TWI252313B - Multiphoton excitation fluorescence microscopy for detecting biochips - Google Patents

Abstract

The invention provides a multiphoton excitation fluorescence microscopy, and also a phosphor analysis device for simultaneously detecting phosphor on biochip with different fluorescent material. The device comprises a biochip, a multiphoton excitation light source, object lens, and plural groups of detecting channels, in which the biochip has phosphor mark thereon through hybridization process to express the biological message after hybridization, multiphoton excitation light source can focus on a light spot after the reaction of object lens through exciting light to excite the phosphor mark bundled with the biochip, and then the phosphor with some different specific wavelength through excitation can be detected via plural groups of detecting channels.

Description

1252313 V. INSTRUCTIONS (1) Field of the Invention: The present invention relates to a fluorescence analysis of a biochip - a multiphoton excitation fluorescence microscopy device, and y is a fluorescent substance of different fluorescent properties to effectively increase enthalpy Bailey's fluorescent analyzer. Bio-positive analysis of the rate of the invention The back of the subject and the best of the protein, the production, the production, and the production of the extremely precise order. Raw and microfluidic wafers: The human base is a tool for understanding the quality of the mass group. The rapid prototyping experiment can be carried out on the bed inspection of the wafer, the century will be the technology of the wafer, the technology is made in a very small factor map of the 10,000-molecular research wafer, and it is necessary to obtain the application of the strain sieve. And according to the carrier used by the film is about to be determined by the cause. The wholeness of the use of biotechnology is covered by the selection and the film is said to be completed in a sequence. The significance of the watch and the object wafer are the main features for the development of the sample sample (parallelization) of the functional environment control. Different kinds of micro-devices require a large amount of paper, glass, or on the eve, to implement the relationship between the various undergraduates, to solve this complexity, and to analyze the accuracy and reagents. Experimental data. & research, new drugs. Undoubtedly, 〇 'scientists use specific biomaterials t biochemical synthesis biochemical test specimens can be divided into DNA wafers (gene wafers), protein wafers, etc., while DNA wafers are currently the fastest growing and most Mature technology. The principle is to use thousands or tens of thousands of points of single-stranded DNA (also known as

1252313 V. INSTRUCTIONS (2) For probes, probes are made in a high-density manner on biomaterials made of glass sheets, nylon films or other materials (generalized wafers). Among them, there are two main sources of single-stranded DNA: oligonucleotides (oligonucleotide) and complementary DNA (complementary DNA; cDNA). Oligonucleotide wafers are mainly manufactured by Affymetrix Biotech Co., Ltd., which use the four bases A, T, C, and G that make up the DNA to form about 20 to 25 (layers) in a stack-like manner. Nucleotides; complementary DNa wafers use known complementary DNA extracted from a patient's specimen or other organism, and then these oligonucleotides or complementary DNA are spotted on the wafer. Next, the message RNA (messenger RNA; mRNA) of the sample to be detected is extracted and 'reverse transcript ion' is used to convert the message r 成 into c DNA and mark the fluorescent substance on the cDNA. The labeled sample cdnA was hybridized with the probe above the wafer, then interpreted using a confocal microscopy device, and the hybridized message was transmitted to a computer for analysis.

Biochips are most commonly used in disease understanding because more than 10% of human diseases are associated with genetic defects or abnormalities. Understanding genes can help clarify disease mechanisms to find ways to prevent or treat them. Therefore, scientists use a series of complicated processes such as blood collection, separation, crushing, extraction, screening, and signal transmission to obtain a certain protein from the biological disease-bearing organism, and then use these genes or proteins as biomaterials to make oyster tablets. (simulated pathogens) 'for medical personnel to use these biofilms with simulated lesions for detection.

1252313

Since the interpretation of traditional biochips is the use of confocal microscopy, you can use it. 〇-, 疋 收 收 一个 一个 以 以 以 以 以 收 收 收 收 收 收 收 收 收 , , , , , , , , , , , , , , , , , 萤 萤 萤 萤 萤 萤 萤 萤 萤 萤 萤 萤 萤 萤 萤 萤It is very close to the fluorescence wavelength. Therefore, there is a limit to the fluorescence analysis of this method applied to multi-color biochips, and it cannot improve the speed disk efficiency of biochip fluorescence analysis; ~ () single photon fluorescence excitation is not only It is limited to the high-intensity area where the beam is focused, so it is necessary to use pinholes as a filter; (3), using a single photon excitation will have more photodamaging and photobleaching effects; 4) Single-photon fluorescent confocal microscopy uses shorter-wavelength visible light. When the sample conforms to lightning scattering, according to the Rayleigh scattering principle, the intensity of the beam in the sample is inversely proportional to the fourth power of the wavelength. The beam excited by a single photon will be greatly attenuated, which will affect the detection effect. OBJECT AND SUMMARY OF THE INVENTION: The main object of the present invention is to provide a fluorescence analysis device for detecting fluorescent marks on a biochip using a multiphoton excitation fluorescence microscopy apparatus. Multi-photon Excitation Fluorescence Another object of the present invention is to provide a 1252313 (4) " '^-microscopic device for simultaneous detection of different fluorescent markers on a biochip. Knife Another object of the present invention is to provide a multiphoton excited fluorescence microscopy apparatus having a complex array detection channel to enhance the fluorescence analysis apparatus for biochip fluorescence detection. The essence consists of a mirror and a point-based substance of the crystal spar laser into a light. With the invention of the objective lens, there is an increase in the number of gene crystal arrays or tens of thousands of sample cDNAs on the chip, and then the system produces the points, after which, after the harvest, the biological information is a multi-photon synchronization to excite different nature organisms. Fluorescence excitation loading for wafer analysis efficiency, titanium sapphire laser system, beam scanning channel. Wherein, a single strand of DNA is spotted on the gene chip, and when the single strand of DNA has been hybridized (hybridization: a biological message with a miscellaneous sum. Then, the raw light source is used to sweep through the beam scanner)瞒 激发 激发 激发 激发 激发 激发 激发 与 与 与 与 与 与 与 与 与 与 与 与 与 与 与 与 与 与 与 与 与 与 与 与 与 与 与 与 与 与 与 与 与 与 与 与 与 与 与 与 与 与The firefly cursor number is described in detail by the gene crystal invention. The disadvantages of the single photon excitation fluorescence are disclosed. The invention discloses the use of multiphoton excitation fluorescence microscopy device to replace the traditional early precursor. Confocal microscopy device, / Fluorescence analysis device S. The following approximately 罝 ^ Ascending efficiency is described below in multiphoton excited fluorescence microscopy

1252313 V. INSTRUCTIONS (5) Centering on the principle of two-photon excitation of fluorescence. Two-photon excitation means that the excited molecules simultaneously absorb two photons with frequencies v 1 and v 2 , and are excited by a single photon with a frequency equivalent to vi + v 2 . Since the excitation of two photons requires absorption of two photons, the transition rate is proportional to the square of the intensity of the incident light and because the two-photon absorption cross section is extremely low, an extremely high instantaneous power is required to effectively generate the excitation. The mechanism by which fluorescence is produced comes mainly from the electronic transition of atoms or molecules. The first picture is the famous Jab lonski diagram, which is used to describe the typical energy level transition in the case of camp light generation, where SO represents the ground state and S1 and S2 respectively represent the first and second excited states of the electron. In the absence of any excitation photons being absorbed, 'according to the Bozeman distribution, the ratio of each excited state to the base, the state of the molecule (R) can be expressed by: R = e - ΔΕ / KT where △ E represents Is the energy difference between the energy levels, k is the Boltzmann constant, and τ is the absolute temperature. At room temperature, since most of the molecules are in the ground state, no fluorescence will occur. However, when an object absorbs a photon of a specific wavelength, the fluorescent molecules therein absorb the excitation photons of the appropriate wavelength and transition to the higher energy level S 1 (arrow 2 ) or S 2 (arrow 1 ). In general, the molecules at S2 will quickly fall back to S1 (Arrow 3) in a non-radiative pattern; while the molecules at S1 will later fall back to the ground state S 0 (Arrow 4) and emit fluorescence. This conversion process takes a very short time' to be completed in about 10-8 seconds. It is worth noting that the excitation of this fluorescent light can also be achieved by absorbing two-photons or multiple photons. Referring to the second figure, the figure discloses a multiphoton fluorescence microscopy device that can synchronously excite fluorescent substances of different fluorescent properties on a biochip, thereby effectively increasing the efficiency of analysis and reducing the consumption of biochips. . First of all

1252313

Thousands or tens of thousands of single-stranded DNA 20 (referred to as probes) are spotted on a biochip in a high-density manner, so that the material of the biochip can be selected from a glass piece of three nylon film or the like. Substance, and the source of single stranded DNA 2〇 can be oligonucleic acid or complementary DNA. Among them, this single-strand dna can also select protein f, antigen or antibody according to the actual needs of I. Next, the RNA of the sample to be detected is extracted from the sample, and the RNA is converted into cDNA by reverse transcription, and the fluorescent substance is labeled on the cDNA. Subsequently, the sample cDNA of the labeled fluorescent substance is hybridized with the probe 20 (i.e., single-stranded DNA) on the wafer 1 (hybridization). After the probe 20 is hybridized with the cDNA of the labeled fluorescent substance on the sample, the wafer 1 has a fluorescent substance bound thereto, i.e., a hybrid and subsequent biological message. This biological message is then detected using a multiphoton excited fluorescence microscopy device. In a multiphoton excited fluorescence microscopy apparatus, multiphoton excitation source 30 produces excitation light for simultaneous excitation of phosphors of different fluorescent properties on biochip 10. In a preferred embodiment, the titanium sapphire laser system can be selected as a multi-photon excitation source for exciting near-infrared light having a wavelength of 70 0 n m~1 〇 〇 〇 n m . / After the excitation light is emitted from the multiphoton excitation light source 30, it is transmitted to the beam scanner 4A via the reflection of the first surface 80 and the second mirror 90. Immediately after this, the light transmitted by the beam scanner 40 will become an amplified parallel beam after passing through the beam mirror 1〇〇. When the beam is focused by the objective lens 5 into a spot and projected onto the sample, the fluorescent mark combined with the biochip can be excited. 1252313

V. INSTRUCTIONS (7) It is worth noting that the beam scanner 4 can enable the bio-message on the aa chip 10 to be scanned one by one according to the setting of the operator. With these excited and specific wavelength fluorescent signals, after receiving by the objective lens 50 and passing through the beam splitter 110, a portion of the fluorescent light is reflected back to the sample' while another portion of the fluorescent light passes through the beam splitter 11 0. The fluorescence of the spectroscope 11 〇* is separately filtered by the complex array filter 60, and then the complex array detection channel 70 corresponding to the complex array filter 60 can be used to detect the complex array filter. 60 transmitted biological messages. In the embodiment of the present invention, the number of complex array filters and detection channels can be four, and - the hard array filter can also select 稜鏡 or raster. Finally, work on data analysis via a computer. ^ On-wafer organisms The use of multiphoton-excited fluorescence microscopy devices to detect biological information has many advantages: (1) Since the excitation wavelength of multiphotons and the wavelength of fluorescence emission are very large, a complete emission spectrum can be easily obtained. Also. Also, since multiphotons can simultaneously excite fluorescent substances of different fluorescent properties, multicolor=fluorescence analysis can be performed simultaneously, which makes more changes in application.

= Significantly improve the efficiency of biochip analysis and reduce the cost of biochips to avoid the cost of wafer south; avoid light damage and photobleaching effects outside the focus point; 2) Multiphoton excitation fluorescence is stimulated by longer wavelengths Light, according to Lei Liyue, principle, the attenuation of light can be significantly reduced, and reach a deeper

1252313 V. Observations of inventions (8). The present invention has been described above by way of a preferred embodiment, and is not intended to limit the spirit of the invention and the invention. Modifications made without departing from the spirit and scope of the invention are intended to be included within the scope of the appended claims.

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Brief Description of the Drawings The following is a detailed description of the above-mentioned contents and the description of the accompanying drawings, which will be able to easily understand the advantages and advantages of the drawings. Among them, the picture of the younger brother is Jablonski. A typical energy level conversion situation when fluorescence is generated is shown; and a white second diagram is a schematic diagram of the multiphoton excitation fluorescence microscopy apparatus of the present invention, showing the connection relationship between the constituent elements. Figure number comparison table: 4 1 0 biochip 30 multiphoton excitation source 5 0 objective lens 70 detection channel 90 second mirror 110 beam splitter 20 single strand DNA 4 0 beam scanner 60 filter 8 0 first mirror 1 0 0 beam mirror set 1 2 0 fluorescence microscopy device

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Claims (1)

ι::? 12523 VI. Patent Application 1 · A multiphoton excited fluorescence microscopy device for detecting optical signals of biochips, wherein the biochip has a glory mark through heterogeneous interactions to express the biological information of the miscellaneous and subsequent The device comprises at least: a multiphoton excitation source for generating excitation light; a beam scanner for scanning the excitation light generated by the multiphoton excitation source, and scanning the biochip one by one according to an operator setting; a mirror for reflecting the excitation light from the beam scanner; an objective lens for focusing the excitation light into a spot to excite the fluorescent mark on the biochip, wherein the fluorescent mark fluorescent signal After receiving through the objective lens and interacting with the beam splitter, a portion of the fluorescent signal passes through the beam splitter; a complex array filter is configured to split the portion of the fluorescent signal that passes through the specific wavelength of fluorescence; and the complex array detection a channel for simultaneously detecting the specific wavelength of fluorescence passing through the complex array filter. 2. The multiphoton excited fluorescence microscopy apparatus as described in claim 1 'The multiphoton excitation light source can be a titanium sapphire laser system and can generate a wavelength of 700 rpm~1 〇〇〇nm Near infrared light. : Mirror filter group norm, re-recovery, please apply for it, 3, install micro-light, light, fluorescing, exciter, light, more than the group, or the grid light mirror, including package, more stocks The number of copies of the group ο degree is close to the height of the 可 可 可 第 其 , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , Excited light Page 14 1252313 6. Patent application scope 5. The multi-photon excited fluorescence microscopy apparatus described in claim 4, the source of the complex array of single-stranded DNA may be an oligonucleotide, a complementary nucleotide or Any combination thereof. a multi-photon excited fluorescence microscopy device for detecting a biological chip optical signal, wherein the biochip has a fluorescent mark by means of a heterogeneous action for expressing a mixed biological message, the device being at least The method includes: a multiphoton excitation light source for generating excitation light; a beam scanner for stimulating the excitation light generated by the multiphoton excitation source to scan the biochip according to an operator setting; a beam mirror for amplifying the excitation light from the beam scanner into a parallel a beam splitter for reflecting the parallel beam from the beam mirror; an objective lens for focusing the parallel beam into a spot to excite the fluorescent mark on the biochip, wherein the fluorescent mark After the fluorescent signal is received by the objective lens and interacts with the beam splitter, a portion of the fluorescent signal passes through the beam splitter; and a plurality of array filters are used to divide the portion of the fluorescent signal passing through into a specific wavelength of fluorescence; The complex array detection channel is used to simultaneously detect the specific wavelength of fluorescence passing through the complex array filter. 7. The multiphoton excitation fluorescence microscopy device as described in claim 6 of the patent application. The multiphoton excitation light source may be a titanium sapphire laser system and may have a wavelength of 70 nm to 1 〇〇〇 nm. Infrared light. 8. Multiphoton excited fluorescent microscopy as described in claim 6 Page 15 1252313 VI. Patent application scope, wherein the complex array filter further comprises 稜鏡, grating or any combination thereof. 9. The multi-photon excited fluorescence microscopy apparatus described in claim 6 of the patent application, the number of the complex array detection channels may be four. I 0. The multiphoton excited fluorescence microscopy apparatus of claim 6, wherein a plurality of complexes of D N A, a protein, an antigen, an antibody, or any combination thereof can be densely arrayed on the biochip. II. The multiphoton excited fluorescence microscopy apparatus of claim 1, wherein the source of the complex array of single strands of DNA may be an oligonucleotide, a complementary nucleotide, or any combination thereof. 1 2. A multiphoton excited fluorescence microscopy apparatus for detecting a biological chip optical signal, wherein the biochip is fluorescently labeled by a heteroduplex to express a mixed biological message, the device At least comprising: a titanium sapphire laser system for generating excitation light to excite the fluorescent mark on the gene wafer; a beam scanner for generating the excitation light by the titanium sapphire laser system Sweeping the gene wafer one by one according to an operator setting, a beam mirror group for amplifying the excitation light from the beam scanner into a parallel beam; a beam splitter for reflecting the parallel beam from the beam mirror; an objective lens And illuminating the parallel light beam into a light spot to excite the fluorescent mark on the biological chip, wherein the fluorescent mark fluorescent signal is received by the objective lens and interacts with the beam splitter to have a portion of the fluorescent light The optical signal passes through the beam splitter; a complex array filter is used to divide the portion of the fluorescent signal passing through into a specific wavelength Page 16 1252313 VI. Patent Application Range Fluorescence; and Complex array detection channel for simultaneous detection of the specific wavelength of fluorescence passing through the complex array filter. 1 3 · If the multiphoton excitation fluorescence microscopy device described in claim 1 of claim 1 is applied, the multiphoton excitation light source may be a titanium sapphire laser system and may generate a wavelength of 70 0 run~1 0 〇〇 Near-infrared light of nm. The multiphoton excited fluorescence microscopy apparatus of claim 12, wherein the multiple array filter further comprises a prism, a grating, or any combination thereof. 1 5 · If the multi-photon excitation fluorescence microscopy device of the 12th item of the patent application is applied, the number of the complex array detection channels can be four. A multiphoton excited fluorescence microscopy apparatus as described in claim 2, wherein a plurality of single-stranded DNA, proteins, antigens, antibodies, or any combination thereof can be densely arrayed on the biochip. 17. The multiphoton excited fluorescence microscopy apparatus as described in claim 6 of the patent application. The source of the complex array of single strands of D N A is an oligonucleotide, a complementary nucleoside 'acid or any combination thereof.