CN109374511B - Light path adjusting device for flow cytometer without liquid path condition - Google Patents
Light path adjusting device for flow cytometer without liquid path condition Download PDFInfo
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- 239000007788 liquid Substances 0.000 title abstract description 17
- 239000004005 microsphere Substances 0.000 claims abstract description 80
- 238000007493 shaping process Methods 0.000 claims abstract description 55
- 230000003287 optical effect Effects 0.000 claims abstract description 54
- 238000001514 detection method Methods 0.000 claims abstract description 51
- 238000006243 chemical reaction Methods 0.000 claims abstract description 33
- 238000001917 fluorescence detection Methods 0.000 claims abstract description 18
- 230000005284 excitation Effects 0.000 claims abstract description 10
- 238000005286 illumination Methods 0.000 claims abstract description 10
- 230000003750 conditioning effect Effects 0.000 claims description 13
- 238000000605 extraction Methods 0.000 claims description 13
- 238000000034 method Methods 0.000 claims description 11
- 238000011282 treatment Methods 0.000 claims description 7
- 230000001678 irradiating effect Effects 0.000 claims description 4
- 239000003086 colorant Substances 0.000 claims description 3
- 230000006835 compression Effects 0.000 claims description 3
- 238000007906 compression Methods 0.000 claims description 3
- 239000012530 fluid Substances 0.000 claims 1
- 238000010586 diagram Methods 0.000 description 19
- 239000000284 extract Substances 0.000 description 5
- 230000001143 conditioned effect Effects 0.000 description 3
- 230000000903 blocking effect Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
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- 238000005259 measurement Methods 0.000 description 1
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- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
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Abstract
The invention provides a light path adjusting device for a flow cytometer without a liquid path, which comprises: the device comprises an irradiation light source, an irradiation light spot shaping light path, a standard microsphere rotating device, a non-forward scattering light beam shaping light path, a non-forward scattering light detection circuit, a forward scattering light and fluorescent light beam shaping light path, a forward scattering light detection circuit, a multi-color fluorescence light splitting light path and a multi-channel fluorescence detection circuit, wherein the irradiation light source provides an excitation light source for fluorescence excitation; the irradiation light spot shaping light path is used for compressing light source light beams into illumination light spots with a certain size; the standard microsphere rotating device is used for rotating the disc loaded with the standard microspheres so as to simulate that single cells pass through the irradiation light spots one by one; the non-forward scattering light beam shaping optical path is used for focusing a non-forward scattering light beam in a certain range; the non-forward scattering light detection circuit is used for carrying out photoelectric conversion on non-forward scattering light and extracting parameters of the generated electric pulse signals.
Description
The invention relates to a split application of a light path adjusting device and a method for a flow cytometer without a liquid path, which has the application number of 201610896195.1 and the application date of 2016.10.14
Technical Field
The invention relates to the field of optical path adjustment and microsphere measurement of a flow cytometer, in particular to an optical path adjusting device without a liquid path.
Background
In the flow cytometer optical path adjusting system, adjustment of an irradiation light spot and spectroscopic collection of an excitation light signal are included. Because the detection area of the microsphere in the flow cytometer is located at the intersection point of the irradiation light spot and the liquid flow direction, and the instability of the liquid path system can cause the difference of the relative positions of the microsphere entering the detection area, the irradiation excitation degrees of the microsphere in the detection area are different, and the forward scattered light, the non-forward scattered light and the fluorescence signal intensity difference of each channel are large. The pulse parameter information obtained by the subsequent detection circuit is inaccurate. The existing optical adjusting system completely depends on a high-precision liquid path control system and has poor independence. The liquid path control system is extremely complex and tedious, and the verification method of the control precision and the laminar flow effect has no standard indexes.
Therefore, there is a need for an optical path adjusting device for a flow cytometer without a liquid path that can effectively solve the above problems.
Disclosure of Invention
According to an aspect of the present invention, there is provided an optical path adjusting apparatus for a flow cytometer without a liquid path, the apparatus comprising: an irradiation light source, an irradiation light spot shaping light path, a standard microsphere rotating device, a non-forward scattering light beam shaping light path, a non-forward scattering light detection circuit, a forward scattering light and fluorescent light beam shaping light path, a forward scattering light detection circuit, a multi-color fluorescence light splitting light path and a multi-channel fluorescence detection circuit, wherein,
the irradiation light source provides an excitation light source for fluorescence excitation;
the irradiation light spot shaping light path is used for compressing light source light beams into illumination light spots with a certain size;
the standard microsphere rotating device is used for rotating the disc loaded with the standard microspheres so as to simulate that single cells pass through the irradiation light spots one by one;
the non-forward scattering light beam shaping optical path is used for focusing a non-forward scattering light beam in a certain range;
the non-forward scattering light detection circuit is used for carrying out photoelectric conversion on non-forward scattering light and realizing parameter extraction on a generated electric pulse signal;
the forward scattering light and fluorescent light beam shaping light path is used for focusing forward scattering light and fluorescent light beams in a certain range;
the forward scattering light detection circuit is used for performing photoelectric conversion on forward scattering light and extracting parameters of the generated electric pulse signals;
the multi-color fluorescence light splitting optical path is used for separating fluorescence signals in each wavelength range and transmitting the fluorescence signals to the corresponding detection circuit, and the multi-channel fluorescence detection circuit is used for performing photoelectric conversion on the fluorescence signals of each channel and extracting parameters of generated electric pulse signals.
Preferably, a laser with certain power and wavelength is used as an irradiation light source, a light beam emitted by the laser is compressed and shaped through an irradiation light spot shaping light path, the irradiation light spot shaping light path realizes the light spot size compression in two orthogonal directions according to the movement direction of the microsphere in a standard microsphere rotating device, and meanwhile, the moving microsphere is irradiated at a light spot focus through adjusting the light spot shaping light path.
Preferably, the standard microsphere rotating device comprises a turntable loaded with standard microspheres, a turntable driving device and a motor motion control circuit, wherein the turntable loaded with the standard microspheres is connected with the motor motion control circuit through the turntable driving device, and the rotation speed of the turntable is controlled by adjusting the rotation speed of the motor, so that the irradiation time of the microspheres by light spots and the irradiation time interval of adjacent microspheres are changed.
Preferably, the turntable loaded with the standard microspheres consists of a bottom layer and a top layer, wherein one layer is provided with a pit for placing the standard microspheres, and the standard microspheres are sealed in the pit through the adhesion of the top layer and the bottom layer to prevent the microspheres from falling off in the rotating process. The standard microspheres form lateral scattered light, forward scattered light and fluorescent signals of various colors one by one through irradiating a light spot area in the rotating process of the turntable loaded with the standard microspheres.
Preferably, the non-forward scattering light beam shaping optical path is placed in a non-forward scattering angle detection area, focuses and shapes non-forward scattering light within a certain angle range, and transmits the shaped beam to the non-forward scattering light detection circuit. The non-forward scattering light detection circuit realizes photoelectric conversion of non-forward scattering light signals and conditioning treatment of electric pulse signals, and extracts electric pulse parameters for representing microsphere characteristics.
Preferably, the forward scattered light and fluorescence beam shaping optical path is placed in the direction of the irradiation beam, focuses and shapes the forward scattered light and the multi-channel fluorescence beam within a certain angle range, and transmits the shaped beam to the forward scattered light detection circuit and the multi-color fluorescence splitting optical path. The forward scattering light detection circuit realizes photoelectric conversion of forward scattering light signals and conditioning treatment of electric pulse signals, and extracts electric pulse parameters for representing microsphere characteristics.
Preferably, the multi-color fluorescence splitting optical path separates multi-channel fluorescence signals through a series of optical devices and transmits the separated color fluorescence light beams to corresponding multi-channel fluorescence detection circuits. The multi-channel fluorescence detection circuit realizes photoelectric conversion of fluorescence signals and conditioning treatment of electric pulse signals, and extracts electric pulse parameters for representing microsphere characteristics.
According to another aspect of the present invention, there is provided a method for adjusting an optical path of a flow cytometer without a liquid path, comprising the steps of:
the irradiation light spot shaping light path compresses light beams generated by the light source and the like to obtain irradiation light spots with a certain size, and the focal length of the light spots is adjusted to enable the microspheres to pass through the focal point;
the standard microsphere rotating device adjusts the movement speed of the microsphere and changes the microsphere detection frequency and the microsphere irradiation time so as to change the frequency and the duration of corresponding optical pulses and electric pulses;
the non-forward scattering light beam shaping light path carries out shaping and other treatments on the non-forward scattering light, and the focal length of the light path is adjusted to enable a detector in the non-forward scattering light detection circuit to be in a focal position;
the non-forward scattered light detection circuit realizes the photoelectric conversion, the electric pulse conditioning, the analog/digital conversion, the parameter extraction and other processing of the non-forward scattered light, and identifies the starting point and the ending point of the electric pulse;
the forward scattering light and fluorescent light beam shaping light path is used for shaping the forward scattering light and fluorescent light beam and transmitting the focused light beam to the multi-color fluorescent light splitting light path;
the multi-color fluorescence light splitting optical path performs light splitting processing on the forward scattering light and the fluorescence light beams of each channel according to wavelength information, and transmits the split light beams to a forward scattering light detection circuit and a corresponding multi-channel fluorescence detection circuit;
the forward scattered light detection circuit realizes the photoelectric conversion, the electric pulse conditioning, the analog/digital conversion, the parameter extraction and other processing of forward scattered light, and identifies the starting point and the ending point of the electric pulse;
the multi-channel fluorescence detection circuit realizes the photoelectric conversion, the electric pulse conditioning, the analog/digital conversion, the parameter extraction and other processing of all fluorescence, and identifies the starting point and the ending point of the electric pulse.
It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed.
Drawings
Further objects, features and advantages of the present invention will become apparent from the following description of embodiments of the invention, with reference to the accompanying drawings, in which:
FIG. 1 is a schematic view of an embodiment of an optical path adjusting device for a flow cytometer without a liquid path;
FIG. 2 is a schematic diagram of the connection of the components of the flow cytometer of the present invention.
Fig. 3a-3c are schematic diagrams of exemplary structures of the illumination light source and the illumination spot shaping optical path shown in fig. 2. Wherein fig. 3a shows the shape change of the spot of the laser light emitted by the irradiation light source after passing through the shaping lens. Fig. 3b-3c are schematic diagrams of the light intensity distribution of the shaped irradiation spot shown in fig. 3 a.
FIGS. 4a-4b show a schematic diagram of an embodiment of a standard microsphere rotation apparatus and standard microspheres.
FIG. 5 is a block diagram of one embodiment of an exemplary non-forward-scattered light beam shaping circuit.
Fig. 6 shows a specific circuit block diagram of the non-forward scattered light detection circuit.
Fig. 7 shows a specific circuit block diagram of the forward scattered light detection circuit.
FIG. 8 is a diagram illustrating an exemplary embodiment of the optical path of the polychromatic fluorescent light-splitting optical path.
FIG. 9 is a block diagram of another exemplary embodiment of the optical path of the polychromatic fluorescent light-splitting optical path.
FIG. 10 shows a block diagram of one embodiment of a multi-channel fluorescence detection circuit.
Detailed Description
Hereinafter, embodiments of the present invention will be described with reference to the accompanying drawings. In the drawings, the same reference numerals denote the same or similar parts, or the same or similar steps.
The objects and functions of the present invention and methods for accomplishing the same will be apparent by reference to the exemplary embodiments. However, the present invention is not limited to the exemplary embodiments disclosed below; it can be implemented in different forms. The nature of the description is merely to assist those skilled in the relevant art in a comprehensive understanding of the specific details of the invention.
While the present invention has been described in detail with reference to the drawings, the cross-sectional views illustrating the structure of the device are not enlarged in general scale for convenience of illustration, and the drawings are only exemplary and should not be construed as limiting the scope of the present invention. In addition, the three-dimensional dimensions of length, width and depth should be included in the actual fabrication.
The present invention provides an optical path adjusting device for a flow cytometer without a liquid path, fig. 1 is a schematic system diagram of the optical path adjusting device for a flow cytometer without a liquid path, as shown in fig. 1, the device includes: an irradiation light source 101, an irradiation spot shaping optical path 102, a standard microsphere rotating device 103, a non-forward scattered light beam shaping optical path 104, a non-forward scattered light detection circuit 105, a forward scattered light and fluorescent light beam shaping optical path 106, a forward scattered light detection circuit 107, a multi-color fluorescent splitting optical path 108 and a multi-channel fluorescent detection circuit 109, wherein,
the irradiation light source 101 provides an excitation light source for fluorescence excitation;
the illumination light spot shaping light path 102 is used for compressing light beams of the light source into illumination light spots with a certain size;
the standard microsphere rotating device 103 is used for rotating the disc loaded with the standard microspheres so as to simulate that single cells pass through the irradiation light spots one by one;
the non-forward scattering light beam shaping optical path 104 is used for focusing a range of non-forward scattering light beams;
the non-forward scattered light detection circuit 105 is used for performing photoelectric conversion on non-forward scattered light and extracting parameters of the generated electric pulse signals;
the forward scattered light and fluorescent light beam shaping optical path 106 is used for focusing forward scattered light and fluorescent light beams in a certain range;
the forward scattered light detection circuit 107 is used for performing photoelectric conversion on forward scattered light and extracting parameters of the generated electric pulse signals;
the multi-color fluorescence splitting optical path 108 is used for separating fluorescence signals in various wavelength ranges and transmitting the fluorescence signals to corresponding detection circuits;
the multi-channel fluorescence detection circuit 109 is used for performing photoelectric conversion on each channel fluorescence signal and extracting parameters of the generated electric pulse signal.
Fig. 2 is a schematic diagram of an optical path adjusting device for a flow cytometer without a liquid path according to a preferred embodiment of the present invention. There are schematically shown parts of an illumination source 101, a standard microsphere rotating means 103, a non-forward scattered light beam shaping optical path 104 and a forward scattered light and fluorescent light beam shaping optical path 106.
Preferably, a laser with certain power and wavelength is used as the irradiation light source 101, and the laser emission beam is compressed and shaped by the irradiation spot shaping light path 102, the irradiation spot shaping light path 102 realizes the spot size compression in two orthogonal directions according to the movement direction of the microsphere in the standard microsphere rotation device 103, and the moving microsphere is irradiated at the spot focus by irradiating the spot shaping light path 102. Fig. 3a-3c are schematic diagrams of exemplary structures of the illumination light source and the illumination spot shaping optical path shown in fig. 2. Wherein fig. 3a shows the shape change of the spot of the laser light emitted by the irradiation light source 101 after passing through the shaping lens. Fig. 3b-3c are schematic diagrams of the light intensity distribution of the shaped irradiation spot shown in fig. 3 a.
Preferably, FIGS. 4a-4b show an embodiment of a standard microsphere rotation device 103 and standard microspheres. The standard microsphere rotating device 103 comprises a turntable loaded with standard microspheres, a turntable driving device and a motor motion control circuit, wherein the turntable loaded with the standard microspheres is connected with the motor motion control circuit through the turntable driving device, and the rotation speed of the turntable is controlled by adjusting the rotation speed of the motor, so that the irradiation time of the microspheres by light spots and the irradiation time interval of adjacent microspheres are changed.
Preferably, the turntable loaded with the standard microspheres consists of a bottom layer and a top layer, wherein one layer is provided with a pit for placing the standard microspheres, and the standard microspheres are sealed in the pit through the adhesion of the top layer and the bottom layer to prevent the microspheres from falling off in the rotating process. The standard microspheres form lateral scattered light, forward scattered light and fluorescent signals of various colors one by one through irradiating a light spot area in the rotating process of the turntable loaded with the standard microspheres.
Fig. 5 is a block diagram of one embodiment of an exemplary non-forward scattered light beam shaping optical path 104. Preferably, the non-forward scattered light beam shaping optical path is placed in a non-forward scattering angle detection area, and the non-forward scattered light within a certain angle range is focused and shaped, as shown in fig. 5, and the non-forward scattered light is collected by a collecting lens, filtered by a blocking filter, and then transmitted to the non-forward scattered light detection circuit 105 by a focusing lens.
The non-forward scattered light detection circuit 105 realizes photoelectric conversion of non-forward scattered light signals and conditioning treatment of electric pulse signals, and extracts electric pulse parameters for representing microsphere characteristics. Fig. 6 shows a specific circuit block diagram of the non-forward scattered light detection circuit 105. As shown in fig. 6, the non-forward scattered light detection circuit 105 includes a sensor, and is configured to convert the collected light signal into an electrical signal, amplify, filter, and the like the electrical pulse signal output by the sensor, transmit the conditioned electrical pulse signal to a subsequent analog-to-digital conversion module to be converted into a digital signal, and input the digital signal to an electrical pulse parameter extraction module for extracting a required electrical signal parameter.
Preferably, the forward scattered light and fluorescence beam shaping optical path 106 is disposed in the direction of the irradiation beam, and focuses and shapes the forward scattered light and the multi-channel fluorescence beam within a certain angle range, and transmits the focused and shaped forward scattered light and fluorescence beam to the forward scattered light detection circuit 107. The forward scattered light detection circuit 107 performs photoelectric conversion of forward scattered light signals and conditioning of electric pulse signals, and extracts electric pulse parameters representing characteristics of the microspheres.
Fig. 7 shows a specific circuit block diagram of the forward scattered light detection circuit 107. As shown in fig. 7, the forward scattered light detection circuit 107 includes a sensor, and is configured to convert the collected optical signal into an electrical signal, amplify, filter, and the like the electrical pulse signal output by the sensor, transmit the conditioned electrical pulse signal to a subsequent analog-to-digital conversion module to be converted into a digital signal, and input the digital signal to an electrical pulse parameter extraction module for extracting a required electrical signal parameter.
Fig. 8 and 9 respectively show a structure diagram of a specific optical path embodiment of the exemplary polychromatic fluorescence splitting optical path 108. As shown in fig. 8 and 9, the forward scattered light is collected by a collecting lens, then is split by a plurality of dichroic beam splitters, is filtered by a band pass filter, and is collected by multiplication of signals by a plurality of photomultiplier tubes (PMT 1-6). Preferably, the multi-color fluorescence splitting optical path 108 separates the multi-channel fluorescence signals through a series of optical devices and transmits the separated color fluorescence light beams to the corresponding multi-channel fluorescence detection circuit 109.
The multi-channel fluorescence detection circuit 109 realizes photoelectric conversion of fluorescence signals and conditioning of electric pulse signals, and realizes extraction of electric pulse parameters representing microsphere characteristics. FIG. 10 shows a specific circuit block diagram of the multi-channel fluorescence detection circuit 109. As shown in fig. 10, the multi-channel fluorescence detection circuit 109 includes multiple fluorescence detection circuits, each including a sensor for converting the collected optical signal into an electrical signal, amplifying, filtering, and the like the electrical pulse signal output by the sensor, and transmitting the conditioned electrical pulse signal to a subsequent analog-to-digital conversion module for conversion into a digital signal, and then inputting the digital signal to an electrical pulse parameter extraction module for extracting the required electrical signal parameters.
In another aspect of the present invention, a method for adjusting an optical path of a flow cytometer without a liquid path is provided, which includes the steps of:
the irradiation light spot shaping light path 102 compresses light beams generated by the light source 101 to obtain irradiation light spots with a certain size, and adjusts the focal length of the light spots to enable the microspheres to pass through the focal point;
the standard microsphere rotating device adjusts the movement speed of the microsphere and changes the microsphere detection frequency and the microsphere irradiation time so as to change the frequency and the duration of corresponding optical pulses and electric pulses;
the non-forward scattering light beam shaping light path carries out shaping and other treatments on the non-forward scattering light, and the focal length of the light path is adjusted to enable a detector in the non-forward scattering light detection circuit to be in a focal position;
the non-forward scattered light detection circuit realizes the photoelectric conversion, the electric pulse conditioning, the analog/digital conversion, the parameter extraction and other processing of the non-forward scattered light, and identifies the starting point and the ending point of the electric pulse;
the forward scattering light and fluorescent light beam shaping light path is used for shaping the forward scattering light and fluorescent light beam and transmitting the focused light beam to the multi-color fluorescent light splitting light path;
the multi-color fluorescence light splitting optical path performs light splitting processing on the forward scattering light and the fluorescence light beams of each channel according to wavelength information, and transmits the split light beams to a forward scattering light detection circuit and a corresponding multi-channel fluorescence detection circuit;
the forward scattered light detection circuit realizes the photoelectric conversion, the electric pulse conditioning, the analog/digital conversion, the parameter extraction and other processing of forward scattered light, and identifies the starting point and the ending point of the electric pulse;
the multi-channel fluorescence detection circuit realizes the photoelectric conversion, the electric pulse conditioning, the analog/digital conversion, the parameter extraction and other processing of all fluorescence, and identifies the starting point and the ending point of the electric pulse.
Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims.
Claims (3)
1. An optical path adjustment device for a flow cytometer without a fluid path, the device comprising: an irradiation light source, an irradiation light spot shaping light path, a standard microsphere rotating device, a non-forward scattering light beam shaping light path, a non-forward scattering light detection circuit, a forward scattering light and fluorescent light beam shaping light path, a forward scattering light detection circuit, a multi-color fluorescence light splitting light path and a multi-channel fluorescence detection circuit, wherein,
the irradiation light source provides an excitation light source for fluorescence excitation;
the irradiation light spot shaping light path is used for compressing light source light beams into illumination light spots with a certain size;
the standard microsphere rotating device comprises a turntable loaded with standard microspheres, a turntable driving device and a motor motion control circuit, wherein the turntable loaded with the standard microspheres is connected with the motor motion control circuit through the turntable driving device, and the rotation speed of the turntable is controlled by adjusting the rotation speed of the motor, so that the irradiation time of the microspheres by light spots and the irradiation time interval of adjacent microspheres are changed;
the standard microsphere rotating device is used for rotating the turntable loaded with the standard microspheres so as to simulate that single cells pass through the irradiation light spots one by one;
the non-forward scattering light beam shaping optical path is placed in a non-forward scattering angle detection area, focuses and shapes non-forward scattering light within a certain angle range, and transmits the shaped light beam to the non-forward scattering light detection circuit;
the non-forward scattering light beam shaping optical path is used for focusing the non-forward scattering light beam in a certain range;
the non-forward scattering light detection circuit is used for performing photoelectric conversion on non-forward scattering light, realizing parameter extraction on a generated electric pulse signal, realizing photoelectric conversion on the non-forward scattering light signal and conditioning treatment on the electric pulse signal, and realizing extraction on electric pulse parameters for representing the characteristics of the microspheres;
the forward scattering light and fluorescent light beam shaping light path is used for focusing forward scattering light and fluorescent light beams in a certain range;
the forward scattering light detection circuit is used for performing photoelectric conversion on forward scattering light and extracting parameters of the generated electric pulse signals;
the multi-color fluorescence light splitting optical path is used for separating fluorescence signals in each wavelength range and transmitting the fluorescence signals to the corresponding detection circuit, and the multi-channel fluorescence detection circuit is used for performing photoelectric conversion on the fluorescence signals of each channel and extracting parameters of generated electric pulse signals.
2. The optical path adjustment device according to claim 1, wherein: the laser with certain power and wavelength is used as an irradiation light source, the laser light beam emitted by the laser is compressed and shaped through an irradiation light spot shaping light path, the irradiation light spot shaping light path realizes the light spot size compression in two orthogonal directions according to the movement direction of the microsphere in a standard microsphere rotating device, and meanwhile, the moving microsphere is irradiated at the focus of the light spot through adjusting the light spot shaping light path.
3. The optical path adjustment device according to claim 1, wherein: the turntable loaded with the standard microspheres consists of a bottom layer and a top layer, wherein a pit for placing the standard microspheres is arranged in one layer, the standard microspheres are sealed in the pit through the adhesion of the top layer and the bottom layer to prevent the microspheres from falling off in the rotating process, and the standard microspheres form side scattered light, forward scattered light and fluorescent signals of various colors through irradiating spot areas one by one in the rotating process of the turntable loaded with the standard microspheres.
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| CN114088606B (en) * | 2021-10-23 | 2023-05-09 | 广州市艾贝泰生物科技有限公司 | Cell analysis device |
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| CN106383082B (en) | 2018-10-19 |
| CN109374511A (en) | 2019-02-22 |
| CN106383082A (en) | 2017-02-08 |
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