CN109991198B - Laser-induced fluorescence detection system with visual real-time imaging focusing - Google Patents
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- 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
- G01N21/6402—Atomic fluorescence; Laser induced fluorescence
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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
- G01N21/645—Specially adapted constructive features of fluorimeters
- G01N21/6456—Spatial resolved fluorescence measurements; Imaging
Abstract
A laser-induced fluorescence detection system with visualized real-time imaging focusing belongs to the technical field of analysis and detection. The detection system adopts a confocal structure and comprises a laser light path, a fluorescence collection light path and an imaging calibration light path. A laser light path is formed by a laser, a neutral density filter, a laser filter, a cylindrical lens, a dichroic mirror and an objective lens; the objective lens, the dichroic mirror, the fluorescence filter, the lens, the slit and the silicon avalanche photodiode form a fluorescence collecting light path; and the Light Emitting Diode (LED), the optical filter, the diaphragm, the objective lens, the dichroic mirror, the reflecting mirror, the lens and the camera form an imaging calibration light path. The imaging calibration light path simplifies the focusing steps of the capillary and the chip in the application of the laser-induced fluorescence detection system, and realizes the visual adjustment and real-time online display of the detection window and the laser spot. The detection system has high sensitivity and good stability, and the detection of the system on the fluorescein sodium can reach the unimol level.
Description
Technical Field
The invention belongs to the technical field of analysis and detection, and particularly relates to a laser-induced fluorescence detection system with visualized real-time imaging focusing.
Background
Laser induced fluorescence detection (LIF) was first proposed by Diebold and Zare in the 70's of the 20 th century in combination with high performance liquid chromatography for the isolation analysis of aflatoxins B1, B2, G1 and G2. The laser has the unique advantages of good monochromaticity, strong light-gathering property and the like, can focus a light beam to a very small area, and has high photon flux, thereby greatly improving the detection sensitivity. At present, the LIF detector is one of the detectors with the highest sensitivity, is suitable for detecting micro-volume samples, and can be used together with a plurality of capillary or microchip separation technologies such as capillary liquid chromatography, capillary electrophoresis, microfluidic chip chromatography and the like. Many samples can generate fluorescence spontaneously or generate fluorescence by derivation to achieve the purpose of fluorescence detection, so the LIF detector becomes one of the first technologies in the fields of analytical chemistry, molecular biology, biomedicine and the like, and has wide application in analytical detection of foods, medicines, environmental pollutants, nucleic acids, proteins, cells and the like.
The LIF detector mainly comprises a light source, an optical system, a detection cell and a light detection element. To achieve highly sensitive detection, LIF detection systems excite fluorescence emission and collect fluorescence signals as efficiently as possible and reduce interference from background signals. Detection cell walls, fluorescence, rayleigh and raman scattering of the eluate, and stray light between optical elements are major factors affecting detection sensitivity. The interference of background stray light is usually reduced by filtering in a spectral, temporal or spatial resolution manner. Spectral filtering is a combination of bandpass and optical filters to remove light outside the emitted fluorescence wavelength range. The time resolution is to eliminate the interference of Rayleigh scattering and Raman scattering light by using pulse laser as an excitation light source and adopting a time resolution technology. The spatial filtering method is to use a pinhole, a slit, etc. to subtract the background stray light outside the detection area and improve the detection selectivity, but the method is cumbersome to operate and has poor applicability.
With the development of micro-nano-scale analysis, the micro-nano-scale separation analysis technology is an urgent problem to be gradually solved, so that the micro-scale development of technologies such as chromatography, electrophoresis and the like is promoted. The capillary and the chip adopt micro-diameter or nano-diameter channels as separation columns, and usually combine with a laser-induced fluorescence detection system to realize quantitative separation and analysis of substances. The separation column with the small inner diameter has the characteristics of high column efficiency, small sample volume consumption, low flow velocity and the like, is widely applied to the field of micro-nano analysis, and has great advantages in separating and analyzing trace substances. The capillary and the chip are combined with laser-induced fluorescence, focusing and other work are required to be realized, usually, a solution containing a fluorescent substance is injected into a channel, the fluorescent substance flowing through a detection window is excited by laser and then emits fluorescence, and the relative position of the detection window and a detection system is adjusted according to the size of a fluorescence signal output by a light detection element to calibrate a light path. However, when the channel size is reduced to the nanometer level, it takes a long time to complete the calibration operation if the position of the detection window cannot be observed. In addition, chromatographic separation is carried out by taking a chromatographic column as a separation medium, the chromatographic column is divided into a filling column, an open column and an integral column according to different stationary phase forms, and the problems of difficult cleaning of a fluorescent reagent during focusing and the like are caused due to the fact that the chromatographic column is filled with a stationary phase or is modified by coating on a wall and the like, so that the detection of a sample is influenced.
Disclosure of Invention
The invention aims to provide a laser-induced fluorescence detection system for visualized real-time imaging focusing, which improves the detection sensitivity of an instrument, reduces the detection limit, simplifies the focusing step, and realizes visualized calibration and real-time online display of the focusing position of a detection window through an imaging calibration light path.
The laser-induced fluorescence detection system adopting the visual real-time imaging focusing adopts a confocal structure and consists of a laser light path, a fluorescence collecting light path and an imaging calibration light path. The Light Emitting Diode (LED) (13), the optical filter (14), the diaphragm (15), the objective lens (5), the first dichroic mirror (6), the second dichroic mirror (7) and the reflecting mirror (16) are on the same longitudinal axis; the optical pump semiconductor laser (1), the neutral density optical filter (2), the laser optical filter (3), the cylindrical lens (4) and the first dichroic mirror (6) are sequentially connected in an optical path and are arranged on a first transverse shaft, and the optical pump semiconductor laser, the neutral density optical filter (2), the laser optical filter (3), the cylindrical lens (4) and the first dichroic mirror (6) form a laser optical path together with the objective lens (5); the silicon avalanche photodiode (11), the slit space optical filter (10), the lens (9), the band-pass optical filter (8) and the second dichroic mirror (7) are sequentially connected in an optical path and are on the second transverse axis, and the silicon avalanche photodiode, the slit space optical filter (10), the lens (9), the band-pass optical filter (8) and the first dichroic mirror (6) jointly form a fluorescence collection optical path; the camera (18), the lens (17) and the reflector (16) are sequentially connected in an optical path and are arranged on a third transverse axis, and an imaging calibration optical path is formed by the camera, the LED (13), the optical filter (14), the diaphragm (15), the objective lens (5), the first dichroic mirror (6) and the second dichroic mirror (7);
the laser light path is used as a laser light source by the optical pump semiconductor laser (1), a laser beam emitted from the laser (1) sequentially passes through optical elements such as a neutral density filter (2), a laser filter (3) and a cylindrical lens (4), is reflected by a first dichroic mirror (6) and then is focused by an objective lens (5), and a sample containing fluorescence is excited at a detection window position to generate a fluorescence signal; the laser light beam is shaped into a linear light spot by a cylindrical lens (4) along a one-dimensional direction from a circular light spot;
the fluorescence collection light path is characterized in that a fluorescence signal is collected through an objective lens (5), passes through a first dichroic mirror (6), is reflected by a second dichroic mirror (7), is filtered by a band-pass filter (8) matched with the emission wavelength of a detection substance to remove stray light except emitted fluorescence, is focused by a lens (9), is further filtered by a slit space filter (10) to remove stray light, enters a silicon avalanche photodiode (11) to be detected, and is connected with a data acquisition card for data storage, wherein the silicon avalanche photodiode (11) module is connected with the data acquisition card.
The imaging calibration light path adopts an LED (13) as a light source, irradiates above a detection window, is filtered by a light filter (14), is collected by an objective lens (5) after the light intensity is adjusted by a diaphragm (15), sequentially passes through two dichroic mirrors, is reflected by a reflector (16), is focused by a lens (17), and then reaches a camera (18) for real-time image acquisition and display.
The laser-induced fluorescence detection system with the visualization real-time imaging focusing function is characterized in that: spectral light splitting can be achieved by using two long-wavelength-pass dichroic mirrors having high reflectance for light shorter than a cutoff wavelength and high transmittance for light longer than the cutoff wavelength. The laser wavelength is represented by lambda 1, the fluorescence wavelength is represented by lambda 2, the LED light wavelength is represented by lambda 3, and lambda 3 is more than lambda 2 and more than lambda 1. The cut-off wavelength of the first dichroic mirror (6) is larger than the laser wavelength lambda 1, the excitation light is reflected by the dichroic mirror, and the emitted fluorescence lambda 2 and the LED light lambda 3 are transmitted, so that the interference of the excitation light on the fluorescence detection is eliminated. The cut-off wavelength of the second dichroic mirror (7) is larger than the fluorescence wavelength lambda 2, the fluorescence lambda 2 is reflected at the same time, the LED light lambda 3 penetrates through the second dichroic mirror, the effect of separating fluorescence from the LED light is achieved, and the LED light does not influence laser incidence and fluorescence collection in the real-time imaging process.
The relative position of the detection window and the objective lens or the laser is adjusted through the x-y-z axis of the three-dimensional translation table of the imaging calibration light path, is displayed through software, and is focused according to the position information and the light intensity distribution curve map displayed in real time in the software. The adjusting direction of the radial (cross section direction) of the separation column channel of the capillary or chip and the relative position of the objective lens (or laser) is defined as an x-axis, the adjusting direction between the axial direction of the separation column and the objective lens is defined as a y-axis, and the adjusting direction of the distance between the separation column and the objective lens is defined as a z-axis. And injecting a solution into the channel of the separation column for imaging calibration, and directly observing the position of the y axis for adjustment. When x and z axes are adjusted, when laser is focused into a channel, a light spot (20) appears in the channel, a vertical line (21) is drawn for the position of the light spot on a capillary or chip channel in software, a curve (VLP) is obtained according to light intensity distribution on the vertical line, when a laser beam is collimated to enter the center of the channel, the brightness of the light spot is maximum, and the height of a spectral peak at a corresponding position in the VLP reaches the maximum. When the position between the laser beam and the channel cross section is shifted, the spot brightness is reduced or disappeared, and the height of the corresponding spectral peak in the VLP is reduced or disappeared.
The laser-induced fluorescence detection system with the visualized real-time imaging focusing has the advantages of obtaining higher detection sensitivity and lower detection limit, simplifying the focusing steps of the capillary and the chip, and realizing the visualized calibration and real-time online display of the focusing position of the detection window.
Drawings
Fig. 1 is a schematic structural diagram of a laser-induced fluorescence detection system for visualizing real-time imaging focusing.
Fig. 2 is a comparison before and after shaping a laser beam by using a cylindrical lens.
FIG. 3 is a diagram of capillary visualization real-time imaging focusing results;
(a) is a schematic diagram defining the x-y-z axis;
(b) is a capillary focusing visualization real-time imaging illustration;
(c) is a light intensity distribution curve spectrogram focused by a capillary tube;
(d) is a capillary focusing x-axis calibration real-time imaging graph and a light intensity distribution curve spectrogram; the method comprises the steps of (i) imaging when a laser beam is collimated and enters the center position of a capillary channel, (ii) imaging when the position between the laser beam and the cross section of the capillary is shifted, and (iv) imaging when the position between the laser beam and the cross section of the capillary is shifted;
(e) is a capillary focusing z-axis calibration real-time imaging graph and a light intensity distribution curve spectrogram.
FIG. 4 is a diagram of a chip visualized real-time imaging focusing result;
(a) the real-time imaging graph and the light intensity distribution curve spectrogram are calibrated by an x axis focused by a chip;
(b) the real-time imaging graph and the light intensity distribution curve spectrogram are calibrated by a z-axis focused by the chip.
FIG. 5 is a graph showing the results of an experiment for detecting fluorescein by capillary chromatography.
In the figure, 1, an optical pump semiconductor laser with the excitation wavelength of 488nm, 2, a neutral density filter, 3, a laser filter with the center wavelength of 488nm, 4, a cylindrical lens, 5, an objective lens, 6, a first dichroic mirror (a dichroic mirror with the cut-off wavelength of 488 nm), 7, a second dichroic mirror (a dichroic mirror with the cut-off wavelength of 600 nm), 8, a band pass filter with the center wavelength of 535nm, 9, a lens, 10, a slit space filter, 11, a silicon avalanche photodiode, 12, a data acquisition card, 13, a Light Emitting Diode (LED), 14, a filter, 15, a diaphragm, 16, a reflector, 17, a lens, 18, a camera, 19, a computer, 20, a light spot, 21, and a vertical line.
Detailed Description
The present invention will be further described with reference to the accompanying drawings, but the present invention is not limited to the following examples.
Example 1
As shown in fig. 1, the structure schematic diagram of the laser-induced fluorescence detection system with visualization real-time imaging focusing is shown, wherein an LED lamp (13), a filter (14), a diaphragm (15), an objective lens (5), a first dichroic mirror (6) with a cutoff wavelength of 488nm, a second dichroic mirror (7) with a cutoff wavelength of 600nm, and a reflector (16) are on the same longitudinal axis. An optical pump semiconductor laser (1) with the excitation wavelength of 488nm, a neutral density filter (2), a laser filter with the center wavelength of 488nm, a cylindrical lens (4) and a first dichroic mirror (6) with the cut-off wavelength of 488nm are on the same horizontal axis, a laser beam emitted by the optical pump semiconductor laser (1) with the excitation wavelength of 488nm is attenuated through the neutral density filter (2) and purified through the laser filter (3) with the center wavelength of 488nm, linear light spots are formed through one-dimensional integration through the cylindrical lens (4), the linear light spots are reflected through the first dichroic mirror (6) with the cut-off wavelength of 488nm and focused through an objective lens (5), and a sample containing fluorescence is excited at a detection window position to generate a fluorescence signal.
A silicon avalanche photodiode (11), a slit space filter (10), a cylindrical lens (9), a band-pass filter (8) with the central wavelength of 535nm and a second dichroic mirror (7) with the cut-off wavelength of 600nm are on the same horizontal axis, generated fluorescent signals are collected by an objective lens (5), pass through a first dichroic mirror (6) with the cut-off wavelength of 488nm and then are reflected by the second dichroic mirror (7) with the cut-off wavelength of 600nm, stray light is filtered by the band-pass filter (8) with the central wavelength of 535nm and focused by the lens (9), the stray light is further filtered by a slit (10) and then enters the silicon avalanche photodiode (11) for detection, and the silicon avalanche photodiode module is connected with a data acquisition card (12) for data storage.
The reflector (16), the lens (17) and the camera (18) are arranged on the same transverse axis, the LED (13) irradiates above the detection window, is filtered by the optical filter (14), is collected by the objective lens (5) after the light intensity is adjusted by the diaphragm (15), sequentially passes through the two dichroic mirrors, is reflected by the reflector (16), and then reaches the camera (18) after being focused by the lens (17), and the camera is connected with ThorCam software to acquire and display real-time images.
As shown in fig. 2, a comparison graph before and after shaping the laser beam by using the cylindrical lens is shown.
The laser beam is focused by the plano-convex cylindrical lens (4), wherein (a) and (b) in fig. 2 are respectively enlarged images of the laser beam before and after being focused by the plano-convex cylindrical lens, and the incident laser beam with a circular spot shape (fig. 2(a)) is focused along a one-dimensional direction and then transformed and shaped into a linear spot (fig. 2(b)) laser beam. The width of the focused linear light spot is reduced by nearly 3.4 times compared with the diameter of a circular light spot, and the linear light spot has higher photon flow in unit area. The linear light spot laser beam is focused by the objective lens and is incident into the capillary channel to detect the lamella area in the channel, thereby improving the spatial resolution capability of the detection system and being more beneficial to the analysis and detection of the micro area on the micro-nano channel.
As shown in fig. 3, it is a diagram of real-time imaging focusing result of capillary visualization, and image acquisition is performed by using ThorCam software.
The capillary detection window is fixed on an x-y-z three-dimensional translation stage, the adjusting direction of the relative position of the capillary in the radial direction (cross section direction) and the objective (or laser) is defined as an x axis, the adjusting direction between the capillary axial direction and the objective is defined as a y axis, and the adjusting direction of the distance between the capillary and the objective is defined as a z axis (see fig. 3 (a)). Where an imaging calibration procedure is performed on a 450nm radius capillary tube (with solution injected into the tube), the y-axis position can be directly observed for adjustment. When the x-axis is adjusted, when the laser is focused into the capillary channel, a light spot (20) in the capillary channel can be observed on the image, a perpendicular line (21) is drawn on the position of the light spot on the capillary in the software as shown in fig. 3(b), and a curve (VLP) is obtained according to the light intensity distribution on the perpendicular line as shown in fig. 3 (c). When the laser beam is collimated and enters the center of the capillary channel, the brightness of the light spot is maximum, and the height of the spectrum peak at the corresponding position in the VLP reaches the maximum. When the position of the laser beam relative to the capillary cross-section shifts, the spot brightness decreases or disappears (iii in fig. 3 (d)), corresponding to a decrease or disappearance of the spectral peak height in the VLP (iv in fig. 3 (d)). Based on the adjustment, the x axis is adjusted, so that the laser beam is collimated and enters the center of the capillary channel to reach the optimal position. When the distance between the capillary and the objective lens is in the optimal position when the z-axis is adjusted, the inner wall of the capillary becomes transparent, defined as 0nm at this time (fig. 3(e)0nm), and the corresponding VLP approaches a smooth straight line. When the distance is decreased or far away, the capillary will appear in different images, (FIG. 3(e)) +10nm and-10 nm images are the images of the capillary corresponding to the increase and decrease in distance of 10nm, respectively, and the corresponding VLP will also fluctuate. The z-axis position is calibrated by this method.
As shown in fig. 4, it is a chip visualization real-time imaging focusing result diagram, and image acquisition is performed by using ThorCam software.
The chip is fixed on an x-y-z three-dimensional translation table, the adjusting direction of the relative position of the radial direction (cross section direction) of the channel and the objective lens (or laser) is defined as an x axis, the adjusting direction between the axial direction of the channel and the objective lens is defined as a y axis, and the adjusting direction of the distance between the chip and the objective lens is defined as a z axis. Here, an imaging calibration operation was performed on a chip with a channel size of 5 μm, and the y-axis position was directly observed to be adjusted, similarly to the capillary calibration operation. When the x-axis and the z-axis are adjusted, the light spot brightness and the VLP spectrum peak are changed along with the relative position and the distance between the channel and the objective lens. When the x axis is adjusted, when the laser beam is collimated and enters the center position of the chip channel, the light spot brightness is maximum, and the height of the spectrum peak at the corresponding position in the VLP reaches the maximum. When the laser beam is shifted from the channel position, the spot brightness decreases or disappears, and the height of the corresponding spectral peak in the VLP decreases or disappears (fig. 4 (a)). When the distance between the chip and the objective lens is changed while adjusting the z-axis, the VLP spectrum peak also fluctuates correspondingly (FIG. 4(b)), thereby focusing the chip channel.
The visual calibration of the detection window and the LIF detection system brings great convenience to on-column detection of the micro-nano scale channel, can avoid the complexity of focusing by using a fluorescent reagent, and also avoids the problem of difficult cleaning caused by fluorescent focusing for the separation column coated with a modified wall (such as a narrow-diameter porous layer open tubular column).
As shown in the figure5Shown is a graph of the experimental result of detecting fluorescein by capillary chromatography.
Sample preparation: a fluorescein sodium solution with the concentration of 10 pmol/L; a chromatographic column: a quartz capillary tube having an inner diameter of 1 μm and an outer diameter of 360 μm, a total length of 34cm and an effective length of 29 cm; buffer solution: 10mmol/L Tris-EDTA solution, pH 8.0; sample introduction conditions are as follows: 100psi, 10 s; driving pressure: 1000 psi; FIG. 3 is a chromatogram obtained by separating a 10pmol/L sample of a sodium fluorescein solution under the present test conditions. The average signal-to-noise ratio of the chromatographic signal peaks was about 3, and the detection limit of the system for sodium fluorescein was 10 pmol/L. According to the sample injection time, the flow rate and the concentration, the detection system can achieve the unimolecular level detection of the fluorescein sodium.
Claims (4)
1. A laser-induced fluorescence detection system with visualized real-time imaging focusing is characterized in that the visualized calibration and real-time online display of the focusing position of a capillary or chip detection window can be realized; the laser-induced fluorescence detection system adopting the visual real-time imaging focusing adopts a confocal structure and consists of a laser light path, a fluorescence collection light path and an imaging calibration light path; the light-emitting diode LED (13), the optical filter (14), the diaphragm (15), the objective lens (5), the first dichroic mirror (6), the second dichroic mirror (7) and the reflecting mirror (16) are on the same longitudinal axis; the optical pump semiconductor laser (1), the neutral density optical filter (2), the laser optical filter (3), the cylindrical lens (4) and the first dichroic mirror (6) are sequentially connected in an optical path and are arranged on a first transverse shaft, and the optical pump semiconductor laser, the neutral density optical filter (2), the laser optical filter (3), the cylindrical lens (4) and the first dichroic mirror (6) form a laser optical path together with the objective lens (5); the silicon avalanche photodiode (11), the slit space optical filter (10), the lens (9), the band-pass optical filter (8) and the second dichroic mirror (7) are sequentially connected in an optical path and are on the second transverse axis, and the silicon avalanche photodiode, the slit space optical filter (10), the lens (9), the band-pass optical filter (8) and the first dichroic mirror (6) jointly form a fluorescence collection optical path; the camera (18), the lens (17) and the reflector (16) are sequentially connected in an optical path and are arranged on a third transverse axis, and an imaging calibration optical path is formed by the camera, the LED (13), the optical filter (14), the diaphragm (15), the objective lens (5), the first dichroic mirror (6) and the second dichroic mirror (7); the laser light path is used as a laser light source by the optical pump semiconductor laser (1), a laser beam emitted from the laser (1) sequentially passes through the neutral density optical filter (2), the laser optical filter (3) and the optical element of the cylindrical lens (4), is reflected by the first dichroic mirror (6) and then is focused by the objective lens (5), and a sample containing fluorescence is excited at the position of the detection window to generate a fluorescence signal; the laser light beam is shaped into a linear light spot by a cylindrical lens (4) along a one-dimensional direction from a circular light spot;
the imaging calibration light path adjusts the relative position of the detection window and the objective lens or laser through the x-y-z axis of the three-dimensional translation table, the relative position is displayed through software, the focusing is carried out according to the position information and the light intensity distribution curve map which are displayed in real time in the software, the radial direction of a capillary or chip channel, namely the adjusting direction of the cross section direction and the relative position of the objective lens or laser is defined as the x axis, the adjusting direction between the axial direction of the channel and the objective lens is defined as the y axis, and the adjusting direction of the distance between the capillary or chip and the objective lens is defined as the z axis; injecting a solution into the channel for imaging calibration, and directly observing the y-axis position for adjustment; when x and z axes are adjusted, when laser is focused into a channel, a light spot (20) in the channel can be observed on an image, a vertical line (21) is drawn for the position where the light spot is positioned on a capillary or chip channel in software, a curve VLP is obtained according to light intensity distribution on the vertical line, when a laser beam is collimated to enter the center of the channel, the brightness of the light spot is maximum, and the height of a spectral peak at a corresponding position in the VLP reaches the maximum value; when the position between the laser beam and the channel cross section is shifted, the spot brightness is reduced or disappeared, and the height of the corresponding spectral peak in the VLP is reduced or disappeared.
2. The laser-induced fluorescence detection system with visualization real-time imaging focusing as claimed in claim 1, wherein the fluorescence collection optical path is to collect fluorescence signals through an objective lens (5), pass through a first dichroic mirror (6), then be reflected by a second dichroic mirror (7), then pass through a band-pass filter (8) matched with the emission wavelength of the detection substance to filter stray light except for emitted fluorescence, then be focused through a lens (9), further pass through a slit spatial filter (10) to filter stray light, and then enter a silicon avalanche photodiode (11) for detection, and the silicon avalanche photodiode (11) module is connected with a data acquisition card for data storage.
3. The laser-induced fluorescence detection system with visualization real-time imaging focusing as claimed in claim 1, wherein the imaging calibration light path employs an LED (13) as a light source, irradiates above the detection window, is filtered by a filter (14), is collected by an objective lens (5) after the light intensity is adjusted by a diaphragm (15), sequentially passes through two dichroic mirrors, is reflected by a reflector (16), is focused by a lens (17), and then reaches a camera (18) for real-time image acquisition and display.
4. The visual real-time imaging focusing laser-induced fluorescence detection system according to claim 1, wherein λ 1 represents a laser wavelength, λ 2 represents a fluorescence wavelength, λ 3 represents an LED light wavelength, λ 3 > λ 2 > λ 1, a cut-off wavelength of the first dichroic mirror (6) is larger than the laser wavelength λ 1, the excitation light is reflected by the dichroic mirror, and the emitted fluorescence λ 2 and LED light λ 3 are transmitted, thereby eliminating interference of the excitation light with fluorescence detection; the cut-off wavelength of the second dichroic mirror (7) is larger than the fluorescence wavelength lambda 2, the fluorescence lambda 2 is reflected at the same time, the LED light lambda 3 penetrates through the second dichroic mirror, the effect of separating the fluorescence from the LED light is achieved, and the laser incidence and the fluorescence collection are not influenced by the LED light in the real-time imaging process.
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