CN101782519B - Device for exciting fluorescent sample by utilizing visible light - Google Patents
Device for exciting fluorescent sample by utilizing visible light Download PDFInfo
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- CN101782519B CN101782519B CN 200910000530 CN200910000530A CN101782519B CN 101782519 B CN101782519 B CN 101782519B CN 200910000530 CN200910000530 CN 200910000530 CN 200910000530 A CN200910000530 A CN 200910000530A CN 101782519 B CN101782519 B CN 101782519B
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Abstract
The invention relates to a device for exciting a fluorescent sample by utilizing visible light, in particular to a device for lightening a fluorescent signal of polyacrylamide protein gel or other similar objects by a backlight-type blue light plate so as to improve a signal-to-noise ratio in the exciting process. The embodiment of the invention shows that the device is suitable for observing a plurality of protein gels dyed by fluorescer, and can ensure that certain weak fluorescent signals can be inspected by naked eyes and a filter lens is not needed. The device for straightly observing the fluorescent signal of the protein gel by utilizing the backlight-type blue light plate comprises a transparent plate for guiding light and at least one light source, wherein the at least one light source is arranged beside the transparent plate so that the fluorescent sample placed on the transparent plate of the device can be excited by light refracted or reflected by the transparent plate according to the Snell's law; and the signal-to-noise ratio in the exciting process is improved through the light refraction effect of the device.
Description
Technical field
The present invention is about utilizing the device of excited by visible light fluorescence sample.In detail, the present invention with the device of excited by visible light with the biological specimen of fluorescer dyeing, improves the signal to noise ratio (S/N ratio) in the excitation process about a kind of application Shi Naier law (Snell ' s Law).
Background technology
It may be one of subject the most attractive in the current bio-science research field that aleuroplast is learned (Proteomics).All have under the situation of rapid progress in high-throughout separation and identification technique, the available protein group picks out and has differential expression between experimental group and control group or pathology sample and healthy sample or through the protein of different modes modification.Protein is that the terminal in the signal transduction process is expressed molecule, can be than the physiological reaction of the more direct reacting cells of its nucleotide precursor, thereby in life science and medical diagnosis, enjoy attention.Yet, some protein (as interferon, lymph medium and hormone acceptor) that is called as rare message target is not only measured less and is difficult to detecting, for modern molecular biology brings very big difficulty, though, there is no the amplifying technique of scalable protein because at present the Polymerase Chain Reaction amplifying techniques such as (PCR) that is applicable to nucleotide is arranged.Because some rare message protein is very important to modern medicine, will help these protein of identification if can develop highly sensitive method for detecting.
Polyacrylamide gel electrophoresis should be the proteosome group of the most widely using and one of learns a skill.In addition, two dimensional electrophoresis method (2-DE) is because of can also having become another common method in aleuroplast recently according to the isoelectric point of indivedual protein and the protein group of molecular weight while separate complex.If desire utilization such as technology identifications such as mass spectroscopy have the protein of differential expression, must use suitable protein gel staining technique that protein belt or point in the polyacrylamide gel are developed the color.Though the Coomassie brilliant blue dye binding method is easy to use, in many cases, and the protein colour band of trace or color dot are developed the color.Though can allow few protein colour band or color dot colour developing to 1 nanogram and carry out high sensitivity decoration method such as negative staining such as silver staining or with the imidazoles zinc salt, the dynamic range of dyeing is more undesirable, and the problem of mass spectrum compatibility is still arranged.Recently, high and dynamic range is wide than colourimetry such as protein gel fluorescein stains such as SYPRO Ruby, Deep Purple, Flamingo and Krypton stains because of susceptibility, be widely used in the range protein body and learned test.Still there is him to plant the usefulness that the protein gel fluorescein stain can be used as the colour developing of fluorescence protein group in addition, for example Pro-QDiamond and Pro-Q Emerald.
For illuminating the fluorescence signal in the protein gel, the fluorophore that must combine with protein molecule with the optical excitation of suitable wavelength.For example, high energy blue laser (488 nanometer) in the Typhoon Trio laser gel scanner (GE Healthcare) just can effectively excite with 4 in the SYPRO Ruby stained gel, 7-diphenyl phenanthroline ruthenium (II) compound (bathophenanthroline complex of ruthenium (II)), wherein this compound is the fluorophore that bimodal phenomenon (excitation wavelength is 280 and 450 nanometers, and emission wavelength is 610 nanometers) appears in excitation curve.The nearest people of having uses the par CCD camera gel imaging system of installing light emitting diode (LED) to replace expensive laser gel scanner, and the example of these imaging systems comprises by the MF-ChemiBIS of DNR (Jerusalem Road,Israel) production, by the LIAS ChemX of Avegene (TaiBei City, Taiwan Province,China) production and the LAS-4000 that is produced by Fujifilm (U.S.'s health is Stamford, Dick state (Stamford) city).In imaging process, has low background for obtaining, a high-quality gel image, suitably the noise that caused because of exciting light transmission or reflection of filtering.For example when scanning is photographed with the SYPRORuby stained gel or for it, all use amber filter (long logical filter of 610 nanometers or the logical filter in broadband) to be blue laser or blue light filtering usually.
In some cases, when for example desiring to detect colour band or color dot with manual type from the protein gel with fluorescer dyeing, fluorescence signal is visual detecting directly.Right most gel imaging equipment, laser gel scanner is for example all adopted the design of closed system, is not suitable for carrying out the artificial real program of doing.Ultraviolet ray (UV) transillumination case be can yet be regarded as and can be satisfied the device of the demand because its long wavelength (UVA) and short wavelength's (UVB) ultraviolet ray all can excite specific fluorescent group.Present many laboratories all use UVB transillumination case to observe fluorescence signal in SYPRO Ruby stained gel in the direct-view mode.Yet ultraviolet transillumination case apparatus has three shortcomings at least.One, the operator directly and long term exposure in the ultraviolet radiation district, even use suitable security protection equipment still to be potentially dangerous.Its two, ultraviolet light and can't effectively excite all fluorophores.Fluorophore in some stain, the fluorophore among Flamingo and the Krypton for example, its maximum excitation wavelength is shorter, is about 270 and 320 nanometers, therefore almost can't inspect colour band or color dot with these stain dyeing with manual type by ultraviolet transillumination case.Its three, most of fluorophore all may produce the phenomenon of photofading because of bringing out of ultraviolet light, for example the half life period of the fluorescence of Deep Purple after UV-irradiation approximately only has six minutes.Fluorescent signals in the gel heals mostly and becomes faint after being exposed to ultraviolet light for a long time, and is last even can't predict with visual.
According to the recent research report, blue light transillumination case can be used as the ideal substitute of ultraviolet transillumination case, for the fluorescence signal of line-of-sighting observation in the protein gel.Have three kinds of blue light transillumination case products at present on the market at least, comprise by the objective Lay suffixation of a nonsyllabic "r" (the Clare Chemical Research of research company, prefecture, Leix, many Lip rivers, Colorado (Dolores)) the Dark Reader that is produced, by liking Safe Imager that little company (Invitrogen, California, USA Ka Ersiba (Carlsbad) city) produced and the Visi-Blue transillumination case of being produced by UVP company (California, USA A Pulan (Upland) city).More than three kinds of blue light transillumination casees all with the blue-light excited fluorophore of high frequency range, and use the blue light of orange or amber filter filtering scattering.These equipment have been used for exciting the fluorophore such as stains such as SYPRO Orange, SYPRO Ruby and SYBR Green.Blue light transillumination case apparatus removes and can still can be designed to lantern or integrated transillumination case-electrophoretic cell for the protein gel of line-of-sighting observation with fluorescer dyeing.Clare Chemical Research company even designed of the running of amber filter glass in order to blue light transillumination case.
If compare with the visual result who obtains via ultraviolet transillumination case, the fluorescence signal that blue light transillumination case is produced mostly a little less than.Its reason is that the stimulation effect of high frequency range blue light is inferior to ultraviolet light.In addition, the blue light of being launched by blue light transillumination case all carries out filtering with orange or amber thick filter (0.5 to 1.0 centimeter) usually, and this filter also can the absorption portion fluorescence signal.Therefore, when using blue light transillumination case, the fluorescent signals in the protein gel is difficult to detect by line-of-sighting observation sometimes, thereby causes being of limited application of blue light transillumination case.
Summary of the invention
The present invention proposes a kind of device that utilizes backlight type blue light plate to illuminate the fluorescence signal in polyacrylamide protein gel or its analog at this, so as to improving the signal to noise ratio (S/N ratio) in the excitation process.Show that according to embodiments of the invention this device is suitable for observing multiple protein gel with fluorescer dyeing, can make some faint fluorescence signal be able to naked eyes and detect, and need not use filter.
This utilizes backlight type blue light plate to make that the device of fluorescence signal comprises in the protein gel to line-of-sight observation:
Transparent panel in order to leaded light; And
At least one light source is located at by this transparent panel, and the optical excitation that causes the fluorescence sample on the transparent panel that is positioned over this device can be subjected to this transparent panel Yi Shinaier law to reflect and reflect is by the signal to noise ratio (S/N ratio) in the anaclasis improved effect excitation process of this device.
Description of drawings
Figure 1A is the synoptic diagram of the structure of demonstration apparatus of the present invention;
Figure 1B to Fig. 1 H is for relatively apparatus of the present invention and other device are observed the effect synoptic diagram of the protein gel that dyes with fluorescer;
Fig. 2 is for showing all possible optical path synoptic diagram in the backlight type blue light panel assembly of the present invention;
Fig. 3 is at SYPRO Ruby stained gel, with the quantification manner comparison its image on the backlight type blue light plate of the present invention image and by the image of laser gel scanner imaging;
Fig. 4 is for showing the application drawing of backlight type blue light plate of the present invention, and wherein this backlight type blue light plate is used to observe the dna gel with SYBR Safe dyeing.
Description of reference numerals
Glass transparent panel 10
Blue light cathode fluorescent tube 30
Black background 40
Plastic cover plate 50
Embodiment
Materials and methods-preliminary program
Obtain commercial standard (CS) protein mixture (GE Healthcare, N.J. Pi Sikatewei (Piscataway) town), it comprises rabbit muscle glycogen phosphorylase b (GP), bovine serum albumin(BSA) (BSA), egg ovalbumin (OVA), ORBC carbonic anhydrase (CA), soybean tryptose Enzyme inhibitor (TI) and cow's milk albumin (LAC).With the continuous twice dilution of this protein mixture, make wherein total egg matter content reduce to 7.8 nanograms from 4,000 nanograms, separate this protein mixture with 15% polyacrylamide gel electrophoresis (SDS-PAGE) then.All electrophoretic procedures are all carried out according to standard criterion, only slightly make an amendment.Then with SYPRORuby, SYPRO Tangerine, SYPRO Orange (Invitrogen, the big island of New York, United States (GrandIsland) town) with Deep Purple (GE Healthcare, N.J. Piscataway town) any stain in is handled the electrophoresis protein gel according to the indication explanation of manufacturer.
Gel imaging and gel image analysing computer
Backlight type blue light plate, it is provided with two each linear blue light cathode fluorescent tubes (CCFL) (2000 luxs, 30 centimeters) of 5 watts.And buy the blue light transillumination case that model is Dark Reader DR-88 from Clare Chemical Research (Dolores prefecture, Colorado).Be the protein gel of Direct observation with fluorescer dyeing, this with amber acrylic plate (1.0 centimeters thick) as filter, with the blue light of filtering transmission or refraction.The digital camera (CanonA700) that identical amber acrylic plate (2.0 centimeters thick) is housed is used to take the gel image, and all uses identical photographic parameter, and promptly the time shutter is 4 seconds, and aperture is 8.Other has ultraviolet transillumination case (TD-2000E, 365 nanometers/312 nanometers, Spectronics, New York, United States Westbury (Westbury) village) to carry out simultaneous test.
In addition, with SYPRO Ruby or Deep Purple stained gel in addition with laser gel scanner (Typhoon Trio, GE Healthcare, N.J. Piscataway town) excitation laser of collocation 488 or 532 nanometers and the logical filter imaging of the band of 610 (30) nanometers.
Backlight type blue light plate assembly of the present invention
Backlight type blue light plate assembly of the present invention is shown in Figure 1A, and it is other as light source to comprise in regular turn that from lower to upper black background 40, glass transparent panel 10 and 30 of 20, two blue light cathode fluorescent tubes of gel are located at glass transparent panel 10.Black background 40 places glass transparent panel 10 belows so that preferable contrast effect to be provided.50 of plastic cover plates place glass transparent panel 10 tops to block unwanted refract light.Above-mentioned backlight type blue light plate can be the visible light source of another photochromic (also promptly white) less than the linear blue light cathode fluorescent tube 30 of 70 dollars price.The present invention is an example with the blue light, but is not limited to use blue light.Moreover transparent panel 10 can be thin glass or plastic plate, and only its thickness should be less than 1 centimeter.This light source can be linear cathode fluorescent tube or light emitting diode, and this light source can be visible light or invisible light.
Use different lighting devices to observe the protein gel that dyes with fluorescer
At first illuminate protein gel respectively with fluorescer dyeing with backlight type blue light plate of the present invention and the blue light transillumination case commonly used, and both results relatively.In evaluation process, select general the most frequently used protein gel fluorescein stain SYPRO Ruby (excitation wavelength/emission wavelength=280 and 450 nanometers/610 nanometers) for use.Find that through test backlight type blue light plate has excellent imaging effect for the protein gel with SYPRO Ruby dyeing, the fluorescence signal of gained image is strong and background value is low.Even the protein content in the colour band is low to moderate 5 nanograms, still can line-of-sight observation these colour bands (referring to Figure 1B, the carbonic anhydrase of triangle 1 expression 4.5 nanograms) by amber filter or filter glass.In comparison, identical gel is by the viewed fluorescence signal of blue light transillumination case less also weak (Fig. 1 C).For example, (is not with the picture of patented claim black and white to the carbonic anhydrase that its minimum protein content of SYPRO Ruby dyeing colour band that can visualization is 18 nanograms in Fig. 1 C? triangle 2 expressions).After test of many times, find, in SYPRO Ruby stained gel, be at least four times by the measured protein signal intensity of blue light transillumination case by the measured protein signal intensity of backlight type blue light plate.
Nearly allly in gel, can all can inspect with naked eyes, prove and to utilize backlight type blue light plate from protein gel, to detect the lower protein of content originally again with SYPRO Ruby dyeing with manual type by the SYPRO Ruby fluorescence signal (Fig. 1 D) of laser gel scanner imaging by backlight type blue light plate.Long term exposure is not only harmless to the operator in visible blue, also harmless to fluorophore, because in process of the test, do not observe the phenomenon that occurs photofading with the protein gel of SYPRO Ruby dyeing, reach one hour on the backlight type blue light plate also multiple so (not showing related data) at this even it is retained in.Find that through test in backlight type blue light panel assembly, the edge of protein gel is the light yellow visible blue all the time, but there is no this phenomenon in blue light transillumination case apparatus.This blue light that appears at the protein gel edge is the result (referring to Fig. 2 B) of blue light in the protein gel inner total reflection that it illuminated, not the significantly observation of interferencing protein colour band.What deserves to be mentioned is equally that at this even do not use filter or filter glass, all SYPRO Ruby fluorescence signals still all can detect with visual type in backlight type blue light panel assembly.But no matter blue light transillumination case or ultraviolet transillumination case all can't reach the effect of this direct-view observation.The characteristic of this very advantageous can allow the researchist by comfortable and safe program, detects colour band or color dot with manual type from some protein gel with fluorescer dyeing.
Use other fluorescence signal in the backlight type blue light plate observing protein gel
Therefore above-mentioned backlight type blue light panel assembly can learn the multiple function of performance in the experiment at aleuroplast for the clear signal of inspecting multiple fluorescein stain.For example, with the running gel that SYPRO Tangerine (excitation wavelength/emission wavelength=300 and 490 nanometers/640 nanometers) and SYPRO Orange (excitation wavelength/emission wavelength=300 and 470 nanometers/570 nanometers) handle, its result all can utilize clear inspect (Fig. 1 E and Fig. 1 F) of backlight type blue light plate.In addition, also can have the low fluorophore that excites coefficient by clear the inspecting of above-mentioned backlight type blue light panel assembly, the fluorophore among the Deep Purple (excitation wavelength/emission wavelength=532 nanometers/610 nanometers, ε=20,000) (in Fig. 1 G, showing) for example in the black and white mode.Nearly allly in gel, can all can directly inspect with naked eyes by the Deep Purple fluorescence signal (in Fig. 1 H, showing) of laser gel scanner imaging in the black and white mode by backlight type blue light plate.So what the reason of not selecting other two kinds of protein gel fluorescein stain Krypton commonly used (excitation wavelength/emission wavelength=518 nanometers/552 nanometers) and Flamingo (excitation wavelength/emission wavelength=515 nanometers/545 nanometers) for use in evaluation process was that its amber filter of using of arranging in pairs or groups in theory may filtering about 550 nanometers of wavelength transmits.In addition, its used high frequency range blue light from cathode fluorescent tube also possibly can't provide enough excitation energies, but produces significant ground unrest.
In brief, with regard to Deep Purple stain, above-mentioned backlight type blue light panel assembly is more clear for the imaging effect of UVA that all generally uses always with the imaging effect (Figure 1B and Fig. 1 G) of the protein gel of these fluorescers dyeing or UVB transillumination case with regard to the SYPRO Ruby that generally uses at present.On the other hand, when using blue light transillumination case, with the integral body observation brightness lower (Fig. 1 C) of the protein gel of these fluorescers dyeing.Perhaps also absorb significant fluorescence signal amount in order to orange or the amber thick filter that the emission blue light is carried out filtering, cause the fluorescent signals in the protein gel in blue light transillumination case, to be difficult to measured.
Utilize backlight type blue light plate to observe the dna gel that dyes with SYBR Safe
Method: length, is separated with 7.5% polyacrylamide gel earlier, again with SYBR after twice is diluted continuously between the dna ladder shape bar tape label between 50bps and the 3Kbps
Safe DNA dyeing cover group (Invitrogen) makes its colour developing.The maximum length of each scalariform bar tape label is listed in Fig. 4 left side.Then by the illumination of backlight type blue light plate, for SYBR
The dna gel photography of Safe dyeing.
Optical path in the backlight type blue light plate of the present invention
Backlight type blue light plate method is based on the Shi Naier law.The method once was used for TOTAL INTERNAL REFLECTION FLUORESCENCE MICROSCOPY (TIRFM), during object on observing microslide, with the ratio optimization of fluorescence signal to ground unrest.Light below will be discussed enter the path of glass transparent panel 10, so that visualization effect provided by the present invention to be described from blue light cathode fluorescent tube 30.
In the application of backlight type blue light plate related critical angle (θ c) comprise following several:
θ c
(glass: air): the critical angle between glass and air;
θ c
(glass: acrylamide): the critical angle between glass and polyacrylamide gel;
θ c
(acrylamide: air): the critical angle between polyacrylamide gel and air.
(1) incident angle θ=＜θ c
(glass: air)
Because the refractive index (n of glass
Glass=1.5) greater than the refractive index (n of air
Air=1.0), therefore, the light that penetrates from glass transparent panel 10 has only at incident angle θ less than critical angle θ c
(glass: air)41.8 ° (=sin
-1(n
Air/ n
Glass)=sin
-1(1/1.5)) situation below is able to refraction angle θ
1 'Refract in the air.For example, if the incident angle θ of blue light
1Be 40 °, then this blue light will refract to (Fig. 2 A, path 1) in the air with 74.6 ° at new refraction angle (sin40 ° * 1.5=sin74.6 ° * 1.0).Because it is other that blue light cathode fluorescent tube 30 is arranged in the glass transparent panel 10 of backlight type blue light panel assembly extremely thin (thickness is less than 5 millimeters), therefore, the position that blue light goes out from glass transparent panel 10 refraction will be not more than 3.33 millimeters (5 millimeters * tan41.8 °) apart from the distance at glass transparent panel 10 edges, and this refract light will be stopped by plastic cover plate 50 (Figure 1A).If the incident angle θ of blue light
2Equal critical angle θ c
(glass: air)41.8 °, then this blue light will reflect fully, and direct of travel is parallel to glass transparent panel 10 (Fig. 2 A, path 2).In glass transparent panel 10, all by 30 emissions of blue light cathode fluorescent tube and incident angle greater than critical angle θ c
(glass: air)41.8 ° blue light all will be reflected, and in glass transparent panel 10, advance, its mode of advancing is identical with the mode of advancing in optical fiber.Therefore, if black background 40 is placed glass transparent panel 10 belows (Figure 1A), even under the situation that starts blue light cathode fluorescent tube 30, it is slightly dark that most glass transparent panel 10 (also promptly apart from the about part more than 3 millimeters in glass transparent panel 10 edges) still shows.Therefore, when observing any object that is positioned on the backlight type blue light plate, the parallel light that all do not have in theory is in observer's sight line.
(2) θ c
(glass: air)＜incident angle θ=＜θ c
(glass: acrylamide)
The main composition of polyacrylamide gel is water and acrylamide, and therefore, the refractive index of specific polyacrylamide gel need be decided on the concentration of gel.Known to the inventor, still there is not the refractometry value of the crystalline powder of acrylamide at present, but known similar substance, promptly the refractive index of poly-2-methyl methacrylate (claiming acryl again) is 1.49.Because the refractive index of water is 1.33, but the refractive index of the most of polyacrylamide gel of reasonable assumption is between 1.33 and 1.49.The refractive index of polyacrylamide gel of clearly not demarcating concentration is through being measured as 1.47.In any case but, the refractive index n of polyacrylamide gel
AcrylamideMust be greater than n
AirAnd less than n
GlassIf with refractive index is that 1.4 polyacrylamide gel places on the glass transparent panel 10, the light that blue light cathode fluorescent tube 30 is launched at incident angle θ less than θ c
(glass: acrylamide)60.1 ° (=sin
-1(n
Acrylamide/ n
Glass)=sin
-1To refract in this gel under the situation (1.4/1.5)).Therefore, in above-mentioned backlight type blue light panel assembly, have only by 30 emissions of blue light cathode fluorescent tube and incident angle θ between 41.8 ° with 60.1 ° between light will refract in the polyacrylamide gel with corresponding refraction angle θ '.For example, in the interface of glass transparent panel 10 and gel 20, if the incident angle θ of blue light
3Be 45 °, then this blue light will be with new refraction angle θ
3 '=49.3 ° (sin45 ° * 1.5=sin49.3 ° * 1.4) refract to (Fig. 2 B, blue path 3) in the gel.Because new incident angle θ
3*(equal its reciprocity interior angle θ
3 ') greater than critical angle θ c
(acrylamide: air)(45.9 °=sin
-1(n
Air/ n
Acrylamide)), therefore, if will be positioned on the backlight type blue light plate with the protein gel of fluorescer dyeing, refract in the gel and not the blue light of fluorescence excitation group will can directly not refract in the air, and advance in gel in the mode of total reflection, till it arrives the vertical side edge of gel.With regard to this interface, above-mentioned blue light can have another incident angle θ
3 "=40.7 ° (90 °-49.3 °=40.7 °), it is worth less than critical angle θ c
(acrylamide: air)45.9 °.Therefore, this blue light refracts in the air with 65.9 ° at new refraction angle (sin40.7 ° * 1.4=sin65.9 ° * 1.0) at last.More than explanation or soluble polyacrylamide gel edge bright blue light (Figure 1B).If the incident angle θ of blue light
4Equal critical angle θ c
(glass: acrylamide)60.1 °, then this blue light will reflect and along the direction that is parallel to glass transparent panel 10 advance (Fig. 2 B, path 4).
Comprehensive speech, if blue light is after blue light cathode fluorescent tube 30 penetrates, its principal angle of incidence θ
3Greater than critical angle θ c
(glass: air)But less than critical angle θ c
(glass: acrylamide), then this blue light will refract in the polyacrylamide gel 20 from glass transparent panel 10, and then by the air total reflection, the edge from gel 20 penetrates at last.All penetrate at above-mentioned all blue lights of this hypothesis without the surface of gel 20.
(3) θ c
(glass: acrylamide)＜incident angle θ
With regard to the interface of glass transparent panel 10 and polyacrylamide gel 20, by 30 emissions of blue light cathode fluorescent tube and incident angle θ greater than critical θ c
(glass: acrylamide)60.1 ° light should be to glass transparent panel 10 by the gel total reflection, the edge via glass transparent panel 10 penetrates at last.For example, if the incident angle θ of blue light
5Be 62 °, then this blue light will be by the gel total reflection, at last again with another incident angle θ
5 '=28 ° (90 °-62 °) inject the edge of glass transparent panel 10, and with refraction angle θ
5 "=44.8 ° (sin28 ° * 1.5=sin44.8 ° * 1.0) penetrate (Fig. 2 C, path 5) from this edge.Part should directly be injected polyacrylamide gel 20 with bigger incident angle θ by blue light cathode fluorescent tube 30 near parallel blue light.This part blue light ought to be by the gel total reflection to glass transparent panel 10, penetrates via the edge of glass transparent panel 10 more at last.For example, if the incident angle θ of blue light
6Be 84 °, then this blue light is the most at last with incident angle θ
6 '=6 ° (90 °-84 °) inject the edge of glass transparent panel 10, and with refraction angle θ
6 "=9 ° (sin6 ° * 1.5=sin9 ° * 1.0) penetrate (Fig. 2 C, path 6) from this edge.
The quality of image assessment of protein gel after photographic imagery on the backlight type blue light plate with fluorescer dyeing
The present invention inspect gel on backlight type blue light plate after photographic imagery the image of gained whether be fit to quantitative test.When carrying out this assessment, with the photographic imagery (Figure 1B) or utilize laser gel scanner imaging (Fig. 1 D) on backlight type blue light plate of SYPRO Ruby stained gel, be 16 GTG erect images (Fig. 3 A and Fig. 3 B) again with the gained video conversion, assessed with one dimension image analysing computer software then.The image of obtaining with this dual mode has close effect, all presents desirable protein staining dynamic range.Between colour band intensity and protein actual content (its scope is between between nanogram and the microgram), there is splendid linear relationship (Fig. 3 C and Fig. 3 D).In addition, the image of obtaining with this dual mode also has similar gray-scale distribution (gray scale histogram) (Fig. 3 E and Fig. 3 F).Can learn from above information, but backlight type blue light plate method or be the efficient apparatus of fluorescence signal in a kind of Direct observation protein gel, and be a kind of economy excitation source reliably, be suitably for the usefulness of gel photography for analysis.
Conclusion
In above-mentioned backlight type blue light panel assembly, have only by 30 emissions of blue light cathode fluorescent tube and principal angle of incidence greater than critical angle θ c
(glass: air)But less than critical angle θ c
(glass: acrylamide)Blue light will refract in the polyacrylamide gel 20 from glass transparent panel 10, then in the gel inner total reflection, penetrate from the edge of gel at last in (Fig. 2, path 3).Other blue light then directly refracts to (Fig. 2, path 1) or final edge ejaculation (Fig. 2, path 2,4,5 and 6) via glass transparent panel 10 in the air.The blue light of being launched by blue light cathode fluorescent tube 30 fully can the direct projection observer eyes.And this be do not need in the test filter or filter glass can be on backlight type blue light plate the most likely reason of visual fluorescence signal.In addition, with the obtained gel image of the present invention, its quality (signal is strong, and ground unrest is than low) also is better than with the obtained person of blue light transillumination case (Figure 1B and Fig. 1 C).Test findings shows that device of the present invention is safety, economic, convenient not only, more can effectively illuminate the protein gel with fluorescer dyeing.
In this mandatory declaration, material changes with light wavelength the refractive index of light.Certain material can and get by following look G equation (Sellmeier equation) derivation the refractive index (n) of the light of different wave length (λ):
n
2(λ)＝1+B
1λ
2/(λ
2-C
1)+B
2λ
2/(λ
2-C
2)+B
3λ
2/(λ
2-C
3)
B wherein
1, B
2, B
3And C
1, C
2, C
3The look G coefficient that the test of serving as reasons obtains.
For example, glass (SiO
2) be that the refractive index of the light of 350 nanometers, 450 nanometers, 550 nanometers and 650 nanometers is respectively 1.56560,1.55257,1.54599 and 1.54210 to wavelength, and air is respectively 1.000284,1.000279,1.000277 and 1.000276 to the refractive index of these light.So commaterial to wavelength the refractive index of long light less than to the wavelength refractive index of short light.Yet the critical angle of light does not significantly change because of the relation of above-mentioned wavelength and refractive index.For example, in the interface of glass and air, wavelength is that the critical angle of the light of 350 nanometers, 450 nanometers, 550 nanometers and 650 nanometers is respectively 39.7 °, 40.1 °, 40.3 ° and 40.4 ° through measurement.Therefore, even input light has different wave length, the application of backlight type blue light panel assembly of the present invention is also unrestricted.Ago-Gel (agarose gel) also can be selected in above-mentioned.
Sum up: by the illumination of backlight type blue light plate, but not only visualization with the protein gel of fluorescer dyeing, but also visualization with SYBR
The dna gel of dyeing.By device of the present invention, can the few DNA (Fig. 4, square represent viewed minimum DNA colour band) of visual inspection to 2 nanograms.Therefore, compared to ultraviolet transillumination case, backlight type blue light plate method not only makes things convenient for safely, has more polynary purposes, can be used for illuminating the fluorescence signal in the multiple biological specimen.
The above is preferred embodiment of the present invention only, is not to be used to limit protection scope of the present invention.
Claims (1)
1. a device that utilizes excited by visible light fluorescence sample is characterized in that, comprising:
In order to the transparent panel of leaded light, the thickness of this transparent panel is less than 1 centimetre; And
At least one visible light source, this light source are installed on by the side of this transparent panel, wherein scribble gel on this transparent panel, this light source launch and principal angle of incidence greater than critical angle θ c
(transparent panel: air)But less than critical angle θ c
(transparent panel: gel)Blue light will refract in this gel from this transparent panel, then in the gel inner total reflection, edge from gel penetrates at last, the optical excitation that causes fluorescence sample on the transparent panel that is positioned over this device can be subjected to this transparent panel Yi Shinaier law (Snell ' s Law) institute to reflect and reflect is by the signal to noise ratio (S/N ratio) in the anaclasis improved effect excitation process of this device.
2, the device that utilizes excited by visible light fluorescence sample according to claim 1 is characterized in that, described fluorescence sample is DNA or the protein with fluorescer dyeing or mark.
3, the device that utilizes excited by visible light fluorescence sample according to claim 1 is characterized in that, described transparent panel below is provided with black background.
4, the device that utilizes excited by visible light fluorescence sample according to claim 1 is characterized in that, described fluorescence sample top is provided with to intercept the filter of irregular refraction light.
5, the device that utilizes excited by visible light fluorescence sample according to claim 1 is characterized in that, described transparent panel is thin glass or plastic plate.
6, the device that utilizes excited by visible light fluorescence sample according to claim 1 is characterized in that, described light source is linear blue light cathode fluorescent tube (CCFL) or light emitting diode (LED).
7, the device that utilizes excited by visible light fluorescence sample according to claim 1 is characterized in that, described gel is polyacrylamide gel or Ago-Gel.
8, the device that utilizes excited by visible light fluorescence sample according to claim 1, it is characterized in that described fluorescence sample is by SYPRO Ruby, Deep Purple, Flamingo, Krypton stain, Pro-Q Diamond, Pro-Q Emerald, SYBR Safe and one of them dyeing of SYPRO Safe.
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| CN102445439B (en) * | 2010-10-09 | 2015-09-02 | 易尚明天科技有限公司 | chemical fluorescence detector |
| CN102455291B (en) * | 2010-10-20 | 2017-04-12 | 纳尔科公司 | Pollution detection method using fluorescent technique |
| CN103245611A (en) * | 2012-02-07 | 2013-08-14 | 鸿林堂科技股份有限公司 | Multiple excitation light source system |
| TWI475213B (en) * | 2012-06-06 | 2015-03-01 | Genereach Biotechnology Corp | A method for detecting the fluorescent |
| CN105283754A (en) * | 2013-06-03 | 2016-01-27 | 文塔纳医疗系统公司 | Fluorescence imaging system for tissue detection |
| CN108107066B (en) * | 2017-12-19 | 2020-08-14 | 北京工业大学 | SEM/ESEM cathode fluorescence imaging method for biological nano probe |
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| JP特开平5-119021A 1993.05.14 |
| Shaobo Zhou ET AL.Measurement of specific radioactivity in proteins separated by two-dimensional gel electrophoresis.《Electrophoresis》.2006,第27卷(第5-6期),1147-1153. * |
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