CN115219466A - Multi-channel fluorescence detection method and device, electronic equipment and storage medium - Google Patents
Multi-channel fluorescence detection method and device, electronic equipment and storage medium Download PDFInfo
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- CN115219466A CN115219466A CN202210821775.XA CN202210821775A CN115219466A CN 115219466 A CN115219466 A CN 115219466A CN 202210821775 A CN202210821775 A CN 202210821775A CN 115219466 A CN115219466 A CN 115219466A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
Abstract
The invention relates to the technical field of fluorescence detection, in particular to a multi-channel fluorescence detection method, a multi-channel fluorescence detection device, electronic equipment and a storage medium, wherein the method comprises the steps of firstly acquiring fluorescence spectra corresponding to all channels; then, analyzing the fluorescence spectrum to obtain a spectrum curve corresponding to each channel; and finally, acquiring fluorescence information corresponding to the channel according to the spectral curve. According to the method, the plurality of channels can obtain a plurality of spectral curves in one-to-one correspondence, and when a certain channel is detected, the spectral curve corresponding to the channel is analyzed, so that the corresponding fluorescence information can be obtained.
Description
Technical Field
The present disclosure relates to the field of fluorescence detection technologies, and in particular, to a multi-channel fluorescence detection method and apparatus, an electronic device, and a storage medium.
Background
The traditional fluorescence PCR optical system adopts the modes of a light filter and a dichroic mirror to realize the dispersion of excitation light and fluorescence, and for an excitation light source, excitation light with a certain spectral bandwidth is obtained through a narrow-band light filter and is used for exciting a fluorescent dye to generate corresponding fluorescence. The spectral bandwidth of the fluorescence generated in the above manner is generally wide due to the physical characteristics, and there is a possibility that a plurality of fluorescent dyes may overlap in spectrum. Therefore, the conventional fluorescent PCR optical system has a problem of channel crosstalk.
Disclosure of Invention
The invention provides a multi-channel fluorescence detection method, a multi-channel fluorescence detection device, electronic equipment and a storage medium, which are used for solving the problem of channel crosstalk during detection of a fluorescence PCR optical system.
In a first aspect, an embodiment of the present invention provides a multichannel fluorescence detection method, including:
acquiring fluorescence spectra corresponding to all channels;
analyzing the fluorescence spectrum to obtain a spectrum curve corresponding to each channel;
and acquiring fluorescence information corresponding to the channel according to the spectral curve.
In the invention, firstly, the fluorescence spectra corresponding to all channels are obtained, and the fluorescence spectra of a plurality of channels are overlapped when possible; then, analyzing the fluorescence spectrum to obtain a spectrum curve corresponding to each channel; and finally, acquiring fluorescence information corresponding to the channel according to the spectral curve. The multiple channels can obtain multiple spectral curves in one-to-one correspondence, and when a certain channel is detected, the spectral curve corresponding to the channel is analyzed, so that corresponding fluorescence information can be obtained.
Optionally, analyzing the fluorescence spectrum to obtain a spectrum curve corresponding to each channel, including:
analyzing the fluorescence spectrum to find the peak of the fluorescence curve corresponding to each channel;
fitting the fluorescence curve according to the wave crest to obtain a spectral curve corresponding to each channel;
and acquiring fluorescence spectrum information corresponding to the channel according to the spectrum curve.
According to the invention, the fluorescence curve corresponding to a single channel is fitted, the fitting mode can only fit the fluorescence curve corresponding to the channel to obtain the spectral curve of the channel, the influence of the fluorescence curves of other channels on the subsequent acquisition of the fluorescence information of the channel is eliminated, then the required spectral information can be directly acquired according to the fitted spectral curve, the independent analysis of each channel can be realized, and the problem of channel crosstalk is reduced. Wherein the peak of the fluorescence curve corresponding to each channel is found to facilitate the determination of the location of the desired fit.
Optionally, fitting the fluorescence curve according to the peak to obtain a spectral curve corresponding to each channel, including:
fitting the fluorescence curve by using a fitting function to obtain a spectral curve corresponding to each channel, wherein the fitting function is as follows:
wherein, c j Is the combined ratio of Lorentzian and Gaussian functions; v. of j Position information of the wave crest; w is a j Is the full width at half maximum information of the peak; i is j Peak intensity information.
In a second aspect, an embodiment of the present invention provides a multi-channel fluorescence detection apparatus, including:
the acquisition module is used for acquiring fluorescence spectra corresponding to all channels;
the processing module is used for analyzing the fluorescence spectrum to obtain a spectrum curve corresponding to each channel;
and the acquisition module is used for acquiring the fluorescence information corresponding to the channel according to the spectral curve.
Optionally, the acquisition module comprises:
the dispersion unit is used for dispersing the fluorescence corresponding to all the channels;
a sensor unit for receiving the dispersed fluorescence to form the fluorescence spectrum.
Optionally, the acquisition module further comprises:
and the collecting unit is used for converging the dispersed fluorescence to the sensor unit.
In a third aspect, an embodiment of the present invention provides an electronic device, including a memory and a processor, where the memory stores a computer program thereon, and the processor implements the method according to any one of the first aspect when executing the program.
In a fourth aspect, an embodiment of the invention provides a computer-readable storage medium on which is stored a computer program which, when executed by a processor, implements the method of any one of the first aspects.
Advantageous effects
The invention provides a multi-channel fluorescence detection method, a multi-channel fluorescence detection device, electronic equipment and a storage medium, wherein the method comprises the steps of firstly, acquiring fluorescence spectra corresponding to all channels; then, analyzing the fluorescence spectrum to obtain a spectrum curve corresponding to each channel; and finally, acquiring fluorescence information corresponding to the channel according to the spectral curve. According to the method, the plurality of channels can obtain a plurality of spectrum curves in one-to-one correspondence, and when a certain channel is detected, the spectrum curve corresponding to the channel is analyzed, so that the corresponding fluorescence information can be obtained, and therefore, the problem of channel crosstalk during fluorescence detection is solved.
It should be understood that the statements herein reciting aspects are not intended to limit the critical or essential features of any embodiment of the invention, nor are they intended to limit the scope of the invention. Other features of the present invention will become apparent from the following description.
Drawings
The above and other features, advantages and aspects of various embodiments of the present invention will become more apparent by referring to the following detailed description when taken in conjunction with the accompanying drawings. In the drawings, the same or similar reference numerals denote the same or similar elements.
FIG. 1 shows a flow diagram of a multi-channel fluorescence detection method of an embodiment of the invention;
FIG. 2 is a schematic diagram of a multi-channel fluorescence detection apparatus according to an embodiment of the present invention;
fig. 3 shows a block diagram of an electronic device according to an embodiment of the present invention.
Detailed Description
In order to make those skilled in the art better understand the technical solutions in one or more embodiments of the present disclosure, the technical solutions in one or more embodiments of the present disclosure will be clearly and completely described below with reference to the drawings in one or more embodiments of the present disclosure, and it is obvious that the described embodiments are only a part of the embodiments of the present disclosure, and not all embodiments. All other embodiments that can be derived by a person skilled in the art from one or more of the embodiments described herein without making any inventive step shall fall within the scope of protection of this document.
It should be noted that, the description of the embodiment of the present invention is only for clearly illustrating the technical solutions of the embodiment of the present invention, and does not limit the technical solutions provided by the embodiment of the present invention.
The principle of the fluorescence PCR optical system is as follows: the light source emits exciting light, the exciting light irradiates the sample cell, so that the reaction substance in the sample cell emits fluorescence, the fluorescence irradiates the detector, and the detector further detects the fluorescence, such as detecting the intensity, energy and the like of the fluorescence.
The fluorescence detection realized by the traditional method specifically comprises the following steps: in the fluorescence detection process, the exciting light emitted by a light source is filtered through narrow-band filtering to obtain exciting light of a corresponding wavelength band, and the exciting light of the corresponding wavelength band is irradiated to a sample reaction pool part under the action of a dichroic mirror (the dichroic mirror can enable the exciting light of certain wavelength to almost completely penetrate through and the exciting light of other wavelength bands to be completely reflected) along a light path channel; then the corresponding fluorescent substance is irradiated to emit fluorescence, and the required fluorescence is irradiated to the detector after the action of the dichroic mirror (the dichroic mirror can enable the fluorescence with certain wavelength to be almost completely transmitted, and the fluorescence with other wavelength bands is completely reflected). In order to solve the problem of channel crosstalk, the bandwidth of the narrow band filter, that is, the width of the wavelength band allowing the excitation light to pass through is usually reduced, but the reduction of the bandwidth of the narrow band filter greatly reduces the capability of the excitation light, further reduces the energy value of exciting the fluorescent substance to emit fluorescence, reduces the energy value of the fluorescence, and further affects the sensitivity of fluorescence detection.
This example fig. 1 shows a flow chart of a multi-channel fluorescence detection method of an embodiment of the invention; referring to fig. 1, the method includes:
s1, acquiring fluorescence spectra corresponding to all channels;
in this embodiment, the excitation light emitted by the light source does not need to pass through the design of the narrow band pass filter, the excitation light emitted by the light source directly irradiates the sample cell, the excitation light of different wavebands irradiates different reaction substances to emit fluorescence of different wavelengths, and the plurality of channels correspond to the fluorescence of a plurality of different wavelength bands. In this embodiment, the light splitting element is adopted to split the fluorescence of different wavelength bands, the light splitting element can disperse the fluorescence, and then the dispersed fluorescence is obtained through the fluorescence detection device, and the fluorescence detection device converts the fluorescence into an electrical signal, thereby forming a fluorescence spectrum. The light splitting element may be a prism, a grating, or other elements, which is not limited in this embodiment. The fluorescence detection device may be a sensor, such as a Charge Coupled Device (CCD). The fluorescence spectrum comprises fluorescence curves corresponding to a plurality of channels, and if there are 4 channels, there are 4 fluorescence curves on the fluorescence spectrum, but there may be overlap between the multiple fluorescence curves.
S2, analyzing the fluorescence spectrum to obtain a spectrum curve corresponding to each channel;
the analysis of the fluorescence spectrum is to analyze the fluorescence curve corresponding to each channel, if 4 channels exist, four fluorescence curves corresponding to the 4 channels are respectively analyzed to form 4 independent and non-overlapping spectrum curves, so as to facilitate independent analysis.
And S3, acquiring fluorescence information corresponding to the channel according to the spectrum curve.
Because the spectral curves of the channels are independent and non-overlapping, the independent spectral curves can be analyzed, specific information of fluorescence corresponding to each channel can be definitely known, the fluorescence spectrum can be really analyzed on a physical layer, and channel crosstalk can be thoroughly deducted.
In summary, the present embodiment provides a multi-channel fluorescence detection method, which first obtains fluorescence spectra corresponding to all channels; then, analyzing the fluorescence spectrum to obtain a spectrum curve corresponding to each channel; and finally, acquiring fluorescence information corresponding to the channel according to the spectral curve. The plurality of channels can obtain a plurality of spectrum curves in one-to-one correspondence, and the spectrum curves corresponding to the channels are analyzed during detection of a certain channel, so that corresponding fluorescence information can be obtained, and therefore the problem of channel crosstalk during fluorescence detection is solved.
Specifically, the analyzing the fluorescence spectrum to obtain a spectrum curve corresponding to each channel includes:
s21, analyzing the fluorescence spectrum to find a peak of a fluorescence curve corresponding to each channel;
the abscissa of the fluorescence spectrum represents the wavelength, the ordinate represents the intensity of fluorescence, find the wave crest of the fluorescence curve corresponding to each channel, in order to position the wave crest of the fitted spectrum curve, find the wave crest through the function of scipy.
S22, fitting the fluorescence curve according to the wave crests to obtain a spectrum curve corresponding to each channel;
during fitting, firstly, the peak of the spectral curve to be fitted is consistent with the peak of the fluorescence curve on the fluorescence spectrum, and then fitting is carried out.
Specifically, the fitting the fluorescence curve according to the peak to obtain a spectrum curve corresponding to each channel includes:
fitting the fluorescence curve by using a fitting function to obtain a spectrum curve corresponding to each channel, wherein the fitting function is as follows:
wherein, c j Is the combined ratio of Lorentzian and Gaussian functions; v. of j Position information of the wave crest; w is a j Is the full width at half maximum information of the peak; i is j Is peak intensity information; n is the number of channels.
When fitting is carried out by using the fitting function, the function F (x) can be better matched with a fluorescence curve by adjusting the combination proportion of the Lorentz function and the Gaussian function, the position information of a peak, the half-height width information of the peak and the peak intensity information, and the fitting is better when the value of the F (x) is larger. Wherein the combined ratio of Lorentz and Gaussian functions is a value between [0,1 ]; during fitting, the half-peak width of the fluorescence curve can be visually seen from the fluorescence spectrum, the half-peak width of the fitted spectrum curve can also be seen, and the size relation between the fluorescence curve and the half-peak width of the spectrum curve can be obviously seen. Therefore, when fitting, if the half-peak width of the spectral curve is larger than that of the fluorescence curve, the half-peak width of the fluorescence curve is appropriately reduced; if the half-peak width of the spectral curve is smaller than the half-peak width of the fluorescence curve, the half-peak width of the fluorescence curve is appropriately increased, and the intensity information of the peak can be adjusted as well, so that the function F (x) can be better matched with the fluorescence curve.
In this embodiment, the curve defined by function F (x) can be fitted using the curve _ fit method included in scipy in Python.
And S23, acquiring fluorescence spectrum information corresponding to the channel according to the spectrum curve.
The fluorescence intensity value of each channel can be corresponded to according to an individual spectrum curve, the fluorescence intensity value is an ordinate value of the spectrum curve, the abscissa represents the wavelength, if the energy of fluorescence needs to be detected, the fluorescence intensity value and the fluorescence wavelength can be used for calculation, and the fluorescence energy on the spectrum curve is the area of the spectrum curve.
In addition, in this embodiment, the fluorescence signals are acquired in a spectrum manner, so that a plurality of channels can acquire the fluorescence spectrum signals simultaneously to obtain a fluorescence spectrum including spectrum curves corresponding to all the channels, and then the spectrum curves corresponding to all the channels are obtained through algorithm fitting and processing. And then fitting the fluorescence spectrum curve of each channel according to a fitting algorithm, thereby realizing the function of simultaneously carrying out fluorescence detection on a plurality of channels besides the function of deducting channel crosstalk.
Based on the same inventive concept, the embodiment of the present invention further provides a multi-channel fluorescence detection apparatus, which can be used to implement the multi-channel fluorescence detection method described in the above embodiments, as described in the following embodiments: because the principle of solving the problems of the multi-channel fluorescence detection device is similar to that of a multi-channel fluorescence detection method, the implementation of the multi-channel fluorescence detection device can be referred to the implementation of the multi-channel fluorescence detection method, and repeated parts are not described again. As used hereinafter, the term "unit" or "module" may be a combination of software and/or hardware that implements a predetermined function. While the system described in the embodiments below is preferably implemented in software, implementations in hardware, or a combination of software and hardware are also possible and contemplated.
FIG. 2 is a schematic diagram of a multi-channel fluorescence detection apparatus according to an embodiment of the present invention; as shown in fig. 2, the apparatus includes:
the acquisition module 10 is used for acquiring fluorescence spectra corresponding to all channels;
the processing module 20 is configured to analyze the fluorescence spectrum to obtain a spectrum curve corresponding to each channel;
and the obtaining module 30 is configured to obtain fluorescence information corresponding to the channel according to the spectral curve.
The embodiment provides a multi-channel fluorescence detection device, which comprises an acquisition model 10, a processing module 20 and an acquisition module 30, wherein the acquisition module 10 is used for acquiring fluorescence spectra corresponding to all channels; the processing module 20 is configured to analyze the fluorescence spectrum to obtain a spectrum curve corresponding to each channel; the obtaining module 30 is configured to obtain fluorescence information corresponding to the channel according to the spectral curve. The processing module 20 analyzes the fluorescence spectra corresponding to the multiple channels into multiple spectral curves corresponding to the multiple channels one to one, and the corresponding fluorescence information can be obtained by detecting the spectral curves corresponding to the channels, so that the problem of channel crosstalk during fluorescence detection is solved in this embodiment.
Specifically, the acquisition module 10 includes:
a dispersion unit 101, configured to disperse the fluorescence corresponding to all channels; the exciting light emitted by the light source is subjected to dispersion after being acted by the dispersion unit so as to meet the requirement that a plurality of channels correspond to the exciting light with different wavelengths. The dispersion unit 101 may be a light splitting element, and the light splitting element may be a prism, a grating, or the like.
A sensor unit 102 for receiving the dispersed fluorescence to form the fluorescence spectrum. The main principle is to convert light energy into an electrical signal for subsequent analysis. The sensor unit may be a sensor itself, or a unit module integrated on the sensor, and the sensor may be a Charge Coupled Device (CCD) or the like, which is not limited in this embodiment.
Specifically, the acquisition module further comprises:
a collecting unit 103 for collecting the dispersed fluorescence to the sensor unit. The fluorescent light dispersed by the dispersing unit 101 is scattered, and the scattered fluorescent light is difficult to receive, so that the collecting unit is used for collecting the scattered fluorescent light to the sensor unit in the embodiment, so that the fluorescent light excited by the excitation light can be completely received by the sensor, and the sensitivity of the fluorescent light detection is further improved. The collecting unit may be an imaging lens, other light-gathering elements, or a unit module integrated on the imaging lens or other light-gathering elements.
An embodiment of the present invention also provides a computer electronic device, and fig. 3 shows a schematic structural diagram of an electronic device to which an embodiment of the present invention can be applied, and as shown in fig. 3, the computer electronic device includes a Central Processing Unit (CPU) 401 that can perform various appropriate actions and processes according to a program stored in a Read Only Memory (ROM) 402 or a program loaded from a storage section 408 into a Random Access Memory (RAM) 403. In the RAM 403, various programs and data necessary for system operation are also stored. The CPU 401, ROM 402, and RAM 403 are connected to each other via a bus 404. An input/output (I/O) interface 405 is also connected to bus 404.
The following components are connected to the I/O interface 405: an input portion 406 including a keyboard, a mouse, and the like; an output section 407 including a display device such as a Cathode Ray Tube (CRT), a Liquid Crystal Display (LCD), and the like, and a speaker; a storage section 408 including a hard disk and the like; and a communication section 409 including a network interface card such as a LAN card, a modem, or the like. The communication section 409 performs communication processing via a network such as the internet. A drive 410 is also connected to the I/O interface 405 as needed. A removable medium 411 such as a magnetic disk, an optical disk, a magneto-optical disk, a semiconductor memory, or the like is mounted on the drive 410 as necessary, so that a computer program read out therefrom is mounted into the storage section 408 as necessary.
The flowchart and block diagrams in the figures illustrate the architecture, functionality, and operation of possible implementations of systems, methods and computer program products according to various embodiments of the present invention. In this regard, each block in the flowchart or block diagrams may represent a module, segment, or portion of code, which comprises one or more executable instructions for implementing the specified logical function(s). It should also be noted that, in some alternative implementations, the functions noted in the block may occur out of the order noted in the figures. For example, two blocks shown in succession may, in fact, be executed substantially concurrently, or the blocks may sometimes be executed in the reverse order, depending upon the functionality involved. It will also be noted that each block of the block diagrams and/or flowchart illustration, and combinations of blocks in the block diagrams and/or flowchart illustration, can be implemented by special purpose hardware-based systems which perform the specified functions or acts, or combinations of special purpose hardware and computer instructions.
The units or modules described in the embodiments of the present invention may be implemented by software or hardware. The described units or modules may also be provided in a processor, and may be described as: a processor includes an acquisition model 10, a processing module 20, and an acquisition module 30, wherein the names of these modules do not in some cases constitute a limitation on the module itself, e.g., the acquisition model 10 may also be described as "a cloud acquisition model 10 for acquiring fluorescence spectra corresponding to all channels".
As another aspect, the present invention further provides a computer-readable storage medium, which may be the computer-readable storage medium included in the multi-channel fluorescence detection apparatus described in the above embodiments; or it may be a computer-readable storage medium that exists separately and is not built into the electronic device. The computer readable storage medium stores one or more programs for use by one or more processors in performing a multi-channel fluorescence detection method described in the present invention.
The foregoing description is only exemplary of the preferred embodiments of the invention and is illustrative of the principles of the technology employed. It will be appreciated by those skilled in the art that the scope of the invention herein disclosed is not limited to the particular combination of features described above, but also encompasses other arrangements formed by any combination of the above features or their equivalents without departing from the spirit of the invention. For example, the above features and the technical features (but not limited to) having similar functions disclosed in the present invention are mutually replaced to form the technical solution.
Claims (8)
1. A multi-channel fluorescence detection method, comprising:
acquiring fluorescence spectra corresponding to all channels;
analyzing the fluorescence spectrum to obtain a spectrum curve corresponding to each channel;
and acquiring fluorescence information corresponding to the channel according to the spectral curve.
2. The multi-channel fluorescence detection method of claim 1, wherein the analyzing the fluorescence spectrum to obtain a spectrum curve corresponding to each channel comprises:
analyzing the fluorescence spectrum to find the peak of the fluorescence curve corresponding to each channel;
fitting the fluorescence curve according to the wave peak to obtain a spectral curve corresponding to each channel;
and acquiring fluorescence spectrum information corresponding to the channel according to the spectrum curve.
3. The multi-channel fluorescence detection method of claim 2, wherein fitting the fluorescence curve according to the peak to obtain a spectral curve corresponding to each channel comprises:
fitting the fluorescence curve by using a fitting function to obtain a spectrum curve corresponding to each channel, wherein the fitting function is as follows:
wherein, c j Is the combined ratio of Lorentzian and Gaussian functions; v. of j Position information of the wave crest; w is a j Is the full width at half maximum information of the peak; I.C. A j Is peak intensity information; n is the number of channels.
4. A multi-channel fluorescence detection device, comprising:
the acquisition module is used for acquiring fluorescence spectra corresponding to all channels;
the processing module is used for analyzing the fluorescence spectrum to obtain a spectrum curve corresponding to each channel;
and the acquisition module is used for acquiring the fluorescence information corresponding to the channel according to the spectrum curve.
5. The multi-channel fluorescence detection device of claim 4, wherein the acquisition module comprises:
the dispersion unit is used for dispersing the fluorescence corresponding to all the channels;
a sensor unit for receiving the dispersed fluorescence to form the fluorescence spectrum.
6. The multi-channel fluorescence detection device of claim 5, wherein the acquisition module further comprises:
and the collecting unit is used for converging the dispersed fluorescence to the sensor unit.
7. An electronic device comprising a memory and a processor, the memory having stored thereon a computer program, characterized in that the processor, when executing the computer program, implements the method according to any of claims 1-3.
8. A computer-readable storage medium, on which a computer program is stored, which, when being executed by a processor, carries out the method according to any one of claims 1-3.
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| CN115792136A (en) * | 2023-01-28 | 2023-03-14 | 清华大学合肥公共安全研究院 | Gas concentration detection method and device, terminal equipment and storage medium |
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