This article will briefly describe how the flow cytometry system works. It then introduces the ADAQ23878, an 18-bit ADC module from Analog Devices, and shows how to use this module to design the detection and conversion stages of a flow cytometer. A related evaluation kit will also be introduced.

Author: Bonnie Baker

Flow cytometry is widely used by clinicians and diagnosticians to analyze cellular properties. They optically assessed properties such as protein content, blood health, granularity and cell size on a cell-by-cell basis. Despite the high sensitivity of the system, designers of flow cytometers are under constant pressure to speed up analysis times, which requires new approaches to designing flow cytometry and its associated electronics.

The flow cytometer irradiates individual cells with laser light, producing scattering and fluorescent signals. To quickly and accurately capture the resulting light and convert it into a digital signal, an avalanche photodiode (APD) and complex electronics are required. It can take a long time to design and implement the circuitry for this process, especially given that flow cytometry data acquisition systems require high-speed, low-noise equipment to ensure system accuracy.

To enable faster flow cytometry analysis and ensure cost-effectiveness, designers can address speed and accuracy issues with a data acquisition solution consisting of an internal amplifier driver and analog-to-digital converter (ADC).

This article will briefly describe how the flow cytometry system works. It then introduces the ADAQ23878, an 18-bit ADC module from Analog Devices, and shows how to use this module to design the detection and conversion stages of a flow cytometer. A related evaluation kit will also be introduced.

Principles of modern flow cytometry

Modern flow cytometry is an automated process that analyzes cells and surface molecules, describing and defining different cell types in heterogeneous cell populations. Not counting the preparation time (which can be over an hour), the instrument can perform three to six feature assessments on 10,000 single cells in less than a minute.

To achieve this, the single-cell preparation step for flow cytometry is critical. The sample is organized in sheath fluid, which hydrodynamically focuses cells or particles into a long, narrow stream of single-cell columns for analysis. After this transformation, the single cell must retain its natural biological characteristics and biochemical composition.

Figure 1 is a schematic diagram of a flow cytometer with a multicellular sample first loaded from the top.

Rapidly implement flow cytometry designs with high-accuracy data acquisition modules
Figure 1: Schematic diagram of a flow cytometer, from sheath fluid focusing to data acquisition. (Image source: Wikipedia, modified by Bonnie Baker)

A flow cytometer contains six main components: flow cell, laser, avalanche photodiode (APD), transimpedance amplifier (TIA), ADC, and a computer for data collection and analysis.

Flow cytometers have either a fluid stream or a sheath fluid, both of which are narrowed to align the cells in a single column across the beam. The laser captures one cell at a time, producing a forward-scattered light (FSC) signal and a side-scattered light (SSC) signal. Fluorescence is sorted by mirrors and filters and then amplified by APD.

Next, the resulting light output is detected, digitized, and analyzed after it hits the APD. For detection, Analog Devices’ LTC6268 500 megahertz (MHz) ultra-low bias current, low voltage noise FET input op amp is ideal for high-speed TIAs required for detection.

Rapidly implement flow cytometry designs with high-accuracy data acquisition modules
Figure 2: The TIA circuit uses an APD (PD1) and a low input current FET op amp to convert the ultra-low photodiode current to the output voltage of IN1+. (Image credit: Bonnie Baker)

When designing this amplifier circuit, the bandwidth must be maximized and parasitic capacitance must be minimized. For example, the parasitic feedback capacitance C affects the circuit stability and bandwidth of Figure 2. Regardless of the resistor package chosen, there will always be parasitic capacitance in the feedback path of the amplifier. However, the 0805 package has a longer end cap pitch and the lowest parasitic capacitance, making it the first choice for high-speed applications.

Increasing the distance between the R1 end caps is not the only way to reduce capacitance. Another way to reduce board-to-board capacitance is to add a ground wire under resistor R1 to shield the electric field path that creates parasitic capacitance (Figure 3).

Rapidly implement flow cytometry designs with high-accuracy data acquisition modules
Figure 3: Adding a ground wire under the feedback resistor can shunt the electric field from the feedback end and dump it to ground. (Image credit: Analog Devices)

In this case, the method specifically involves placing a short ground wire under and between the resistor pads close to the TIA output. This method results in a parasitic capacitance of 0.028 picofarads (pF) and a TIA bandwidth of 1/(2π*RF*CPARASITIC), which is equivalent to 11.4 MHz.

The light signal is directed to several avalanche diodes with appropriate filters. APD, TIA and ADC systems convert these signals to their digital representation and send the data to a microprocessor for further analysis.

Modern cytometers are often equipped with multiple lasers and APDs. Current commercial equipment has 10 lasers and 30 avalanche photodiodes. Increasing the number of laser and photomultiplier detectors enables multiple antibody labeling to precisely identify target cell populations through phenotypic markers.

However, analysis speed depends on a delicate balance of:

・ Sheath fluid flow rate
・ Ability to form single-cell arrays during hydrodynamic focusing
・ Tunnel diameter
・ Ability to maintain cellular integrity
· Electronic component

flow cytometry acoustic focusing

While the addition of multiple lasers and APDs can speed up analysis and identification, at best the latest modern single-cell flow cytometry can collect data on up to a million cells per minute. In many applications, such as the detection of circulating tumor cells in blood as low as 100 per milliliter, this treatment is not sufficient. In clinical applications of rare cells, assays often require analysis of billions of cells and are time-consuming.

The sonic focusing process is an alternative to the hydrodynamic focusing cell preparation process. In this process, a piezoelectric material, such as lead zirconate titanate (PZT), is attached to a glass capillary to convert electrical pulses into mechanical vibrations (Figure 4a). By using PZT to vibrate the sidewall of the glass capillary at the resonant frequency of the rectangular flow cell, the system generates various acoustic standing waves with different numbers of pressure nodes.

Rapidly implement flow cytometry designs with high-accuracy data acquisition modules
Figure 4: Schematic (a) of an acoustic flow cell made of rectangular glass capillaries. Location of the first three pressure nodes of the fixed-width capillary (b). (Image credit: National Center for Biotechnology Information)

These PZT frequency nodes arrange the flowing particles into discrete streamlines (Fig. 4b). Acoustic flow cells use linear standing acoustic waves, tuned to various wavelengths by generating single or multiple harmonics. As predicted by the simple linear standing wave model, the cells in the sample generate single or multiple single-cell columns in the flow cell.

With this precise cellular organization, the width of the sheath fluid tunnel can be expanded to accelerate the flow through the laser beam (Figure 5).

Rapidly implement flow cytometry designs with high-accuracy data acquisition modules
Figure 5: For the hydrodynamic sample flow (c. and d.), as the width of the sheath fluid increases, the cellular sample scatters, making the optical measurement process difficult. The acoustically focused sample stream (a. and b.) maintains a single column of cells regardless of sheath fluid width. (Image credit: Thermo Fischer Scientific)

Traditional hydrodynamic focusing (Figure 5c.) aligns single cells in preparation for laser scanning. While the wider the funnel in the sample flow core, the faster the sheath fluid material (Figure 5d.), this also causes dispersion of the single-cell column, which can create signal changes and affect data quality.

Acoustic focusing (Fig. 5a.) aligns biological cells and other particles closely together, even with wide tunnels. This precise arrangement of cells allows for higher sampling rates while maintaining data quality (Fig. 5b.).

In practice, flow cytometry acoustic focusing increases the cell sampling frequency by a factor of approximately 20 (Figure 6).

Rapidly implement flow cytometry designs with high-accuracy data acquisition modules
Figure 6: Comparison of sampling times for various flow cytometry devices based on fluid flow cytometry (A, B, C) versus sonic focus cytometry (D). (Image credit: Thermo Fischer Scientific)

In Figure 6, devices in A, B, and C employ hydrodynamic techniques, while D employs sonic focusing flow cytometry.

Acoustic Focused Flow Cytometry Data Acquisition

The electronic design of the acoustic focusing flow cytometry device requires high-speed photosensitive electronics to accommodate the velocity of blood cells and sheath fluid through the larger diameter nozzle. The previously mentioned 600 MHz high speed LTC6268 combined with a dedicated 0805 resistor package layout enables light sensing rates up to 11.4 MHz (Figure 7 left). The output of the LTC6268 is fed into an Analog Devices ADAQ23878 ADC for digitization.

Rapidly implement flow cytometry designs with high-accuracy data acquisition modules
Figure 7: The ADAQ23878 ADC digitizes the optical signal from the photodiode (PD1) and TIA circuit (left). (Image credit: Bonnie Baker)

The ADAQ23878 is an 18-bit, 15 megasamples per second (MSPS) precision high-speed system-in-package (SIP) data acquisition solution. It shifts the design challenges faced by designers, such as input-driven component selection, optimization, and placement, to the device, dramatically shortening the development cycle for precision measurement systems.

This modular approach to SIP reduces end-system component count by combining multiple general-purpose signal processing and conditioning blocks with a high-speed, 18-bit, 15 MSPS successive approximation register (SAR) ADC in a single device. These modules contain a low noise, fully differential ADC driver amplifier and a stable reference buffer.

The ADAQ23878 also integrates key passive components using Analog Devices’ iPassive technology to minimize temperature-dependent error sources and optimize performance. The ADC’s fast settling driver stage helps it ensure fast data acquisition.

Evaluating the ADAQ23878 μModule

To evaluate the ADAQ23878, Analog Devices offers the EVAL-ADAQ23878FMCZ evaluation board (Figure 8). Demonstrating the performance of the ADAQ23878 μModule, this evaluation board is a versatile tool for evaluating flow cytometry front-end designs and various other applications.

Rapidly implement flow cytometry designs with high-accuracy data acquisition modules
Figure 8: The EVAL-ADAQ23878FMCZ evaluation board for the ADAQ23878 has on-board circuitry with associated software for control and data analysis and is SDP-H1 compatible. (Image credit: Analog Devices)

The EVAL-ADAQ23878FMCZ evaluation board requires a personal computer running Windows 10 or later, a low noise precision signal source, and a bandpass filter suitable for 18-bit testing. The evaluation board requires the ADAQ23878ACE plug-in and SPD-H1 driver.

Epilogue

While the use of standard hydrodynamic focused flow cytometry to examine biological cells one by one has been successful, the need to speed up analysis has led to a switch to sonic focused flow-based methods. However, to support more advanced flow cytometry, electronics must also improve while minimizing space, cost, and development time.

As described in this article, the LTC6268 high-speed op amp can be combined with the ADAQ233878 precision, high-speed, μModule data acquisition solution to create a complete data acquisition system for advanced flow cytometry equipment.

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