Tuning of flow cytometers

Abstract

Methods for tuning the illumination assembly of a flow cytometer are described. One method of tuning the illumination assembly of the flow cytometer includes receiving a first alignment error of a first laser diode of the illumination assembly, mounting a tuning device in the flow cytometer, and adjusting a first switch associated with the first laser diode. Another method of tuning the illumination assembly of the flow cytometer includes inserting an oscillating wave signal into the drive circuitry of the illumination assembly. The oscillating wave signal provides frequency modulation of the drive circuitry.

Description

[0001] Cross-references to related applications

[0002] This application, filed as a PCT international application on January 31, 2025, claims the benefit and priority of U.S. Provisional Patent Application No. 63 / 558,176, filed on February 27, 2024, the disclosure of which is incorporated herein by reference in its entirety. Background Technology

[0003] In flow cytometry, particles are arranged in a sample stream and typically pass one after another through one or more excitation beams that interact with the particles. The light scattered or emitted by the particles upon interaction with the one or more excitation beams is collected and analyzed to characterize and differentiate the particles. In sorting flow cytometry, particles can be extracted from the sample stream after being characterized by their interactions with one or more excitation beams, and thus sorted into different groups. Summary of the Invention

[0004] Generally, this disclosure relates to tuning an illumination assembly for a flow cytometer. In one possible configuration, the tuning device is used to adjust parameters associated with one or more laser diodes of the illumination assembly. In another possible configuration, the tuning device inserts an oscillating wave signal into the drive circuitry of the illumination assembly to provide frequency modulation of the laser diodes. Various aspects are described in this disclosure, including but not limited to the following.

[0005] One aspect relates to a method of tuning an illumination assembly of a flow cytometer, the method comprising: receiving a first alignment error of a first laser diode of the illumination assembly; mounting a tuning device in the flow cytometer; and adjusting a first switch associated with the first laser diode.

[0006] On the other hand, an apparatus for tuning an illumination assembly of a flow cytometer is provided, the apparatus comprising: a connector for connection to the illumination assembly; and one or more switches for adjusting one or more laser diodes of the illumination assembly, the switches being adjustable between different settings for tuning each of the one or more laser diodes.

[0007] Various other aspects will be set forth in the following description. These aspects may involve individual features and combinations of features. It should be understood that the foregoing general description and the following detailed description are merely exemplary and explanatory, and do not limit the broad inventive concept on which the embodiments disclosed herein are based. Attached Figure Description

[0008] The following drawings, which form part of this application, are illustrations of the described techniques and are not intended to limit the scope of this disclosure in any way.

[0009] Figure 1 An example of a system for performing flow cytometry is shown, which includes a flow cytometer and a workstation.

[0010] Figure 2 Examples Figure 1 An example of the illumination component in a flow cytometer.

[0011] Figure 3 It shows the function of tuning Figure 2 An example of a tuning device for a lighting component.

[0012] Figure 4 Schematic illustration of using Figure 3 The tuning device is used for tuning. Figure 2 An example of a method for lighting components.

[0013] Figure 5 It shows Figure 1 An example of the thermoelectric cooler (TEC) control panel inside a flow cytometer.

[0014] Figure 6 It shows the insertion into Figure 5 In the TEC control board Figure 3 An example of a tuning device.

[0015] Figure 7 It shows the insertion into Figure 5 In the TEC control board Figure 3 Another view of the tuning device.

[0016] Figure 8 The illustration shows the use of Figure 2 An example of a driving circuit for a lighting component.

[0017] Figure 9 The diagram illustrates, graphically, the insertion of the oscillating wave signal into... Figure 8 The curve of the previous half-peak coefficient of variation (HPCV) in the driving circuit.

[0018] Figure 10 The diagram illustrates, graphically, the insertion of the oscillating wave signal into... Figure 8 The HPCV curve after that in the drive circuit. Detailed Implementation

[0019] Various embodiments will be described in detail with reference to the accompanying drawings, in which the same reference numerals denote the same parts and components in several views. Reference to the various embodiments does not limit the scope of the appended claims. Furthermore, any examples set forth in this specification are not intended to be limiting, and merely illustrate some of the many possible embodiments of the appended claims.

[0020] Figure 1 An example of a system 10 that can be used to perform flow cytometry is illustrated. System 10 includes a flow cytometer 100 and a workstation 110. Generally, the flow cytometer 100 is an analytical instrument for detecting the physical and chemical properties of cellular or particulate samples. In some examples, the flow cytometer 100 is designed to capture robust and high-quality data for characterizing biologically relevant nanoparticles. The flow cytometer 100 is an instrument that provides simultaneous assessment of nanoparticle size, concentration, and cargo to understand biological mechanisms of action and nanoparticle origin. The flow cytometer 100 can collect data from millions of particles or cells within minutes and display it in various formats on a display monitor 114 of the workstation 110.

[0021] The flow cytometer 100 includes a housing 101 having a multi-disc loader 104 that can receive containers of cells and / or particulate samples. In some examples, the containers contain nanoparticles, such as extracellular vesicles (EVs). The user of the system 10 manually loads the containers into the multi-disc loader 104. Once loaded, the flow cytometer 100 extracts samples from the containers to perform flow cytometry experiments.

[0022] Workstation 110 is connected to flow cytometer 100 via a wired or wireless connection to receive data from flow cytometer 100 for display on display monitor 114. Workstation 110 includes one or more user input devices, such as mouse 116 and keyboard 118, thereby allowing the user of system 10 to input data and information, control flow cytometer 100, and change the data displayed on display monitor 114.

[0023] Workstation 110 also includes a computing device 112. In some examples, workstation 110 utilizes computing device 112 to process raw data received from flow cytometer 100. Alternatively or additionally, flow cytometer 100 may include computing device to process data collected from flow cytometry. In such examples, flow cytometer 100 sends processed data to workstation 110 for display on display monitor 114.

[0024] Figure 2 An example of an illumination assembly 200 inside a flow cytometer 100 is illustrated. The illumination assembly 200 includes one or more laser diodes 202. Figure 2In the illustrated example, the illumination assembly 200 includes a first laser diode 202a, a second laser diode 202b, and a third laser diode 202c. Each of the first laser diode 202a, the second laser diode 202b, and the third laser diode 202c emits an excitation beam for projection onto particles flowing through the interrogation zone in the flow chamber 204.

[0025] The first laser diode 202a, the second laser diode 202b, and the third laser diode 202c each emit an excitation beam of a specific wavelength. As an illustrative example, the first laser diode 202a emits an excitation beam in the red visible spectrum (e.g., 625 nm to 825 nm), the second laser diode 202b emits an excitation beam in the blue visible spectrum (e.g., 450 nm to 490 nm), and the third laser diode 202c emits an excitation beam in the violet visible spectrum (e.g., 300 nm to 450 nm).

[0026] exist Figure 2 In the example shown, the first laser diode 202a, the second laser diode 202b, and the third laser diode 202c are arranged parallel to each other. It should be understood that the number, type, and arrangement of the laser diodes 202 are not limited to the examples shown and described herein and can be varied as needed. For example, the system may include four, five, six, or any other suitable number of laser diodes.

[0027] The lighting assembly 200 also includes one or more dichroic mirrors 206. Figure 2 In the example shown, the illumination assembly 200 includes a first dichroic mirror 206a, a second dichroic mirror 206b, and a third dichroic mirror 206c disposed between a laser diode 202 and a flow chamber 204. The dichroic mirrors 206 are configured according to the wavelength of the light beam emitted by the respective laser diode 202. Each dichroic mirror 206 is configured to reflect the light beam of a corresponding laser diode among the laser diodes 202 and transmit the light beam of the other laser diodes. For example, the first dichroic mirror 206a reflects light of the wavelength emitted by the first laser diode 202a toward the flow chamber 204, the second dichroic mirror 206b reflects light of the wavelength emitted by the second laser diode 202b toward the flow chamber 204 and transmits light of the wavelength emitted by the first laser diode 202a, and the third dichroic mirror 206c reflects light of the wavelength emitted by the third laser diode 202c toward the flow chamber 204 and transmits light of the wavelengths emitted by the first laser diode 202a and the second laser diode 202b.

[0028] The excitation beam emitted by the laser diode 202 is reflected or transmitted through the dichroic mirror 206 to form a collinear beam. The collinear beams share the optical axis and provide a common focal point for multiple light sources by focusing on the same point of inquiry. The dichroic mirrors 206 are adjustable in their position or orientation, allowing them to be used to adjust the position of the beam's focal point, particularly in a plane perpendicular to the optical axis.

[0029] The lighting assembly 200 also includes one or more lens assemblies 208. Figure 2 In the example shown, the illumination assembly 200 includes a first lens assembly 208a, a second lens assembly 208b, and a third lens assembly 208c arranged between a respective laser diode 202 and a respective dichroic mirror 206.

[0030] The position or orientation of the lens assembly 208 is adjustable to adjust the position of the focal point of the excitation beam, particularly its position in a plane perpendicular to the optical axis. Generally, the dichroic mirror 206 can be used to coarsely adjust the position of the focal point of the excitation beam, while the lens assembly 208 can be used to finely adjust the position of the focal point of the excitation beam.

[0031] It should be understood that the number, type, and arrangement of the dichroic mirror 206 and the lens assembly 208 can be changed as needed and are not limited to the examples illustrated herein. Furthermore, the dichroic mirror 206 and the lens assembly 208 can be replaced with other optical elements having similar functions.

[0032] The wavelengths of the excitation beams emitted by the first laser diode 202a, the second laser diode 202b, and the third laser diode 202c are affected by the temperature of the laser diodes. During the operation of the flow cytometer 100, it is desirable to maintain a constant temperature for the first laser diode 202a, the second laser diode 202b, and the third laser diode 202c so that the wavelengths of the excitation beams emitted by the laser diodes are constant, in order to record consistent flow cytometry experiments using the flow cytometer 100.

[0033] The lighting assembly 200 includes a thermoelectric cooler (TEC) control board 216 that regulates the temperature of a first laser diode 202a, a second laser diode 202b, and a third laser diode 202c. The TEC control board 216 controls the temperature of the laser diodes 202a by delivering current and voltage to thermal elements, such as a Peltier cooler, a heater, or a thermoelectric heat pump, which are regulated by the TEC control board 216 based on temperature feedback readings from temperature sensors. Each of the first laser diode 202a, the second laser diode 202b, and the third laser diode 202c may include a temperature sensor for providing temperature feedback readings to the TEC control board 216, and a thermal element controlled by the TEC control board 216 for regulating the temperature of the laser diode.

[0034] Alignment verification of the illumination assembly 200 is typically performed to ensure quality control of flow cytometry experiments performed by the flow cytometer 100. In some cases, alignment verification is performed daily. Alignment verification typically involves collecting control data from control beads (such as fluorescent microspheres) and analyzing the control data to ensure they are within the maximum permissible value of the half-peak coefficient of variation (HPCV). Small HPCV values ​​(e.g., less than 2%) indicate correct alignment of the optics on the illumination assembly 200, while large HPCV values ​​(e.g., greater than 2%) indicate misalignment of the optics.

[0035] When the control data is outside the maximum allowed value of HPCV, the alignment verification returns an alignment error. Otherwise, when the control data is within the maximum allowed value of HPCV, the flow cytometer 100 passes the alignment verification, and the user can continue to use the flow cytometer 100 to run flow cytometry experiments.

[0036] Large HPCV values ​​can be caused by instabilities in the excitation beam emitted by the laser diode 202 of the illumination assembly 200. Furthermore, different laser diode temperatures can make alignment of the laser diode 202 difficult, causing alignment verification to return alignment errors. Typically, replacing the laser diode 202 to correct alignment errors is expensive and wasteful of resources. In view of the foregoing, it is desirable to develop a technique that can be used to correct alignment errors of the laser diode 202 of the illumination assembly 200 without replacing the laser diode 202.

[0037] Figure 3 An example of a tuning device 300 for tuning an illumination assembly 200 is shown. As will be described in more detail, the tuning device 300 can be used to tune the illumination assembly 200 such that the illumination assembly passes alignment verification without replacing the first laser diode 202a, the second laser diode 202b, or the third laser diode 202c.

[0038] The tuning device 300 includes a body 302 and a cable tether 304 extending from the body 302 and terminating at a first connector 306. The tuning device 300 also includes a power harness 308 comprising a second connector 310 at one end and a power socket 311 at the opposite end. The power harness 308 further includes a cable tether 312 connected to internal electrical components housed within the body 302 of the tuning device 300. As will be described in more detail, the second connector 310 is inserted into a power socket of the TEC control board 216, and the power socket 311 receives the power harness of the flow cytometer 100. In this example, the cable tether 312 taps voltage from the power harness of the flow cytometer 100 to supply voltage to power the tuning device 300.

[0039] The tuning device 300 includes one or more switches 314 for adjusting the lighting assembly 200. Figure 3 In the example shown, the tuning device 300 includes a first switch 314a for adjusting the operation of a first laser diode 202a, a second switch 314b for adjusting the operation of a second laser diode 202b, and a third switch 314c for adjusting the operation of a third laser diode 202c. The number of switches 314 on the tuning device 300 may vary based on the number of laser diodes 202 on the illumination assembly 200.

[0040] The first switch 314a, the second switch 314b, and the third switch 314c are each adjustable between different temperature settings to tune the temperatures of the first laser diode 202a, the second laser diode 202b, and the third laser diode 202c, respectively. As described above, the temperature of the laser diode 202 is adjusted by the TEC control board 216. The tuning device 300 interfaces with the TEC control board 216 to individually adjust the temperatures of the first laser diode 202a, the second laser diode 202b, and / or the third laser diode 202c. As an illustrative example, each switch 314 is adjustable between five different temperature value positions (such as 20°C, 27°C, 35°C, 42°C, and 50°C).

[0041] Figure 4 An example of a method 400 for tuning an illumination assembly 200 is schematically illustrated. Method 400 uses a tuning device 300 to correct for instability in the excitation beam emitted by the laser diode 202 and alignment errors caused by the temperature of the laser diode, without replacing the laser diode 202.

[0042] Method 400 includes an operation 402 of receiving alignment errors during alignment verification performed on flow cytometer 100. Alignment errors may be caused by instability of the excitation beam and / or the laser diode temperature of the laser diode 202 on illumination assembly 200. Operation 402 may include receiving alignment errors of a first laser diode 202a of illumination assembly 200. Alternatively or additionally, operation 402 may include receiving alignment errors of a second laser diode 202b of illumination assembly 200. Alternatively or additionally, operation 402 may include receiving alignment errors of a third laser diode 202c of illumination assembly 200. Alignment errors received in operation 402 may be displayed on display monitor 114 of workstation 110.

[0043] Method 400 includes operation 404 of mounting the tuning device 300 in the flow cytometer 100. Operation 404 may include powering off the flow cytometer 100 and opening the housing 101, such as by removing the cover to access the illumination assembly 200.

[0044] Figure 5 An example of the TEC control panel 216 inside the flow cytometer 100 is shown. Figure 6 An example of a tuning device 300 inserted into a TEC control panel 216 is shown. Figure 7 Another view shows the tuning device 300 inserted into the TEC control panel 216. Now refer to... Figures 5 to 7 Operation 404 of method 400 may include disconnecting connector 322 of power harness 324 from power socket 320 of TEC control board 216 (see [link]). Figure 5 As an example, power outlet 320 can be power from a J10 connector port.

[0045] Operation 404 may also include connecting the second connector 310 of power harness 308 to the power socket 320 of TEC control board 216, and connecting the connector 322 of power harness 324 to the power socket 311 of power harness 308 (see...). Figure 6 In this way, the power harness 308 of the tuning device 300 is connected between the power socket 320 and the power harness 324 of the TEC control board 216.

[0046] When the tuning device 300 is installed, connector 322 of the power harness 324 of the flow cytometer 100 is inserted into power socket 311 of the power harness 308 of the tuning device 300. A second connector 310 of the tuning device 300 is inserted into power socket 320 of the TEC control board 216. The wire between the second connector 310 and power socket 311 is a straight-through cable, and cable tie 312 receives power (e.g., 5V) from the power harness 324 of the flow cytometer 100. In this way, the TEC control board 216 receives power from the power harness 324, and the tuning device 300 receives power (e.g., 5V) from the cable tie 312 to power its operation.

[0047] Operation 404 may also include connecting the first connector 306 at the end of the cable tether 304 of the tuning device 300 to a second socket 318 of the TEC control board 216. As an example, the second socket 318 may be a 9-pin serial J12 connector port, such that the first connector 306 of the tuning device 300 may be a DB9 (9-pin) connector. In some examples, the first connector 306 of the tuning device 300 is secured to the second socket 318 of the TEC control board 216 using one or more screws 307 (see [link to documentation]). Figure 7 ).

[0048] During installation, the tuning device 300 should set switch 314 to the intermediate temperature position (e.g., 35°C), which is the default temperature setting for the laser diode 202. After the tuning device 300 is installed, the flow cytometer 100 is powered on, and the user or field technician can log in to the system software on the workstation 110.

[0049] See Figure 4 Method 400 includes operation 406, which involves adjusting the position of switch 314 on tuning device 300 to adjust the laser diode temperature of at least one laser diode in laser diode 202. As discussed above, each switch 314 can be adjusted between five different temperature value positions (such as 20°C, 27°C, 35°C, 42°C, and 50°C). As described above, an intermediate temperature value position (e.g., 35°C) is the default temperature setting, such that operation 406 includes adjusting the position of switch 314 on tuning device 300 to a position lower than the intermediate temperature value position (e.g., 20°C or 27°C) or to a position higher than the intermediate temperature value position (e.g., 42°C or 50°C). In some examples, operation 406 includes adjusting the position of switch 314 on tuning device 300 to the lowest temperature value position (e.g., 20°C).

[0050] In an alternative example, the switches 314 on the tuning device 300 do not include an intermediate temperature value position, which is the default temperature setting of the laser diode 202 on the illumination assembly 200, because the flow cytometer 100 will increase HPCV at the default temperature setting whenever the tuning device 300 is used on the flow cytometer 100. In such examples, each switch of the switches 314 on the tuning device 300 may have adjustable temperature value positions of 20°C, 25°C, 30°C, 40°C, and 45°C. In another example, the tuning device 300 may include a logging function, enabling the tuning device 300 to operate as a temperature logger.

[0051] Operation 406 may include adjusting a first switch 314a associated with the first laser diode 202a to a temperature value position other than an intermediate temperature value position (e.g., 20°C, 27°C, 42°C, or 50°C). As another example, operation 406 may include adjusting a second switch 314b associated with the second laser diode 202b to a temperature value position other than an intermediate temperature value position. As yet another example, operation 406 may include adjusting a third switch 314c associated with the third laser diode 202c to a temperature value position other than an intermediate temperature value position.

[0052] Method 400 includes operation 408 for determining whether the half-peak variation coefficient (HPCV) of the laser diode 202, which was tuned in operation 406 by adjusting switch 314, is less than a permissible maximum value for HPCV. For example, operation 408 may include rerunning alignment verification on flow cytometer 100.

[0053] When the HPCV of the laser diode 202, tuned by adjusting switch 314 in operation 406, is not less than the maximum permissible HPCV, causing alignment verification to continue returning alignment errors (i.e., "No" in operation 408), method 400 includes returning to operation 406 to further adjust switch 314 to tune the laser diode. For example, when switch 314 is initially adjusted to a first temperature value position, operation 406, when repeated, may include further adjusting switch 314 to a different temperature value position. Operations 406 and 408 may be repeated multiple times as needed to tune the illumination assembly 200 until the HPCV of the laser diode 202 is less than the maximum permissible HPCV, causing alignment verification on the flow cytometer 100 to no longer return alignment errors.

[0054] When the HPCV of the laser diode 202, tuned by adjusting switch 314 in operation 406, is less than the maximum acceptable HPCV such that alignment verification does not return an alignment error (i.e., "Yes" in operation 408), method 400 includes operation 410 of closing the housing 101 of the flow cytometer 100. When an acceptable HPCV is obtained at both the high-temperature position (42°C and 50°C) and the low-temperature position (20°C and 27°C) on switch 314, the lower temperature position should be selected because the laser diode 202 has a longer lifetime at the lower temperature.

[0055] Figure 8 An example of a drive circuit 800 for a lighting assembly 200 is schematically shown. Figure 8 As shown, the drive circuit 800 includes a direct current (DC) driver 802 for powering the laser diode 202 of the lighting assembly 200. The drive circuit 800 also includes an oscillator 806 that inserts an oscillating wave signal into the drive circuit 800.

[0056] The oscillating wave signal provides frequency modulation for the drive circuitry to mitigate instabilities in the excitation beam emitted by the laser diode 202 of the illumination component 200. In some examples, the oscillating wave signal is alternating current (AC). In other examples, the oscillating wave signal provides frequency modulation between approximately 10 MHz and approximately 300 MHz.

[0057] like Figure 8 As shown, the oscillation signal is inserted into a bias T-type circuit 804 connected to the DC driver 802 and the laser diode 202. The bias T-type circuit 804 ensures that the oscillation signal does not interfere with the DC driver 802.

[0058] Figure 9 The graph 900 is illustrated graphically, showing the half-peak variation coefficient (HPCV) before the oscillating wave signal is inserted into the drive circuit 800. Figure 10 A graph 1000 is illustrated graphically, showing the HPCV after the oscillating wave signal is inserted into the drive circuit 800. Figure 10 In the example shown, the oscillating wave signal has a frequency of 50 MHz at 10 volts. Figure 8 and Figure 9 As shown, the oscillating wave signal significantly reduces the variability of the wavelength emitted by the laser diode 202. For example, graph 900 shows an HPCV with two peaks, while graph 1000 shows an HPCV with a Gaussian shape due to the smaller variability of the wavelength emitted by the laser diode 202.

[0059] The various embodiments described above are provided by way of illustration only and should not be construed as limiting in any way. Various modifications may be made to the above embodiments without departing from the true spirit and scope of this disclosure.

Claims

1. A method for tuning the illumination assembly of a flow cytometer, the method comprising: Receive the first alignment error of the first laser diode of the lighting assembly; A tuning device is installed in the flow cytometer; as well as Adjust the first switch associated with the first laser diode.

2. The method of claim 1, further comprising: Receive the second alignment error of the second laser diode of the lighting assembly; Adjust the second switch associated with the second laser diode.

3. The method of claim 2, further comprising: The third alignment error of the third laser diode of the lighting assembly is received; Adjust the third switch associated with the third laser diode.

4. The method of claim 3, wherein the first laser diode emits red visible light, the second laser diode emits blue visible light, and the third laser diode emits violet visible light, and wherein the illumination component guides the red visible light, the blue visible light, and the violet visible light to project onto the interrogation area inside the flow cytometer.

5. The method of claim 4, wherein the first switch, the second switch, and the third switch are each adjustable between five different temperature settings to tune the first laser diode, the second laser diode, and the third laser diode, respectively.

6. The method according to any one of claims 1 to 5, the method further comprising: An oscillating wave signal is inserted into the driving circuit of the lighting component, the oscillating wave signal providing frequency modulation for the driving circuit.

7. The method of claim 6, wherein the oscillating wave signal provides the frequency modulation between about 10 MHz and about 300 MHz.

8. The method of claim 6 or 7, wherein the oscillating wave signal is inserted into a bias T-type circuit, the bias T-type circuit being connected to a DC driver and to the lighting assembly.

9. An apparatus for tuning an illumination assembly of a flow cytometer, the apparatus comprising: Connector, the connector being used to connect to the lighting assembly; and One or more switches are provided for adjusting one or more laser diodes of the lighting assembly, the switches being adjustable between different settings to tune each of the one or more laser diodes.

10. The apparatus of claim 9, wherein the one or more switches comprise: A first switch, the first switch being used to tune the first laser diode of the lighting assembly; A second switch, the second switch being used to tune the second laser diode of the lighting assembly; and A third switch is used to tune the third laser diode of the lighting assembly.

11. The apparatus of claim 10, wherein the first laser diode emits red visible light, the second laser diode emits blue visible light, and the third laser diode emits violet visible light, and wherein the illumination component directs the red visible light, the blue visible light, and the violet visible light to project onto the interrogation area inside the flow cytometer.

12. The apparatus of claim 10 or 11, wherein the first switch, the second switch, and the third switch are each adjustable between five different temperature settings to tune the first laser diode, the second laser diode, and the third laser diode, respectively.

13. The apparatus of any one of claims 9 to 12, wherein the connector is inserted into the drive circuit of the lighting assembly for inserting an oscillating wave signal to provide frequency modulation of the drive circuit.

14. The apparatus of claim 13, wherein the oscillating wave signal provides the frequency modulation between about 10 MHz and about 300 MHz.

15. The apparatus of claim 13 or 14, wherein the oscillating wave signal is inserted into a bias T-type circuit, the bias T-type circuit being connected to a DC driver and to the one or more laser diodes of the illumination assembly.

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