Fluorescence collecting device for flow cytometer

Abstract

The utility model relates to the field of flow cytometry and fluorescence collection, in particular to a fluorescence collection device for a flow cytometry, which comprises a flow cell, a collection component, a light splitting component and a detection component, the flow cell is used for guiding and focusing the fluorescence-labeled test particles; the collecting assembly is used for collecting fluorescent light and scattered light excited by laser irradiating the central particles of the flow cell; the light splitting assembly is used for dispersing multiple kinds of fluorescence collected by the collection assembly to different receivers through the long-pass optical filter, the dichroscope and the band-pass optical filter, and the detection assembly is used for measuring fluorescence signals collected on the receivers. The first long-pass optical filter is placed behind the receiving lens to suppress the short-wavelength laser scattering light on the test particles, so that the requirement of all optical filters on the suppression capability of short-wavelength exciting light is reduced, and then the second long-pass optical filter is used for suppressing the long-wavelength laser scattering light on the test particles, so that the suppression capability of all optical filters on the short-wavelength exciting light is reduced. And the requirements of all optical filters on the inhibition capability of the long-wavelength exciting light are reduced.

Description

Technical Field

[0001] The utility model relates to the fields of flow cytometers and fluorescence collection, in particular to a fluorescence collection device used on a flow cytometer. Background Art

[0002] Currently, a flow cytometer is an instrument for analyzing and sorting single columns of cells or particles. The cell diversion and focusing module can use hydrodynamic or acoustic focusing technology to align cells in a single column and maintain sequential movement along the center of the axis. Flow cytometers typically use lasers as light sources. The two laser light sources used in this application have central wavelengths of 488nm and 638nm, respectively. The excitation light source illuminates the cells passing sequentially through the diversion chamber in a vertical direction. When the cells pass through the excitation light source, the fluorescent markers on the cell surface are excited to produce fluorescence.

[0003] In addition to reflecting the laser light source, the cell surface also stimulates fluorescence. The light signal is typically collected perpendicular to the laser light source. Therefore, the light signal collected by the fluorescence collection device is a mixture of fluorescence and laser light, and the reflected laser light signal is generally stronger than the excited fluorescence signal. Therefore, the bandpass filter of the fluorescence channel has high requirements for its ability to suppress the corresponding wavelength of the laser light source.

[0004] Flow cytometers typically require multiple excitation light sources. By arranging these lasers in a spatially separated pattern, cells are sequentially passed through different excitation light sources. Each laser source can typically produce several types of fluorescence. Each fluorescence source requires a separate receiver. As the number of laser sources increases, the number of fluorescence receiving channels also increases, leading to a linear increase in hardware costs. Summary of the Invention

[0005] In order to solve the problem of high cutoff performance requirements for fluorescence channel bandpass filters in current flow cytometers, and the problem of high overall cost caused by a large number of receivers in multi-laser light source scenarios, the present application proposes a fluorescence collection device for use on a flow cytometer.

[0006] A fluorescence collection device for a flow cytometer, comprising a flow cell, a collection component, a spectroscopic component and a detection component;

[0007] The flow cell is used to guide and focus the fluorescently labeled test particles;

[0008] The collecting component is used to collect the fluorescence and scattered light excited by the laser irradiating the central particle of the flow cell;

[0009] The light splitting component is used to disperse the multiple fluorescences collected by the collecting component to different receivers through a long-pass filter, a dichroic mirror and a band-pass filter;

[0010] The detection component is used to measure the fluorescent signal collected on the receiver.

[0011] Furthermore, the spectroscopic component includes a fluorescent collimating lens, a first long-pass filter, a first dichroic mirror, a second long-pass filter, a second dichroic mirror, a second band-pass filter and a second receiver distributed in sequence; a first band-pass filter and a first receiver are sequentially arranged on one side of the second dichroic mirror; a third dichroic mirror, a fourth band-pass filter and a fourth receiver are sequentially arranged on one side of the first dichroic mirror; and a third band-pass filter and a third receiver are sequentially arranged on one side of the third dichroic mirror.

[0012] Furthermore, the objective lens in the collecting assembly collects the incident fluorescence and performs collimation, and the diameter of the light spot after collimation is smaller than the receiving surfaces of all receivers.

[0013] Furthermore, the first long-pass filter and the second long-pass filter are placed perpendicular to the collimated light, the cutoff wavelength of the first long-pass filter is greater than the center wavelength of the excitation light with a shorter wavelength, and the cutoff wavelength of the second long-pass filter is greater than the center wavelength of the excitation light with a longer wavelength.

[0014] Furthermore, the first dichroic mirror, the second dichroic mirror and the third dichroic mirror are all placed at an angle of 45° to the collimated light.

[0015] Furthermore, the first band-pass filter, the second band-pass filter, the third band-pass filter and the fourth band-pass filter are all placed perpendicular to the collimated light.

[0016] Furthermore, the cutoff wavelength of the first long-pass filter is 505nm~510nm, the cutoff wavelength of the first dichroic mirror is 650nm~655nm, and the cutoff wavelength of the second long-pass filter is 655nm~660nm.

[0017] The advantages of this application are:

[0018] 1. Because the scattered light signal generated by the excitation light on the test particles is very strong and affects each receiving channel, in practical application scenarios, the filter of each receiving channel must have a strong cutoff capability for this scattered light. This application addresses the problem of high cutoff performance requirements for excitation light scattered light by bandpass filters. First, a first long-pass filter is placed after the receiving lens to suppress short-wavelength laser scattered light from the test particles, thereby reducing the requirements for the short-wavelength excitation light suppression capability of all filters. Then, a second long-pass filter is used to suppress long-wavelength laser scattered light from the test particles, thereby reducing the requirements for the long-wavelength excitation light suppression capability of all filters.

[0019] 2. To address the high cutoff performance requirements for bandpass filters, this application first places a first longpass filter after the receiving lens to suppress 488nm laser light scattered from the test particles, thereby reducing the requirements for the 488nm wavelength suppression capabilities of the third and fourth bandpass filters. Secondly, a first dichroic mirror is used to separate the fluorescence signal. A second longpass filter is then placed after the first dichroic mirror to suppress 638nm laser light scattered from the test particles, reducing the requirements for the 638nm suppression capabilities of the first and second bandpass filters. Furthermore, because the third and fourth bandpass filters share the first longpass filter 3, one longpass filter can be saved compared to adding a longpass filter to each channel.

[0020] 3. In order to solve the problem of a large number of receivers, this application adopts a time-division multiplexing method for receivers, and collects fluorescence signals excited by different lasers and with similar wavelengths onto the same receiver. Time-division multiplexing means that the excitation light of the laser does not overlap in space. When the particles pass through the flow pool, they are excited by different lasers in turn, and there is an excitation sequence. For example, the particles are first excited by a 488nm laser and then by a 638nm laser. Then there are two fluorescence signals at the receiving end, the first one is excited by a 488nm laser, and the second one is excited by a 638nm laser. In this way, the time-division multiplexing function is realized. Therefore, the wavelengths of PerCP and APC are similar and the signals are sequential in time, so filters can be shared, and the receiver receives the two signals through time-division multiplexing. Improve integration while reducing costs. The same is true for PE_CY7 and APC_CY7. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a physical diagram of the fluorescence collection device structure without the receiver.

[0022] Figure 2 This is the physical structure diagram of the receiver.

[0023] Figure 3 This is a top view of the optical path structure of the fluorescence collection device used in the flow cytometer of the present application.

[0024] Figure 4 This is a plan view of the optical path structure of the fluorescence collection device used in the flow cytometer of the present application.

[0025] Figure 5 It is the receiving channel parameter of the fluorescence collection device used in the flow cytometer in this application.

[0026] In the figure: 1-flow cell, 2-fluorescence collimating lens, 3-first long-pass filter, 4-first dichroic mirror, 5-second long-pass filter, 6-first receiver, 7-first band-pass filter, 8-second dichroic mirror, 9-second band-pass filter, 10-second receiver, 11-third receiver, 12-third band-pass filter, 13-fourth receiver, 14-third dichroic mirror, 15-fourth band-pass filter. DETAILED DESCRIPTION

[0027] The following is a detailed description of an embodiment of the present invention. This embodiment is implemented based on the technical solution of the present invention, and provides a detailed implementation method and specific operation process. However, the protection scope of the present invention is not limited to the following embodiment.

[0028] It should be pointed out that all directional indications in the embodiments of the present invention (such as two sides, edges, up, down, left, right, front, back, middle, top, bottom, tail, axial, radial...) are only used to explain the relative position relationship, movement state, etc. between the various components under a certain specific posture (as shown in the accompanying drawings). If the specific posture changes, the directional indication will also change accordingly.

[0029] Example 1

[0030] like Figure 1 As shown, a fluorescence collection device for a flow cytometer includes a flow cell 1, a collection component, a spectroscopic component and a detection component;

[0031] The flow cell 1 is used to guide and focus the fluorescently labeled test particles;

[0032] The collecting component is used to collect the fluorescence and scattered light excited by the laser irradiating the central particle of the flow cell 1;

[0033] The spectroscopic component is used to disperse the multiple fluorescences collected by the collecting component to different receivers through long-pass filters, dichroic mirrors and band-pass filters, so as to measure the fluorescence intensities respectively;

[0034] The detection component is used to measure the fluorescent signal collected on the receiver.

[0035] The spectroscopic component includes a fluorescence collimating lens 2, a first long-pass filter 3, a first dichroic mirror 4, a second long-pass filter 5, a second dichroic mirror 8, a second band-pass filter 9 and a second receiver 10 which are distributed in sequence; a first band-pass filter 7 and a first receiver 6 are sequentially arranged on one side of the second dichroic mirror 8; a third dichroic mirror 14, a fourth band-pass filter 15 and a fourth receiver 13 are sequentially arranged on one side of the first dichroic mirror 4; a third band-pass filter 12 and a third receiver 11 are sequentially arranged on one side of the third dichroic mirror 14.

[0036] The first receiver 6 and the second receiver 10 can realize time-division multiplexing under the condition of spatially separated excitation of dual lasers, which is equivalent to four receivers; therefore, a total of six channels of fluorescence signals can be received; the long-pass filter is used to enhance the collection device's suppression effect on the laser light source wavelength.

[0037] Example 2

[0038] A fluorescence collection device for a flow cytometer comprises a flow cell 1, a collection component, a spectroscopic component and a detection component;

[0039] The flow cell 1 is used to guide and focus the fluorescently labeled test particles;

[0040] The collecting component is used to collect the fluorescence and scattered light excited by the laser irradiating the central particle of the flow cell 1;

[0041] The spectroscopic component is used to disperse the multiple fluorescences collected by the collecting component to different receivers through a long-pass filter, a dichroic mirror and a band-pass filter, so as to measure the fluorescence intensities respectively;

[0042] The detection component is used to measure the fluorescent signal collected on the receiver.

[0043] The spectroscopic component includes a fluorescence collimating lens 2, a first long-pass filter 3, a first dichroic mirror 4, a second long-pass filter 5, a second dichroic mirror 8, a second band-pass filter 9 and a second receiver 10 which are distributed in sequence; a first band-pass filter 7 and a first receiver 6 are sequentially arranged on one side of the second dichroic mirror 8; a third dichroic mirror 14, a fourth band-pass filter 15 and a fourth receiver 13 are sequentially arranged on one side of the first dichroic mirror 4; a third band-pass filter 12 and a third receiver 11 are sequentially arranged on one side of the third dichroic mirror 14.

[0044] The first receiver 6 and the second receiver 10 can realize time-division multiplexing under the condition of spatially separated excitation of dual lasers, which is equivalent to four receivers; therefore, a total of six channels of fluorescence signals can be received; the long-pass filter is used to enhance the collection device's suppression effect on the laser light source wavelength.

[0045] The objective lens in the collection assembly collects the incident fluorescence and collimates it. The diameter of the collimated light spot is smaller than the receiving surface of all receivers, ensuring that the fluorescence signal is completely received by the receivers.

[0046] The first longpass filter 3 and the second longpass filter 5 have different cutoff wavelengths. They are positioned perpendicular to the collimated light. The cutoff wavelength of the first longpass filter 3 is slightly greater than the center wavelength of the shorter excitation light, while the cutoff wavelength of the second longpass filter 5 is slightly greater than the center wavelength of the longer excitation light. The receiver receives fluorescence signals, which require laser excitation. The excitation light is generated by lasers, specifically two lasers with center wavelengths of 488 nm and 638 nm, respectively. There are two principles of light splitting: splitting by gradually increasing wavelengths or by gradually decreasing wavelengths. The function of the long-pass filter is to split the optical signal into two. Light with a wavelength higher than the cut-off wavelength of the long-pass filter can pass through, while light with a wavelength shorter than the cut-off wavelength will be reflected. The purpose of using two long-pass filters is to eliminate the influence of the laser excitation light on the receiving channel. Combined with the received fluorescence wavelength, the receiving wavelength of the first fluorescence channel is 525 / 20nm, so the cut-off wavelength of the first long-pass filter 3 needs to be between 488nm and 515nm (525 / 20nm means The central wavelength is 525nm and the bandwidth is 20nm, that is, the conduction wavelength range is between 515~535nm). At the same time, considering the cutoff ability of the filter at 488nm, a long-pass filter with a cutoff wavelength of 505nm is selected. This not only takes into account the suppression effect on 488nm, but also ensures that the FITC (525 / 20nm) channel can pass the signal well. Similarly, the second long-pass filter 5 is selected with a cutoff wavelength of 650nm, which also takes into account the wavelengths of the 638nm laser and the PerCP (675 / 20nm) and APC (675 / 20nm) channels.

[0047] The first dichroic mirror 4 separates the receiving ranges of the two longer wavelength first receivers 6 and the second receiver 10 and the receiving ranges of the two shorter wavelength third receivers 11 and the fourth receiver 13. On this basis, the second dichroic mirror 8 further separates the receiving wavelength ranges of the first receiver 6 and the second receiver 10, and the receiving ranges of the third receiver 11 and the fourth receiver 13, and finally divides all the light into four wavelength ranges. Specifically, the dichroic mirror can divide the wavelength range of light into two. There are a total of four receivers that receive light signals in different wavelength ranges. Therefore, the first dichroic mirror 4 needs to separate the receiving ranges of the two longer wavelength first receivers 6 and the second receiver 10 and the receiving ranges of the two shorter wavelength third receivers 11 and the fourth receiver 13. On this basis, the second dichroic mirror 8 further divides into two, and separates the receiving wavelength ranges of the first receiver 6 and the second receiver 10, and the receiving ranges of the third receiver 11 and the fourth receiver 13, and finally divides all the light into four wavelength ranges. In summary, The first dichroic mirror 4 splits the light into two parts: less than 650 nm and greater than 650 nm. The second dichroic mirror 8 further splits the part greater than 650 nm into parts less than 700 nm and greater than 700 nm. That is, after the second splitting, the light becomes 650-700 nm and greater than 700 nm. Similarly, the third dichroic mirror 1414 splits the light into 505-550 nm (because the first long-pass filter 33 has blocked light less than 505 nm) and 550-650 nm (light greater than 650 nm is transmitted by the second long-pass filter 55 and the second dichroic mirror 88).

[0048] The first dichroic mirror 4, the second dichroic mirror 8 and the third dichroic mirror 14 are all placed at an angle of 45° to the collimated light. The function of the dichroic mirror is to separate the fluorescence of the laser light source with a spectrum longer than the wavelength from the fluorescence of the laser light source with a spectrum shorter than the wavelength.

[0049] The first bandpass filter 7, the second bandpass filter 9, the third bandpass filter 12 and the fourth bandpass filter 15 are all placed perpendicular to the collimated light. The transmission bands of the first bandpass filter 7, the second bandpass filter 9, the third bandpass filter 12 and the fourth bandpass filter 15 are all different. The wavelength of the transmission band is determined by the emission wavelength of the fluorescent dye and must be compatible with the wavelength range of the light split by the dichroic mirror. Figure 5 The channel wavelengths in the image are related, and must also be consistent with the splitting results of the first dichroic mirror 44, the second dichroic mirror 88, and the third dichroic mirror 1414.

[0050] Under the dual laser excitation condition, the fluorescence spectra generated by the fluorescently labeled test particles excited at the two laser focal points have similar wavelengths and can both be imaged on the receiving surfaces of the first receiver 6 and the second receiver 10, thereby realizing time-division multiplexing.

[0051] The cutoff wavelength of the first long-pass filter 3 is 505nm~510nmnm. The cutoff wavelength of the filter indicates the wavelength corresponding to when the transmittance is reduced to 50%. Therefore, there is still some distance from 50% transmittance to 1% transmittance and 95% transmittance. For example, for the first long-pass filter 3 with a cutoff wavelength of 505nm, the wavelength at which 1% transmittance is about 492nm and the wavelength at which 95% transmittance is about 515nm. Therefore, the cutoff wavelength needs to take into account the cutoff effect of 488nm and the transmittance effect of 525 / 20nm. The cutoff wavelength of the first dichroic mirror 4 is 650nm~655nm. Because the long-pass filter needs Considering the effect of suppressing the laser wavelength while preventing excessive loss of effective signals, a balance needs to be struck. The farther the cutoff wavelength is from the laser wavelength to be suppressed, the easier it is to achieve the suppression effect. However, the optical signal that needs to pass will also be suppressed. For example, if the cutoff wavelength of the long-pass filter is set to 670nm to suppress 638nm and transmit 660nm, the long-pass filter with a cutoff wavelength of 670nm may have a better suppression effect on 638nm than the cutoff wavelength of 655nm. However, this will also suppress the 660nm band that needs to be transmitted, so a model with a cutoff wavelength of 650-655nm is preferred. The cutoff wavelength of the second long-pass filter 5 is 655nm-660nm.

[0052] Example 3

[0053] See also Figure 3 The present invention provides a fluorescence collection device for a flow cytometer, comprising a flow cell 1 for a fluorescently labeled test particle guide and focusing module, an objective lens for collecting fluorescence excited by laser irradiation onto the central particle of the flow cell 1, a spectroscopic component and a receiver;

[0054] The dichroic mirrors are all long-wave pass and short-wave reflective, with the cutoff wavelength of the first dichroic mirror 4 being approximately 650nm, the cutoff wavelength of the second dichroic mirror 8 being approximately 700nm, and the cutoff wavelength of the third dichroic mirror 14 being approximately 550nm;

[0055] The cutoff wavelength of the first long-pass filter 3 is approximately 505 nm, and the cutoff wavelength of the second long-pass filter 5 is approximately 655 nm;

[0056] The center wavelength and bandwidth of the first band-pass filter 7 are 675 / 20 nm, the center wavelength and bandwidth of the second band-pass filter 9 are 780 / 60 nm, the center wavelength and bandwidth of the third band-pass filter 12 are 525 / 20 nm, and the center wavelength and bandwidth of the fourth band-pass filter 15 are 575 / 20 nm;

[0057] pass Figure 3After light is split by the spectroscopic assembly, the four fluorescence channels excited by the 488nm laser light source—FITC (525 / 20nm), PE (575 / 20nm), PerCP (675 / 20nm), and PE_CY7 (780 / 60nm)—all benefit from the added effect of the first long-pass filter 3. Meanwhile, the two fluorescence channels excited by the 638nm laser light source—APC (675 / 20nm) and APC_CY7 (780 / 60nm)—both benefit from the added effect of the second long-pass filter 5. Therefore, this fluorescence receiving device achieves better suppression of laser light sources than one without a long-pass filter. Alternatively, while meeting the ultimate performance requirements, the performance requirements of the bandpass filter can be appropriately reduced, thereby reducing system costs.

[0058] Further, through Figure 4 The visible spectrum analyzer collects PE_CY7 (780 / 60nm) and APC_CY7 (780 / 60nm) onto the same second receiver 10. Similarly, PerCP (675 / 20nm) and APC (675 / 20nm) are collected onto the same first receiver 6. Because PE_CY7 and PerCP are excited by a 488nm laser source, and APC_CY7 and APC are excited by a 638nm laser source, when particles are excited successively by the two laser sources in flow cell 1, the first receiver 6 and the second receiver 10 receive two signals, respectively. This enables time-division multiplexing of the first and second receivers 6 and 10, allowing them to receive four fluorescence signals using two receivers. This reduces the number of receivers, size, and cost.

[0059] A fluorescence collection device for a flow cytometer of the present invention is applied to the company's flow cytometer. The instrument uses laser light sources with central wavelengths of 488 nm and 638 nm, respectively, and has 6 receiving channels. The 488 nm laser light source excites the fluorescence of 4 channels, namely FITC (525 / 20 nm), PE (575 / 20 nm), PerCP (675 / 20 nm), and PE_CY7 (780 / 60 nm). The remaining 2 fluorescence channels are excited by the 638 nm laser light source, namely APC (675 / 20 nm) and APC_CY7 (780 / 60 nm).

Claims

1. A fluorescence collection device for a flow cytometer, characterized in that: It includes a flow cell (1), a collection component, a spectroscopic component and a detection component; The flow cell (1) is used to guide and focus the fluorescently labeled test particles; The collecting component is used to collect the fluorescence and scattered light excited by the laser irradiating the central particle of the flow cell (1); The light splitting component is used to disperse the multiple fluorescences collected by the collecting component to different receivers through a long-pass filter, a dichroic mirror and a band-pass filter; The detection component is used to measure the fluorescent signal collected on the receiver.

2. A fluorescence collection device for a flow cytometer according to claim 1, characterized in that: The light splitting component comprises a fluorescence collimating lens (2), a first long-pass filter (3), a first dichroic mirror (4), a second long-pass filter (5), a second dichroic mirror (8), a second band-pass filter (9) and a second receiver (10) which are sequentially arranged; a first band-pass filter (7) and a first receiver (6) are sequentially arranged on one side of the second dichroic mirror (8); a third dichroic mirror (14), a fourth band-pass filter (15) and a fourth receiver (13) are sequentially arranged on one side of the first dichroic mirror (4); and a third band-pass filter (12) and a third receiver (11) are sequentially arranged on one side of the third dichroic mirror (14).

3. The fluorescence collection device for a flow cytometer according to claim 2, characterized in that: The objective lens in the collecting assembly collects the incident fluorescence and performs collimation, and the diameter of the light spot after collimation is smaller than the receiving surfaces of all receivers.

4. The fluorescence collection device for a flow cytometer according to claim 2, characterized in that: The first long-pass filter (3) and the second long-pass filter (5) are placed perpendicular to the collimated light, the cut-off wavelength of the first long-pass filter (3) is greater than the center wavelength of the excitation light with a shorter wavelength, and the cut-off wavelength of the second long-pass filter (5) is greater than the center wavelength of the excitation light with a longer wavelength.

5. The fluorescence collection device for a flow cytometer according to claim 4, characterized in that: The first dichroic mirror (4), the second dichroic mirror (8) and the third dichroic mirror (14) are all placed at an angle of 45° to the collimated light.

6. The fluorescence collection device for a flow cytometer according to claim 2, characterized in that: The first band-pass filter (7), the second band-pass filter (9), the third band-pass filter (12) and the fourth band-pass filter (15) are all placed perpendicular to the collimated light.

7. The fluorescence collection device for a flow cytometer according to claim 2, characterized in that: The cut-off wavelength of the first long-pass filter (3) is 505 nm to 510 nm, the cut-off wavelength of the first dichroic mirror (4) is 650 nm to 655 nm, and the cut-off wavelength of the second long-pass filter (5) is 655 nm to 660 nm.

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