluorescence imaging analysis system of the present invention, described excitation apparatus is provided with scattering part further, and it is arranged between described excitation source and described exciter filter.The light that described excitation source sends forms uniform light beam irradiation to described exciter filter after described scattering part.
In fluorescence imaging analysis system of the present invention, described scattering part is in-plane scatter mirror, and the light that described excitation source sends forms uniform beam through described in-plane scatter mirror and exposes to described exciter filter, then is irradiated on sample stage.
In fluorescence imaging analysis system of the present invention, described scattering part can also comprise plane mirror and cambered surface catoptron, described plane mirror is used for the light reflection of described excitation source to described cambered surface catoptron, described cambered surface catoptron forms uniform beam and reflexes to described exciter filter, then is irradiated on sample stage.
In fluorescence imaging analysis system of the present invention, the bottom of described sample stage is provided with slide rail, for described sample stage is shifted out described shell.
In fluorescence imaging analysis system of the present invention, " image unit " refers to the device catching sample excitation light, such as, and digital camera.
In fluorescence imaging analysis system of the present invention, described imaging device comprises shooting analytic unit further, and it is connected with image unit, for measuring and analyze the concentration of described fluorescent material.In one embodiment, described image unit and described shooting analytic unit also can be wholely set, its concentration also measuring for catching fluorescence and analyze the fluorescent material of catching.
In fluorescence imaging analysis system of the present invention, described shooting analytic unit comprises:
Digital image acquisition units, it obtains digital picture for the fluorescence caught through described filter glass;
Graphics processing unit, it is connected with digital image acquisition units, for analyzing the amount of fluorescence etc. in described digital picture;
Image-display units, it is connected with described graphics processing unit, for showing described digital picture, amount of fluorescence; And described image-display units and graphics processing unit carry out interactive operation etc.
Described shooting analytic unit comprises data storage cell further, and it is connected with described graphics processing unit, for storing the information such as described digital picture, amount of fluorescence.
In fluorescence imaging analysis system of the present invention, described excitation source is adjustable excitation source, and it comprises the excitation source of at least more than one different spectral ranges; Described exciter filter is adjustable exciter filter, and it comprises the exciter filter of at least more than one different spectral ranges.In one embodiment, multiple filter array of different spectral range are arranged on draw-in groove or support, need by actual conditions, the optical filter of different spectral range can be selected by rotation draw-in groove or support.The installation of described adjustable light source in fluorescence imaging analysis system can with reference to the usual method of prior art.
Similarly, described filter glass is turnable filter sheet, and it comprises the filter glass of at least more than one different spectral ranges.In one embodiment, multiple filter glasss of different spectral range being arranged on draw-in groove or support, needing by actual conditions, different spectral range can be selected by rotating draw-in groove or support.
In the present invention, the physical constructions such as adjustable exciter filter, turnable filter sheet all by existing usual technical design, can be arranged in fluorescence imaging analysis system with reference to usual method.
The invention allows for a kind of fluorescence imaging analysis method, comprise the steps:
Step 1: the detected material carrying fluorescent material is placed on sample stage, sample stage is positioned over enclosure;
Step 2: select to determine excitation source, exciter filter and filter glass by the optical characteristics of described fluorescent material, described excitation source and described exciter filter are arranged in excitation apparatus, described filter glass are arranged on the lead to the hole site be positioned on described shell;
Step 3: open described excitation source, exciter filter described in the light therethrough of described excitation source forms monochromatic exciting light, and described exciting light is radiated on the detected material on described sample stage; Fluorescent material in described detected material excites generation fluorescence by described exciting light;
Step 4: the veiling glare of described filter glass filtering except fluorescence, catches the fluorescence through described filter glass by image unit, obtains fluoroscopic image and fluorescence values.
In fluorescence imaging analysis method of the present invention, utilize described fluorescence imaging analysis system to realize detecting the actual concentrations of detected material, comprise the steps: further after described step 4
Step 5: with detecting gained fluorescence values and detected material concentration drawing standard curve, obtain equation of linear regression;
Step 6: will carry fluorescent material and the tested substance of concentration the unknown is placed on described sample stage, is positioned over described enclosure by described sample stage, repeats fluoroscopic image and fluorescence values that step 2 ~ 4 obtain described detected material;
Step 7: utilize described shooting analytic unit gained fluorescence values to be substituted into the actual concentrations calculating detected material in described equation of linear regression.
Beneficial effect of the present invention comprises:
The excitation source adopted in the present invention has multiple advantage: LED monochromaticity is relatively good, and ripple is wide narrow, and alternative large.Such as green fluorescent protein exciting light crest 470nm, if select peak to be the LED of 475nm, has very little a part of optical wavelength and reaches 500nm, can leak through color filter film.In order to avoid impact measures, peak is selected to be the light that the LED of 453nm does not just have to leak through color filter film.LED stable luminescence, stable light source is extremely important for mensuration.And LED has the advantages such as power saving is inexpensive.
The present invention adopts excellent optical design, and first, the monochromaticity of exciting light, decreases such as with ultraviolet light exciting other fluorescence of sample.Secondly, the application of scattering light transmission piece makes illumination uniformity on sample stage.The black box design of present system inside, get rid of entering of veiling glare, all these are designed to mensuration and provide extraordinary optical environment.
In the present invention, in shooting analysis component, the digital picture utilizing digital image acquisition units to obtain can utilize graphics processing unit carry out real time readouts and store.Compared with the image unit (as camera) of simple function, utilize image unit/parts and shooting analytic unit/parts can obtain the quantitative information (such as, the concentration of fluorescent material) of fluoroscopic image and fluorescent material simultaneously.In addition, digital image acquisition units is used can also to record dynamic image.
In the present invention, power electric needed for LED light source and imaging identification system is forced down, energy consumption is little, coordinates instrument built-in lithium battery can not have out of doors etc. the local work of power supply for a long time.
Expensive fluorescent instrument of the prior art is compared, and the present invention, without the need to adopting expensive grating and interference filter etc., without the need to adopting photomultiplier and measuring electronic circuit etc., and can realize splendid qualitative and quantitative Detection results.The structural design of fluorescence imaging analysis system of the present invention is exquisite, forms simple, and be easy to safeguard, accessory is easily changed, and cost is low, is suitable for widespread use.
Accompanying drawing explanation
Fig. 1 is the structural representation of Image analysis system.
Fig. 2 is the structural representation of Image analysis system in another embodiment.
Fig. 3 is the structural representation of Image analysis system in another embodiment.
Fig. 4 (a) is the sectional view of shell, and Fig. 4 (b) is the external view of shell.
Fig. 5 is the schematic diagram of shooting analytic unit.
Fig. 6 is the schematic diagram of hardware circuit in shooting analytic unit.
Fig. 7 is the image shown by image-display units.
Fig. 8 (a) is the schematic diagram of draw-in groove, and Fig. 8 (b) is the schematic diagram of driver plate.
Fig. 9 is the rectilinear of the present invention and prior art the data obtained in embodiment 1.
Figure 10 is the scatter diagram of the corresponding reading of variable concentrations in embodiment 3.
Figure 11 is the image in a specific embodiment shown by image-display units.
Figure 12 is the image in a specific embodiment shown by image-display units.
Figure 13 is mean value and the standard error of 5 readings in a pipe position in eight unions in this specific embodiment.
Embodiment
In conjunction with following specific embodiments and the drawings, the present invention is described in further detail.Implement process of the present invention, condition, experimental technique etc., except the following content mentioned specially, be universal knowledege and the common practise of this area, the present invention is not particularly limited content.
As in Fig. 1 ~ 10,10-shell, 11-sample stage, 12-light hole, 13-pulley, 14-door, 15-draw-in groove, 16-driver plate 20-excitation apparatus, 21-excitation source, 22-scattering part, 221-plane mirror, 222-cambered surface catoptron, 23-exciter filter, 30-imaging device, 31-filter glass, 32-image unit, 33-makes a video recording analytic unit, 331-digital image acquisition units, 332-graphics processing unit, 333-image-display units, 334-data storage cell.
As depicted in figs. 1 and 2, fluorescence imaging analysis system of the present invention comprises shell 10, is arranged at least one excitation apparatus 20 of shell 10 inside and imaging device 30.
Shell 10 is the hollow housing closed, its inner formation darkroom, and the inside of shell 10 is provided with sample stage 11, for placing the detected material carrying fluorescent dye/fluorescin; The surface of shell 10 is provided with a light hole 12, and light hole 12 is right against sample stage 11, for observing samples platform 11.
See Fig. 1, in this specific embodiment, the quantity of excitation apparatus 20 is one, and this excitation apparatus 20 is arranged on the inside of shell 10, and it comprises excitation source 21 and exciter filter 23; The light that excitation source 21 sends forms monochromatic exciting light through exciter filter 23 and is irradiated to sample stage 11, sends fluorescence after detected material is stimulated.In the present embodiment, shell 10 inside is provided with excitation apparatus 20, and excitation apparatus 20 is separately positioned on the both sides of light hole 12, and its monochromatic excitation light exported is radiated on the detected material of sample stage 11 obliquely.See Fig. 2, the quantity of excitation apparatus 20 is two, and two excitation apparatus 20 are arranged on the inside of shell 10 symmetrically.The quantity of the excitation apparatus 20 in the present invention is not limit, and comprise 1,2 or multiple, the setting position of excitation apparatus 20 is not limit, and can adjust according to actual installation situation.
Imaging device 30 is right against the outside that sample stage 11 is arranged on shell 10, and it comprises filter glass 31 and image unit 32; Filter glass 31 is right against light hole 12 and arranges, and for the veiling glare except fluorescence except of filtering through light hole 12, image unit 32 is right against filter glass 31 and arranges, for catching the fluorescence through filter glass 31.The excitation wavelength of common Green fluorescent dye and the emission wavelength of fluorescence are see with following table 1.
The common Green fluorescent dye of table 1. excites and emission wavelength
| Dyestuff title | Excitation wavelength (nm) | Emission wavelength (nm) |
| SYBR Green I/II | 497 | 520 |
| Gel Green | 500 | 520 |
| SYTO 13 | 488 | 509 |
| SYTO 16 | 488 | 518 |
| FITC(Cy3) | 490-495 | 525-530 |
| AlexaFluor | 495 | 519 |
| GFP | 483 | 509 |
| Calcein | 486 | 509 |
In order to form the exciting light of above-mentioned respective wavelength, excitation source 21 and exciter filter 23 need be adopted to form the monochromatic excitation light of respective wavelength scope.Excitation source 21 adopts LED light source, and its spectral range is 445 ~ 475nm.Excitation source 21 inside can be provided with organizes LED more, and comprise blue led, red LED and White LED etc. and often organize LED, shades of colour light source is independently controlled by different switches, thus selects the light source of different wavelength range.Such as, the centre wavelength of blue led lamp is 453nm, is applicable to and excites the such as Green fluorescent dye/fluorescin GFP such as SRBY Breen I/II, SYTO 13, FITC (Cy3), Calcein.
Before the detected material containing above-mentioned fluorescent dye/fluorescin is detected, need by fluorescent dye/fluorescin or other fluorescent material to determine the model of excitation source 21, exciter filter 23 and filter glass 31.Specifically, if for green fluorescence material, the transmittance spectrum scope of exciter filter 23 is 325nm ~ 500nm, and the transmittance spectrum scope of filter glass 31 is 500nm ~ 2500nm.Such as, if exciting light is blue excitation light, blue wavelength is 476-495nm, and the selectable model of corresponding filter glass 31 is JB470 (λ tj470 ± 10nm); If exciting light is green exciting light, green wavelength is generally 495-570nm, the selectable model of corresponding filter glass 31 is golden yellow cut-off type glass (JB510, Shanghai organic optical glass factory, λ tj510 ± 10nm) or JB490 (λ tj490 ± 10nm), can the veiling glare of the following wavelength of filtering 500nm, coordinate blue led lamp to can be used for detecting Green fluorescent dye/fluorescin; If exciting light is yellow exciting light, yellow wavelengths is generally 570-590nm, and the selectable model of corresponding filter glass 31 is CB565 (λ tj565 ± 10nm) or CB580 (λ tj580 ± 10nm); Red light wavelength is 620-750nm, and respective filter can select HB610/620/630.
In the present invention, the optical property relation between fluorescent material, excitation source, exciter filter, filter glass as shown in the following Table 2.
Optical property relation between table 2 fluorescent material, excitation source, exciter filter, filter glass
As shown in Figure 2, scattering part 22 is provided with further between excitation source 21 and exciter filter 23, scattering part 22 is a plane mirror, the light that excitation source 21 sends forms uniform light beam irradiation to exciter filter 23 after scattering part 22, thus generate uniform exciting light and be irradiated to sample stage 11, the detected material that its top is placed is subject to intensity of illumination everywhere and irradiates uniformly.
In the another preferred embodiment of the present invention, scattering part 22 is plane mirror 221 and cambered surface catoptron 222, as Fig. 3, plane mirror 221 for by the light reflection of excitation source 21 to cambered surface catoptron 222, cambered surface catoptron 222 forms uniform beam reflection to exciter filter 23.
As shown in Fig. 4 (a) He Fig. 4 (b), shell 10 is closed darkrooms, conveniently place detected material on sample stage 11, offering at the side of shell 10 can the door 14 of folding, the bottom of sample stage 11 is provided with pulley 13, and the chute (not shown) cooperatively interacted with pulley 13.Sample stage 11 utilizes pulley 13 to move along chute, can shift out shell 10 be convenient to place detected material from this door 14.After being placed detected material, more inner along slide to shell 10, close door make 14 shell 10 keeps a closed darkroom.
As shown in Figure 5, imaging device 30 also comprises shooting analytic unit 33.Shooting analytic unit 33 is connected with image unit 32, and for catching fluorescence, also mensuration and analysis of fluorescence material comprise the concentration of fluorescent dye or fluorescin to this shooting analytic unit 33.See Fig. 5, this shooting analytic unit 33 comprises digital image acquisition units 331, graphics processing unit 332 and image-display units 333.Digital image acquisition units 331 obtains digital picture for catching fluorescence.Graphics processing unit 332 is connected with digital image acquisition units 331, for the amount of fluorescence in analysing digital image.Image-display units 333 is connected with graphics processing unit 332, and image-display units 333 can show digital picture and amount of fluorescence thereof simultaneously.Further setting data storage unit 334 in this shooting analytic unit 33, data storage cell 334 is connected with graphics processing unit 332, has obtained digital picture and amount of fluorescence for storing.
In present pre-ferred embodiments, that digital image acquisition units 331 adopts is the OV7670 of OV (Omni Vision) company.Consult Fig. 6, graphics processing unit 332 adopts the micro-chip processor of the STM32F103RCT6 model of ARM V7 framework, hardware platform is ALIENTEK MiniSTM32 development board, graphics processing unit 332 control OV 7670 carries out digital image acquisition, and the image of collection is processed, draw the amount of fluorescence of fluorescent material in digital picture.Image-display units 333 is ALIENTEK3.5 cun of TFT LCD module.See Fig. 7, the digital picture that image-display units 333 Graphics Processing obtains and amount of fluorescence (rgb value), the final sample readings with reference to gathering in the past, display Yin/Yang.Data storage cell 334 is Kingston 4GB SD card.
This shooting analytic unit 33 and the integral setting of image unit 32, be arranged on the outside of shell 10.Measurement waiting status can be entered after opening shooting analytic unit 33, press " beginning " button can automatically measure, measure about 3 seconds consuming time, can select after measurement completes whether to preserve this measurement result in data storage cell 334, select "Yes" then can preserve measurement result in Kingston 4GB SD card, enter measurement waiting status next time afterwards; If be unsatisfied with this measurement result, select "No" then to abandon this measurement result, shooting analytic unit 33 enters measurement waiting status next time automatically.After measurement completes, the measurement result of preservation can search from the Kingston 4GB SD card of this device, and can import on other any memory devices.
About adjustable excitation source, it comprises the LED light source of at least one different spectrum property, and LED light source is integrated in and excites in excitation apparatus 20, by the open and close of switch control rule respective color LED light source.
About adjustable exciter filter, it utilizes the mode such as draw-in groove 15 or driver plate 16 to regulate required exciter filter.The adjustable exciter filter of what Fig. 8 (a) showed is card slot type, extracts exciter filter during replacement, is inserted by the exciter filter of required spectral range in draw-in groove 15 fixing from draw-in groove 15.The adjustable exciter filter of what Fig. 8 (b) showed is dial-type, multiple exciter filter is arranged on circular driver plate 16 by it, rotate this driver plate 16 by modes such as rotating shafts, the exciter filter of corresponding light spectral limit is rotated to the below of excitation source 21.Turnable filter sheet utilizes the mode such as draw-in groove 15 or driver plate 16 to realize too.The structure of adjustable exciter filter, turnable filter sheet all can design by prior art.
Embodiment 1
1. build and cultivate the Escherichia coli carrying fluorescence protein expression carrier, the name of fluorescin is called GFP (green fluorescent protein), 395nm and 475nm is maximum and secondary large excitation wavelength respectively.
2. the quantitative measurement of exciting light:
Step 1: by concentration known and a series of Escherichia coli of carrying fluorescence protein expression carrier of gradient dilution be placed on successively on sample stage, each detect a sample.Detected material carries fluorescent material, and sample stage is positioned over enclosure;
Step 2: excitation source 21 is blue led lamp; Exciter filter 23 is ZB1 model, and its transmittance spectrum scope is 300 ~ 500nm; Filter glass 31 is JB510 model, and its transmittance spectrum scope is 510nm ± 10nm ~ 2500nm.Select to determine excitation source, exciter filter and filter glass by the optical characteristics of fluorescent material, excitation source and exciter filter are arranged in excitation apparatus, filter glass are arranged on the lead to the hole site be positioned on shell;
Step 3: open excitation source, the light therethrough exciter filter of excitation source forms monochromatic exciting light, and exciting light is radiated on the detected material on sample stage; Fluorescent material stimulated luminescence in detected material excites generation fluorescence;
Step 4: the veiling glare of filter glass filtering except fluorescence, catches the fluorescence through filter glass by image unit, obtains fluoroscopic image and fluorescence values;
Meanwhile, the Escherichia coli of carrying fluorescence protein expression carrier are placed in Fluorescence spectrophotometer of the prior art; The fluorospectrophotometer selected is Varian Cary Eclipse, and the exciting light of employing is blue, and wavelength coverage is 485nm ± 2.5nm.
3. the correlation analysis of quantitative data and fluorospectrophotometer testing result:
See the green fluorescent protein representing variable concentrations with the point in following table 3 and Fig. 9, Fig. 9, straight line represents the linear regression relation between two kinds of instrument readings, and two kinds of detection methods present good correlativity (R
2=0.98).As can be seen here, testing result and the existing apparatus of fluorescence imaging analysis system of the present invention have same linear relation, and checking effectively can be applied to fluoroscopic examination experiment.
Table 3: two kinds of pick-up units are to the detected value of fluorescence signal
Embodiment 2
Image unit 32 and shooting analytic unit 33 is make use of in the present embodiment.Image unit 32 adopts digital camera, utilizes the shooting analytic unit 33 be connected with digital camera to carry out Real-time measuring and analyzing.The object of the present embodiment is the relation in order to determine green fluorescent protein concentration and reading in larger concentration range.The measuring condition of the present embodiment, the shooting condition (comprising aperture and time shutter) of digital camera, green fluorescent protein kind and corresponding optical filter thereof are all identical with embodiment 1, equation of linear regression wherein for measuring actual concentrations is recorded by above embodiment 1, equation of linear regression is: y=0.0532x+16.628, R
2=0.9803.If equation of linear regression is unknown, then repeats step 2-4 and obtain equation of linear regression with drawing standard curve.
The present embodiment regulates green fluorescent protein concentration and green fluorescence under measuring variable concentrations wears white reading to verify the relation of concentration and reading, and detailed process is as follows:
Step 5: utilize above embodiment 1 to detect gained fluorescence values and this e. coli concentration drawing standard curve, obtain equation of linear regression;
Step 6: will carry fluorescent material and the Escherichia coli matter of concentration the unknown is placed on sample stage, is positioned over enclosure by sample stage, repeats step 2-4 and obtains this colibacillary fluoroscopic image and fluorescence values.
Step 7: utilize shooting analytic unit 33 that gained fluorescence values is substituted into equation of linear regression, calculate this colibacillary actual concentrations.
By above step 5 ~ 7, carry out imaging analysis to the detected material of variable concentrations (GFP amount), the data of acquisition are as shown in following table 4 and Figure 10.Result shows that green fluorescent protein concentration and reading are good linear relationship.Fluorescence imaging analysis system of the present invention, needed to carry out linear determination before measuring each fluorescent dye, to determine the range of linearity of different fluorescent dye.If the conventional sense of the fluorescent material for fixed test condition, then step 2-4 need not adjust after setting first, and the equation of linear regression obtained also need not adjust.
Table 4 is in the relation of identical shooting condition green fluorescent protein reading and concentration
| Reading | Concentration |
| 32 | 1 |
| 62 | 2 |
| 84 | 3 |
| 102 | 4 |
| 116 | 5 |
| 130 | 6 |
| 139 | 7 |
Embodiment 3
In the present embodiment, adopt the shooting analytic unit 33 of image unit 32 and Highgrade integration to catch fluorescence and measure and analysis of fluorescence dyestuff/fluorescent protein concentration.Image unit 32 and the integral setting of shooting analytic unit 33, substituted for the working method of digital camera and computing machine combination in the past.The image unit 32 of integrated integrated setting and shooting analytic unit 33, greatly reduce the volume and weight of present system, reduce its cost of manufacture under meeting the prerequisite of its performance, easy to use.
Adopt SYBR Green I to send fluorescence analysis in conjunction with DNA in the present embodiment, the eppendorf pipe containing SYBR Green I+DNA solution is placed in fluorescence imaging analysis system of the present invention.Wherein, excitation source 21 is blue led lamp; Exciter filter 23 is ZB1 model, and its transmittance spectrum scope is 300 ~ 500nm; Filter glass 31 is JB510 model, and its transmittance spectrum scope is 510nm ± 10nm ~ 2500nm; That digital image acquisition units 331 adopts is the OV7670 of OV (Omni Vision) company.Graphics processing unit 332 adopts the micro-chip processor of the STM32F103RCT6 model of ARM V7 framework, hardware platform is ALIENTEK MiniSTM32 development board, graphics processing unit 332 control OV 7670 carries out digital image acquisition, and the image of collection is processed, draw the average RGB value of digital picture, the final sample readings MARK=149 with reference to setting, this rgb value is higher than MARK, and display is positive.Image-display units 333 is ALIENTEK 3.5 cun of TFT LCD module.The digital picture that process obtains by image-display units 333 and rgb value show.As shown in figure 11, the highlight regions in figure is the exciting light that detected material Green fluorescent material produces, with reference to the sample readings " 149 " of setting as MARK, the Yin/Yang demonstrating testing result for positive (in Figure 11 with yang! Represent).
Embodiment 4
In the present embodiment, detection hardware platform, with embodiment 3, adopts SYBR Green I to send fluorescence in conjunction with DNA and carries out multisample analysis.Eppendorf eight union containing SYBR Green I+DNA solution is placed in fluorescence imaging analysis system of the present invention.The image collected is divided into 8 parts by graphics processing unit 332 automatically, calculates the fluorescence signal of each part respectively and provides fluorescence values.As shown in figure 12, the highlight regions in figure is the exciting light that detected material Green fluorescent material produces, and with reference to the sample readings set as MARK, demonstrates the Yin/Yang of testing result.Do 5 duplicate readings to same eight unions, result is see the mean value of 5 readings in a pipe position in some expression eight union in table 5 and Figure 13, Figure 13, and the groove on point represents the standard error of 5 readings.5 times, each pipe position result is the positive, shows good repeatability in qualitative analysis.
Same eight union of table 55 reading result statistics
Protection content of the present invention is not limited to above embodiment.Under the spirit and scope not deviating from inventive concept, the change that those skilled in the art can expect and advantage are all included in the present invention, and are protection domain with appending claims.
Claims (14)
1. a fluorescence imaging analysis system, is characterized in that, comprises shell (10), at least one excitation apparatus (20) and imaging device (30);
Described shell (10) is the hollow housing closed, its inner formation darkroom, and the inside of described shell (10) is provided with sample stage (11), for placing the detected material carrying fluorescent material; Described shell (10) is provided with light hole (12), described light hole (12) is right against described sample stage (11), for observing described sample stage (11);
Described excitation apparatus (20) is arranged on the inside of described shell (10), and it comprises excitation source (21) and exciter filter (23); The light that described excitation source (21) sends forms monochromatic exciting light through described exciter filter (23) and is irradiated to described sample stage (11); Fluorescence is sent after described detected material is stimulated;
Described imaging device (30) comprises filter glass (31) and image unit (32); Wherein, described filter glass (31) is right against described light hole (12) and arranges, for the veiling glare except fluorescence except of filtering through described light hole (12); Described image unit (32) is right against described filter glass (31) and arranges, for catching the fluorescence through described filter glass (31).
2. fluorescence imaging analysis system as claimed in claim 1, it is characterized in that, described fluorescent material comprises fluorescent dye, fluorescin.
3. fluorescence imaging analysis system as claimed in claim 1, it is characterized in that, determine to select corresponding described excitation source (21), described exciter filter (23) and described filter glass (31) according to the optical property of described fluorescent material; Wherein, the cutoff wavelength of described exciter filter (23) is between the excitation wavelength and emission wavelength of described fluorescent material.
4. fluorescence imaging analysis system as claimed in claim 1, it is characterized in that, when fluorescent material is green fluorescence material, the spectral range of described excitation source (21) is 445 ~ 475nm; The transmittance spectrum scope of described exciter filter (23) is 325nm ~ 500nm; The transmittance spectrum scope of described filter glass (31) is 500nm ~ 2500nm; Or,
When fluorescent material is red fluorescent material, the spectral range of described excitation source (21) is 585nm ± 29nm; The transmittance spectrum of described exciter filter (23) is 500nm ~ 620nm; The transmittance spectrum scope of described filter glass (31) is 620nm ~ 2500nm; Or,
When fluorescent material is yellow fluorescent substance, the spectral range of described excitation source (21) is 531nm ± 40nm; The transmittance spectrum of described exciter filter (23) is 350nm ~ 580nm; The transmittance spectrum scope of described filter glass (31) is 580nm ~ 2500nm; Or,
When fluorescent material is blue fluorescent substance, the spectral range of described excitation source (21) is 357nm ± 44nm; The transmittance spectrum of described exciter filter (23) is 280nm ~ 410nm; The transmittance spectrum scope of described filter glass (31) is 410nm ~ 2500nm.
5. fluorescence imaging analysis system as claimed in claim 1, is characterized in that, arrange scattering part (22) in described excitation apparatus (20) further; The light that described excitation source (21) sends forms uniform beam after described scattering part (22), exposes to described exciter filter (23).
6. fluorescence imaging analysis system as claimed in claim 5, it is characterized in that, described scattering part (22) is in-plane scatter mirror.
7. fluorescence imaging analysis system as claimed in claim 5, it is characterized in that, described scattering part (22) comprises plane mirror (221) and cambered surface catoptron (222), described plane mirror (221) for by the light reflection of described excitation source (21) to described cambered surface catoptron (222), described cambered surface catoptron (222) forms uniform beam reflection to described exciter filter (23).
8. fluorescence imaging analysis system as claimed in claim 1, it is characterized in that, the bottom of described sample stage (11) is provided with slide rail, and it is for shifting out described shell (10) by described sample stage (11).
9. fluorescence imaging analysis system as claimed in claim 1, it is characterized in that, described imaging device (30) comprises shooting analytic unit (33) further, described shooting analytic unit (33) is connected with described image unit (32), for measuring and analyze the concentration of described fluorescent material.
10. fluorescence imaging analysis system as claimed in claim 9, it is characterized in that, described shooting analytic unit (33) comprising:
Digital image acquisition units (331), it obtains digital picture for the fluorescence caught through described filter glass (31);
Graphics processing unit (332), it is connected with described digital image acquisition units (331), for analyzing the amount of fluorescence in described digital picture;
Image-display units (333), it is connected with described graphics processing unit (332), for showing described digital picture, amount of fluorescence.
11. fluorescence imaging analysis systems as claimed in claim 10, it is characterized in that, described shooting analytic unit (33) comprises data storage cell (334) further, it is connected with described graphics processing unit (332), for storing described digital picture, amount of fluorescence.
12. fluorescence imaging analysis systems as claimed in claim 1, is characterized in that, described excitation source (21) is adjustable excitation source, and it comprises the excitation source of at least more than one different spectral ranges; Described exciter filter (23) is adjustable exciter filter, and it comprises the exciter filter of at least more than one different spectral ranges; Described filter glass (31) is turnable filter sheet, and it comprises the filter glass of at least more than one different spectral ranges.
13. 1 kinds of fluorescence imaging analysis methods, utilize the fluorescence imaging analysis system as described in any one of claim 1-12, it is characterized in that, comprise the steps:
Step 1: the detected material carrying fluorescent material is placed on sample stage (11);
Step 2: determine corresponding excitation source (21), exciter filter (23), filter glass (31) by described fluorescent material;
Step 3: open described excitation source (21), exciter filter (23) described in the light therethrough of described excitation source (21) forms monochromatic exciting light, and described exciting light is radiated on described detected material; Fluorescent material in described detected material is stimulated generation fluorescence;
Step 4: the veiling glare of described filter glass (31) filtering except described fluorescence, catches the fluorescence through described filter glass (31) with image unit (32), obtains fluoroscopic image and fluorescence values.
14. fluorescence imaging analysis methods as claimed in claim 13, is characterized in that, described method, for realizing the Concentration Testing of detected material, comprises the steps: after described step 4 further
Step 5: with detecting gained fluorescence values and detected material concentration drawing standard curve, obtain equation of linear regression;
Step 6: will fluorescent material be carried and the tested substance of concentration the unknown is placed on described sample stage (11), described sample stage (11) is positioned over described shell (10) inner, repeats fluoroscopic image and fluorescence values that step 2 ~ 4 obtain described detected material;
Step 7: utilize described shooting analytic unit (33) gained fluorescence values to be substituted into the actual concentrations calculating detected material in described equation of linear regression.
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