TWM548790U - Device utilizing light refraction for fluorescent sample excitation - Google Patents

Description

Device for exciting a fluorescent sample by light refraction

The present invention relates to a device for exciting a fluorescent sample by means of light refraction, in particular to a device for applying a Snell's law to excite a biological sample dyed with a fluorescent agent, and to improve the signal-to-noise ratio during the excitation process.

Proteomics is probably one of the most attractive disciplines in the field of biological science research. In the case of high-throughput separation and identification techniques, we have been able to use proteomics to identify proteins with differences in expression or modification between the experimental and control groups or between pathological and healthy samples. Proteins are terminal expression molecules in the process of signal transmission, which can directly reflect the physiological responses of cells compared with their nucleotide precursors, and thus have received much attention in life science research and medical diagnosis. However, some proteins known as rare message targets (such as interferons, lymphatic mediators, and hormonal receptors) are not only small but difficult to detect, which poses great difficulties for modern molecular biology because they are currently suitable for nucleosides. Amplification techniques such as acidase chain reaction (PCR) of acid, but there is no amplification technique that can amplify proteins. Since some rare message proteins are extremely important for modern medicine, developing highly sensitive detection methods will help identify these proteins.

Polyacrylamide gel electrophoresis is one of the most widely used proteomics technologies. In addition, two-dimensional electrophoresis (2-DE) has recently become a common method in proteomics because it can simultaneously separate complex proteomes based on the isoelectric point and molecular weight of individual proteins. If a protein such as mass spectrometry is to be used to identify proteins with differential expression, the protein ribbon or color point in the polypropylene amide gel must be colored using appropriate protein gel staining techniques. The Coomassie Brilliant Blue Dye Binding method, while convenient to use, does not, in many cases, allow for the development of smaller protein bands or color points. High-sensitivity staining methods such as silver staining or negative staining with imidazole zinc salt can produce protein bands or color points as small as 1 ng, but the dynamic range of dyeing is less than ideal, and there is still mass spectrometry compatibility. Sexual problem. Recently, protein gel fluorescent dyes such as SYPRO Ruby, Deep Purple, Flamingo and Krypton dyes have been widely used in various proteomics tests due to their high sensitivity and wide dynamic range compared to colorimetric methods. In addition, there are other protein gel fluorescent dyes that can be used as fluorescent proteomics, such as Pro-Q Diamond and Pro-Q Emerald.

In order to illuminate the fluorescent signal in the protein gel, it is necessary to excite the fluorescent group bound to the protein molecule with light of a suitable wavelength. For example, a high-energy blue laser (488 nm) in a Typhoon Trio laser gel scanner (GE Healthcare) can effectively excite 4,7-diphenyl phenanthrene in a gel dyed with SYPRO Ruby. a hopophenanthroline complex of ruthenium (II), wherein the complex exhibits a bimodal phenomenon (excitation wavelengths of 280 and 450 nm, emission wavelength of 610 nm). group. Recently, expensive laser CCD cameras have been replaced by inexpensive CCD camera gel imaging systems equipped with light-emitting diodes (LEDs), examples of which include MF-ChemiBIS produced by DNR (Jerusalem, Israel). LIAS ChemX manufactured by Avegene (Taiwan) and LAS-4000 manufactured by Fujifilm (Stamford, Connecticut). In the imaging process, in order to obtain a gel image with low background and high quality, it is necessary to properly filter out the noise caused by the transmission or reflection of the excitation light. For example, when scanning or photographing a gel stained with SYPRO Ruby, an amber filter (610 nm long pass filter or broadband pass filter) is usually used for blue laser or blue light filtering.

In some cases, such as when it is desired to manually detect a good band or color point from a protein gel dyed with a fluorescent agent, the fluorescent signal must be visually detectable. However, most gel imaging devices, such as laser gel scanners, are designed with closed systems and are not suitable for manual implementation. The ultraviolet (UV) transillumination box can excite a specific fluorescent group due to its long wavelength (UVA) and short wavelength (UVB) ultraviolet rays, and it is a device that can meet the above requirements. Many laboratories currently use a UVB transillumination box to view fluorescent signals in SYPRO Ruby-stained gels in a direct view. However, the ultraviolet transillumination box device has at least three disadvantages. First, the operator's direct and long-term exposure to the UV radiation zone is potentially dangerous even with appropriate safety equipment. Second, ultraviolet light does not effectively excite all fluorophores. Fluorescent groups in some dyes, such as the luminescent clusters in Flamingo and Krypton, have a maximum excitation wavelength of about 270 and 320 nm, so they are almost impossible to manually view through the UV transillumination box. A dyed ribbon or color point. Third, most of the fluorophores may be photobleached by ultraviolet light. For example, the half-life of Deep Purple's fluorescent light after exposure to ultraviolet light is only about six minutes. The weak fluorescent signal in the gel is mostly weak after being exposed to ultraviolet light for a long time, and finally it cannot be visually detected.

According to recent research reports, the Blu-ray Transillumination Box is an ideal replacement for UV transillumination boxes for direct observation of fluorescent signals in protein gels. There are currently at least three Blu-ray transillumination box products on the market, including Dark Reader produced by Clare Chemical Research (Dolores County, Colorado, USA), Safe Imager manufactured by Invitrogen (Carlsbad, Calif.), and UVP (Upland, California, USA). City) produced Visi-Blue transillumination box. All of the above three blue transillumination boxes excite the fluorophores with high-bandwidth blue light and filter out the scattered blue light using an orange or amber filter. These devices have been used to ignite fluorophores in dyes such as SYPRO Orange, SYPRO Ruby and SYBR Green. In addition to the laser gel dyed protein gel, the Blu-ray transillumination device can be designed as a hand lamp or an integrated transillumination box-electrophoresis unit. Clare Chemical Research has even designed amber filter glass to facilitate the operation of the Blu-ray Transillumination Box.

The fluorescent signal generated by the blue transillumination box is mostly weaker than the visual result obtained by the ultraviolet transillumination box. The reason is that the excitation effect of high-frequency wide blue light is inferior to that of ultraviolet light. In addition, the blue light emitted by the blue transillumination box is usually filtered with a thick orange or amber filter (0.5 to 1.0 cm), and the filter also absorbs some of the fluorescent signals. Therefore, when a blue light transillumination box is used, the weak fluorescent signal in the protein gel is sometimes difficult to detect by direct observation, which results in a limited application range of the blue transillumination box.

SUMMARY OF THE INVENTION The object of the present invention is to provide a device for illuminating a fluorescent signal in a polypropylene amide protein gel or the like using a backlit blue plate to improve the signal to noise ratio during excitation.

In order to achieve the foregoing object, the apparatus for utilizing photorefractive to excite a fluorescent sample includes a transparent plate for guiding light, wherein the fluorescent sample is disposed thereon; and a back plate disposed under the transparent plate; a cover plate disposed above the transparent plate; and at least one illuminant as an excitation light source disposed between the transparent plate and the back plate and opposite to an edge of the transparent plate to enable the illuminant The light is incident from the edge of the transparent plate, so that the fluorescent sample on the transparent plate is excited by receiving the refraction and reflection of the light source according to Snell's law, thereby improving the signal-to-noise ratio during the excitation process. To block the refracted light that penetrates the transparent plate.

According to one embodiment of the present invention, the illuminator is a blue cold cathode fluorescent lamp.

Through the illumination of the novel backlit Blu-ray panel, not only the protein gel stained with a fluorescent agent but also the DNA gel stained with SYBR® can be visually observed. With the device of the present invention, as little as 2 ng of DNA can be visually observed. Therefore, compared with the ultraviolet transillumination box, the novel backlight blue plate method is not only safe and convenient, but also has multiple uses, and can be used to illuminate fluorescent signals in various biological samples.

Materials and Methods - Preparation: A commercial standard protein mixture containing rabbit muscle glycogen phosphorylase b (GP), bovine serum albumin (BSA), egg ovalbumin (OVA), bovine erythrocyte carbonic anhydrase (CA) ), soybean tryptone inhibitor (TI) and bovine milk albumin (LAC). The protein mixture was serially diluted twice, reducing the total protein content from 4,000 ng to 7.8 ng, and then separating the protein mixture by 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). . All electrophoresis procedures were performed according to standard specifications with minor modifications. The electrophoresis protein gel was then processed according to the manufacturer's instructions using any of the SYPRO Ruby, SYPRO Tangerine, SYPRO Orange and Deep Purple dyes.

Gel Imaging and Gel Image Analysis: A backlit Blu-ray panel is provided with two 5 watt linear blue cold cathode fluorescent tubes (CCFL) (2000 lux, 30 to 60 cm). A Blu-ray transillumination box with the Dark Reader DR-88 is also available. To directly observe the protein gel stained with a fluorescent agent, an amber acrylic plate (1.0 cm thick) was used as a filter to filter out the transmitted or refracted blue light. A digital camera equipped with the same amber acrylic plate (2.0 cm thick) was used to capture the gel image and all used the same photographic parameters, ie an exposure time of 4 seconds and an aperture of 8. An ultraviolet transillumination box (TD-2000E, 365 nm / 312 nm, Spectronics) was also available for simultaneous testing.

In addition, the gel dyed with SYPRO Ruby or Deep Purple is also equipped with a laser gel scanner (Typhoon Trio, GE Healthcare) with 488 or 532 nm excitation laser and 610 (30) nano bandpass. Filter imaging.

The backlight type blue-plate assembly of the present invention, as shown in FIG. A, includes a back plate 40 from bottom to top, which has a black surface, which is used as a background, and two illuminants 30. A blue cold cathode fluorescent lamp tube, a glass transparent plate 10, and a gel as a fluorescent sample 20 are provided. The two illuminants 30 (i.e., two blue cold cathode fluorescent lamp tubes) are located outside the edge of the glass transparent plate 10 as a light source. The back panel 40 of the black background is placed under the glass transparent panel 10 to provide a better contrast. A plastic cover 50 is placed over the glass transparent plate 10 to block unwanted refracted light. The illuminant 30 of the linear blue cold cathode fluorescent lamp can be another visible light source (ie, white). This new type takes Blu-ray as an example. Furthermore, the transparent plate 10 can be a thin glass or a plastic plate, but the thickness should be less than 1 cm, so as to avoid the long distance of light refraction and affect the excitation effect. The illuminant 30 can be a linear cold cathode fluorescent tube or a light emitting diode, and the light source can be visible or invisible.

Use a different illuminating device to observe the protein gel stained with a fluorescent agent: firstly illuminate the protein gel stained with the fluorescent agent with the backlight blue light plate of the present invention and the conventional blue light transillumination box, and compare the results of the two . In the evaluation process, the most commonly used protein gel fluorescent dye SYPRO Ruby (excitation wavelength / emission wavelength = 280 and 450 nm / 610 nm) was selected. It has been found through experiments that the novel back-illuminated blue plate has excellent imaging effect on the protein gel dyed by SYPRO Ruby, and the obtained image has strong fluorescent signal and low background value. Even if the protein content in the ribbon is as low as 5 ng, the ribbons can be viewed directly through an amber filter or filter glass (see Figure B, red triangle 1 for 4.5 ng carbonic anhydrase). In contrast, the fluorescence signal observed by the same gel through the blue transillumination box is less and weaker (Fig. 1C). For example, in the first panel C, the SYPRO Ruby stained ribbon can be visually observed to have a minimum protein content of 18 ng carbonic anhydrase (indicated by red triangle 2). After repeated tests, it was found that in the gel dyed with SYPRO Ruby, the intensity of the protein signal measured by the backlit Blu-ray plate was at least four times the intensity of the protein signal measured by the blue transillumination box.

Almost all SYPRO Ruby fluorescent signals (Fig. D) that can be imaged by laser laser scanners in the gel can be visually inspected through the new backlit Blu-ray plate, demonstrating that the backlit Blu-ray plate can be manually used. The protein with a lower content was re-detected from the protein gel stained with SYPRO Ruby. Long-term exposure to visible blue light is not only harmless to the operator, but also harmless to the fluorophore, because no photobleaching of the protein gel stained with SYPRO Ruby was observed during the test, even if it was left in the backlit blue light. This is also the case for an hour on the board (the relevant data is not shown here). It has been found through experiments that in the novel backlit blue panel device, the edge of the protein gel is always bright blue, but this phenomenon is not present in the blue transillumination box device. This is the result of total reflection of the blue-based blue light appearing at the edge of the protein gel in the protein gel it illuminates (see Figure B), without significantly interfering with the observation of the protein band. It is also worth mentioning that all SYPRO Ruby fluorescent signals can be visually detected in a backlit Blu-ray panel device even without the use of filters or filter glasses. However, this direct view observation cannot be achieved by either the Blu-ray Transillumination Box or the UV Transillumination Box. This highly advantageous feature allows researchers to manually detect excellent bands or color points from certain protein gels dyed with a fluorescent agent through a comfortable and safe procedure.

Use the backlit Blu-ray plate to observe other fluorescent signals in the protein gel: The above-mentioned new backlit Blu-ray device can clearly view the signals of various fluorescent dyes, so it can perform various functions in proteomics experiments. For example, an electrophoresis gel treated with SYPRO Tangerine (excitation wavelength / emission wavelength = 300 and 490 nm / 640 nm) and SYPRO Orange (excitation wavelength / emission wavelength = 300 and 470 nm / 570 nm), The results can be clearly examined using the novel backlit Blu-ray panel (Fig. E and Fig. F). In addition, the luminescent group having a low excitation coefficient, such as a fluorophore in Deep Purple (excitation wavelength / emission wavelength = 532 nm / 610 nm, ε = 20,000) can be clearly observed through the above-mentioned backlight type blue panel device. Displayed in black and white in the first picture G). Almost all of the Deep Purple fluorescent signals (shown in black and white in Figure H) that can be imaged by a laser gel scanner in the gel can be viewed directly by the naked eye through a backlit Blu-ray plate. In the evaluation process, the other two commonly used protein gel fluorescent dyes Krypton (excitation wavelength / emission wavelength = 518 nm / 552 nm) and Flamingo (excitation wavelength / emission wavelength = 515 nm / 545) were not selected. The reason for the nano) is that the amber filter used with it can theoretically filter out the emission signal with a wavelength of about 550 nm. In addition, the high-frequency wide blue light from the cold cathode fluorescent tube may not provide sufficient excitation energy, but it produces significant background noise.

In short, for the currently used SYPRO Ruby and Deep Purple dyes, the imaging effect of the novel backlit blue plate device for protein gel dyed with these phosphors (Fig. B and Fig. 1) G) is more clear than the commonly used UVA or UVB transillumination box. On the other hand, when a conventional blue transillumination box is used, the overall observed brightness of the protein gel dyed with the phosphors is low (Fig. C). The orange or amber thick filter used to filter the blue light may also absorb significant amounts of fluorescent signal, making the weak fluorescent signal in the protein gel difficult to detect in the blue transillumination box.

Using the novel backlit blue light plate to observe the DNA gel stained with SYBR Safe: Method: The DNA ladder mark with a length between 50bps and 3Kbps was double-diluted and then condensed with 7.5% polypropylene guanamine. The gel was separated and stained with a SYBR® Safe DNA staining kit (Invitrogen). The maximum length of each ladder strip mark is listed on the left side of the fourth figure. The DNA gel stained with SYBR® Safe was then photographed by illumination of a backlit Blu-ray plate.

The optical path in the new backlit Blu-ray panel: the backlit Blu-ray panel method is based on Snell's law. This method was used for total internal reflection fluorescence microscopy (TIRFM), which optimizes the ratio of fluorescent signals to background noise when observing objects on a slide. The path from the illuminant 30 of the blue cold cathode fluorescent lamp to the glass transparent plate 10 will be discussed below to illustrate the visual observation provided by the present invention.

The critical angle (θc) involved in the application of the novel backlit blue panel includes the following: θc (glass: air): critical angle between glass and air; θc (glass: acrylamide): glass and poly Critical angle between acrylamide gels; θc (acrylamide: air): the critical angle between polypropylene amide gel and air.

(1) Incident angle θ = < θc (glass: air): Since the refractive index of glass (n glass = 1.5) is larger than the refractive index of air (n air = 1.0), the light emitted from the glass transparent plate 10 is only Refraction angle θ1' is refracted into the air under the condition that the incident angle θ is smaller than the critical angle θc (glass: air) 41.8° (=sin-1 (n air / n glass) = sin-1 (1/1.5)) . For example, if the incident angle θ1 of a blue light is 40°, the blue light will be refracted into the air with a new refraction angle of 74.6° (sin40°×1.5=sin74.6°×1.0) (Fig. 2, Path 1). Since the illuminant 30 of the blue cold cathode fluorescent lamp tube is located beside a very thin (less than 5 mm thick) glass transparent plate 10 in the backlight type blue plate device, the blue light is refracted from the glass transparent plate 10. The position will be no more than 3.33 mm (5 mm x tan 41.8 °) from the edge of the glass transparent plate 10, and the refracted light will be blocked by the plastic cover 50 (Fig. 1A). If the incident angle θ2 of a blue light is equal to the critical angle θc (glass: air) 41.8°, the blue light will be completely refracted, and the traveling direction is parallel to the transparent plate 10 made of glass (second diagram A, path 2). In the glass transparent plate 10, all blue light emitted by the blue cold cathode fluorescent lamp tube 30 and having an incident angle greater than a critical angle θc (glass: air) of 41.8° will be reflected and traveled inside the glass transparent plate 10. It travels in the same way as it does in an optical fiber. Therefore, if the black background back plate 40 is placed under the glass transparent plate 10 (Fig. A), even in the case where the blue cold cathode fluorescent lamp 30 is activated, most of the glass transparent plate 10 ( That is, the portion which is about 3 mm or more from the edge of the glass transparent plate 10 is still slightly dark. Therefore, when observing any object placed on a backlit blue panel, there should theoretically be no light parallel to the observer's line of sight.

(2) θc (glass: air) <incident angle θ=<θc (glass: acrylamide): The main component of the polypropylene guanamine gel is water and acrylamide, therefore, a specific polypropylene guanamine gel The refractive index depends on the concentration of the gel. However, there is currently no refractive index measurement of the acrylamide crystalline powder, but there is a similar substance, that is, poly-2-methyl methacrylate (also known as acrylic) has a refractive index of 1.49. Since the refractive index of water is 1.33, it is reasonable to assume that the refractive index of most polypropylene guanamine gels is between 1.33 and 1.49. The refractive index of a polypropylene guanamine gel, which was not clearly calibrated, was measured to be 1.47. In any case, the refractive index n acrylamide of the polypropylene guanamine gel must be greater than n air and less than n glass. If a polypropylene amide gel having a refractive index of 1.4 is placed on the glass transparent plate 10, the light emitted by the blue cold cathode fluorescent lamp 30 is less than θc (glass: acrylamide) 60.1° at an incident angle θ. (=sin-1 (n acrylamide / n glass) = sin-1 (1.4/1.5)) will be refracted into the gel. Therefore, in the backlight type blue panel device described above, only the light emitted by the blue cold cathode fluorescent lamp tube 30 and having an incident angle θ between 41.8° and 60.1° will have a corresponding refraction angle θ' Refractive into a polypropylene amide gel. For example, in the interface between the glass transparent plate 10 and the gel 20, if the incident angle θ3 of a blue light is 45°, the blue light will have a new angle of refraction θ3'=49.3° (sin45°×1.5=sin49. 3° x 1.4) is refracted into the gel (Fig. 2, path 3). Since the new incident angle θ3* (equal to its equivalent internal angle θ3') is greater than the critical angle θc (acrylamide: air) (45.9° = sin-1 (n air / n acrylamide)), A protein gel stained with a fluorescent agent is placed on a backlit blue light plate, and the blue light that is refracted into the gel and does not excite the fluorophore will not be directly refracted into the air, but is totally reflected in the gel. Travel inside until it reaches the vertical side of the gel. For this interface, the above blue light may have another incident angle θ3"=40.7° (90°-49.3°=40.7°), and its value is less than the critical angle θc (acrylamide:air) 45.9°. Therefore, the blue light is finally It will be refracted into the air at a new angle of refraction of 65.9° (sin40.7° × 1.4 = sin 65.9 ° × 1.0). The above description may explain the blue light emitted by the edge of the polypropylene amide gel (Fig. 1B). If the incident angle θ4 of a blue light is equal to the critical angle θc (glass: acrylamide) 60.1°, the blue light will refract and travel in a direction parallel to the glass transparent plate 10 (second image B, path 4).

In summary, if a blue light is emitted from the blue cold cathode fluorescent lamp 30, the main incident angle θ3 is greater than the critical angle θc (glass: air) but less than the critical angle θc (glass: acrylamide), then the blue light will It is refracted from the glass transparent plate 10 into the polypropylene amide gel 20, which is then totally reflected by the air and finally ejected from the edge of the gel 20. It is assumed here that all of the above blue light is not emitted through the surface of the gel 20.

(3) θc (glass: acrylamide) <incident angle θ: at the interface between the glass transparent plate 10 and the polypropylene amide gel 20, emitted by the blue cold cathode fluorescent lamp 30 and the incident angle θ Light greater than the critical θc (glass: acrylamide) 60.1° should be totally reflected by the gel into the glass transparent plate 10, and finally emitted through the edge of the glass transparent plate 10. For example, if the incident angle θ5 of a blue light is 62°, the blue light will be totally reflected by the gel, and finally injected into the glass transparent plate at another incident angle θ5′=28° (90°-62°). The edge of 10 is emitted from the edge at a refraction angle θ5" = 44.8° (sin28° × 1.5 = sin 44.8 ° × 1.0) (second figure C, path 5). Partially close parallel blue light should be blue light The cold cathode fluorescent lamp tube 30 is directly incident on the fluorescent sample 20 of the polypropylene amide gel at a larger incident angle θ. This part of the blue light should be totally reflected by the gel into the glass transparent plate 10, and finally through the glass. For example, if the incident angle θ6 of a blue light is 84°, the blue light will eventually be incident on the glass transparent plate at an incident angle of θ6'=6° (90°-84°). The edge of 10 is emitted from the edge at a refraction angle θ6" = 9° (sin6° × 1.5 = sin9° × 1.0) (Fig. 2C, path 6).

Image quality evaluation after photographic imaging of fluorescent protein dyed gel on backlit blue light panel: Whether the image obtained by photographic imaging of the new inspection gel on a backlit blue light panel is suitable for quantitative analysis. In this evaluation, the gel stained with SYPRO Ruby was imaged on a backlit Blu-ray plate (Fig. B) or imaged using a laser gel scanner (Fig. D), and the resulting image was taken. Converted to a 16-bit grayscale positive image (Fig. 3A and Fig. B), and then evaluated with a one-dimensional image analysis software. The images obtained in the two ways have similar effects and all exhibit ideal protein dyeing dynamic range. There is an excellent linear relationship between ribbon intensity and actual protein content (between Nike and micrograms) (third panel C and third panel D). In addition, the images obtained in the two ways also have a similar gray scale distribution (gray histogram) (third graph E and third graph F). It is known from the above information that the backlit blue light plate method is an effective device for directly observing the fluorescent signal in the protein gel, and is an economical and reliable excitation light source suitable for gel photography for analysis.

Conclusion: In the above-mentioned backlight blue-plate device, only the blue cold cathode fluorescent lamp 30 is emitted and the main incident angle is larger than the critical angle θc (glass: air) but smaller than the critical angle θc (glass: acrylamide) The blue light is refracted from the glass transparent plate 10 into the polypropylene amide gel 20, which is then totally reflected in the gel and finally ejected from the edge of the gel (Fig. 2, path 3). The other blue light is directly refracted into the air (Fig. 2, path 1) or finally exited through the edge of the glass transparent plate 10 (second figure A~C, paths 2, 4, 5 and 6). The blue light emitted by the blue cold cathode fluorescent lamp 30 does not directly hit the observer's eyes. This is the most likely reason for visually viewing the fluorescent signal on a backlit Blu-ray panel without the need for a filter or filter glass. In addition, the quality of the gel image obtained by the present invention (the signal is strong and the background noise ratio is low) is also superior to those obtained by the blue transillumination box (Fig. B and Fig. C). The test results show that the device of the present invention is not only safe, economical and convenient, but also effectively illuminates the protein gel dyed with the fluorescent agent.

It must be stated here that the refractive index of the material to light varies with the wavelength of the light. The refractive index (n) of a particular material for light of different wavelengths (λ) can be derived from the following Selmeier equation: n2(λ)=1+B1λ2/(λ2-C1)+B2λ2/ (λ2-C2)+B3λ2/(λ2-C3), where B1, B2, B3 and C1, C2, and C3 are the Meyer Meyer coefficients obtained by the test. For example, the refractive index of glass (SiO2) for light having wavelengths of 350 nm, 450 nm, 550 nm, and 650 nm is 1.56560, 1.55257, 1.54599, and 1.54210, respectively, and the refractive index of air to the light is They are 1.000284, 1.000279, 1.000277 and 1.000276 respectively. Therefore, the refractive index of the same material for light having a longer wavelength is smaller than the refractive index of light having a shorter wavelength. However, the critical angle of light does not change significantly due to the relationship between the above wavelength and the refractive index. For example, in the interface between glass and air, the critical angles of light with wavelengths of 350 nm, 450 nm, 550 nm, and 650 nm are measured at 39.7°, 40.1°, 40.3°, and 40.4°, respectively. . Therefore, even if the input light has different wavelengths, the application of the novel backlit Blu-ray panel device is not limited. The above agarose gel can also be selected.

In summary, through the illumination of the novel backlit Blu-ray panel, not only the protein gel stained with a fluorescent agent but also the DNA gel stained with SYBR® can be visually observed. With the device of the present invention, as little as 2 ng of DNA can be visually observed (fourth image, red squares indicate the smallest amount of DNA band observed). Therefore, compared with the ultraviolet transillumination box, the novel backlight blue plate method is not only safe and convenient, but also has multiple uses, and can be used to illuminate fluorescent signals in various biological samples.

The above detailed description is intended to be illustrative of a preferred embodiment of the present invention, which is not intended to limit the scope of the present invention, and other equivalents and modifications may be made without departing from the spirit of the invention. Changes are to be included in the scope of patents covered by this new model.

10‧‧‧Transparent board
20‧‧‧Fluorescent samples
30‧‧‧Lights
40‧‧‧ Backboard
50‧‧‧ cover

The first panel A shows the construction of the novel apparatus and compares the effect of the novel apparatus and other apparatus on the observation of a protein gel dyed with a fluorescent agent (Fig. B to Fig. H). The second graph A to the second graph C respectively show all possible optical paths in the novel backlit blue panel device. The third panel A to the third panel F are for quantitative comparison of the images imaged on the novel backlit blue panel and the images imaged by the laser gel scanner for the gel dyed with SYPRO Ruby. The fourth panel shows the application of the novel backlit Blu-ray panel, which is used to observe DNA gel dyed with SYBR Safe.

10‧‧‧Transparent board

20‧‧‧Fluorescent samples

30‧‧‧Lights

40‧‧‧ Backboard

50‧‧‧ cover

Claims (6)

A device for exciting a fluorescent sample by using light refraction, comprising: a transparent plate for guiding light, wherein the fluorescent sample is disposed; a back plate disposed under the transparent plate; a cover plate disposed on the Above the transparent plate; and at least one illuminant is used as an excitation light source, and is disposed between the transparent plate and the back plate, and is located outside the edge of the transparent plate, so that the light of the illuminant is made of the transparent plate The edge is incident, causing the fluorescent sample on the transparent plate to be excited by receiving the refraction and reflection of the light source according to Snell's law, thereby improving the signal-to-noise ratio during the excitation process, and the cover plate is used to block the penetration The refracted light of the transparent plate. The device of claim 1, wherein the fluorescent sample is DNA, protein or any other biological sample that can be excited by light, which is stained or labeled with a fluorescent agent. The device of claim 1, wherein the transparent plate is made of glass or plastic material, and the transparent plate has a thickness of less than 1 cm. The device of claim 1, wherein the back sheet has a black surface for use as a background to provide a better contrast effect, and the cover is made of a plastic material. The device of claim 1, wherein the illuminant is a blue cold cathode fluorescent lamp. The device of claim 5, wherein the illuminant has an illuminance of 2000 lux.