Accuri cytometers revolutionise flow cytometry

Millennium Science Pty Ltd
By LabOnline Staff
Wednesday, 19 May, 2010

The Accuri C6 Flow Cytometer System represents a dramatic step forward in flow cytometry and cell analysis.

For the first time, researchers have access to a high capability analytical cytometer at an affordable price. Sized to fit on any benchtop, the C6 Flow Cytometer System makes a powerful research tool available to a much wider range of life scientists. Accuri’s user-driven design has resulted in a system that offers both performance and simplicity. Combined with the intuitive CFlow software, Accuri’s product makes flow cytometry equally accessible to experts and new users alike.

This article addresses how Accuri can sell a high performance flow cytometer for a fraction of the price of existing systems and illustrates the company’s approach to product development.


Life science researchers have used flow cytometry in an expanding set of applications for close to four decades. This technology is used to study the cell cycle, apoptosis, cellular and molecular profiles, acellular molecular detection, cell proliferation, transfection, and cell viability. Interest in flow cytometry has been further boosted with the current growth in proteomics and high throughput cell-based drug screening as well as the increasing use of biomarkers in drug development.

The premise behind flow cytometry technology is straightforward. Particles or cells are suspended in a liquid stream and flow past a laser light source. An optical detector collects scattered laser light and fluorescence, and electronics measure and digitise these light emissions for analysis on a computer. The data provide information about the particles or cells, such as size, shape and surface features. The ability to identify cells based on the presence of fluorescent tags, such as dye-labeled antibodies, dramatically enhances the analytical potential of flow cytometry, and the technology is commonly used to explore a wide range of cellular properties.

By the 2000s, this easy-to-understand technology had not yet translated into an easy-to-use instrument. Flow cytometry has never enjoyed the mainstream acceptance afforded other technologies, such as microscopy or PCR (polymerase chain reaction) analysis, primarily due to the cost, size, and complexity of existing instruments. Conventional flow cytometers are expensive, large, high-maintenance instruments that require extensive training to use correctly. As a result, most flow cytometry users must share an instrument or submit samples to a core facility for analysis, a less than ideal situation for users with a high number of samples. Historically, the flow cytometry industry has prioritized development of new features based on existing designs, as opposed to simplifying designs to reduce the complexity and cost. This focus on conventional hardware and software has further restricted growth and mainstream acceptance of the technology.

Accuri's founders, Jennifer Baird and Collin Rich.

The Accuri Cytometers’ biolab in Ann Arbor.

Sensing an opportunity to innovate, in 2004, Accuri’s founders began talking to life science researchers to find out if there was a need for a less expensive, more intuitive, high capability flow cytometer. The answer was a resounding yes! In 2005, they founded Accuri Cytometers with a single objective: to revolutionize flow cytometry.

What do researchers want?

The company’s first step was to solicit input from hundreds of researchers who use flow cytometers to find out what they cared about most.

They wanted:

  • High quality data
  • The ability to detect forward and side scatter and up to 4 fluorescent colors
  • Both blue and red laser excitation
  • An affordable system
  • Easy-to-use software
  • A low-maintenance product that was quick to install and took up little space in their labs

Based on these findings, members of Accuri’s Research and Development Team started to build an entirely new type of flow cytometer. They created their designs from scratch, taking advantage of leading edge technology when possible, and inventing new technology when necessary. In December 2006, Accuri reached their goal and previewed the C6 Flow Cytometer with CFlow software.

Design philosophy

Accuri believes it is possible to build powerful analytical tools that are accessible and easy-to-use. Researchers should be able to focus on their experiments and not have to spend weeks becoming instrumentation experts.

Accuri’s design philosophy is based on several core principles:

  1. Customer first: Researchers<0x2019> needs and requirements are the main focus
  2. Simplicity: Use the simplest design that can possibly work
  3. Performance: Product specs should meet or exceed those of more expensive instruments
  4. Innovation: Question all existing design assumptions; build new products from the ground up using advanced engineering theory
  5. Advanced technology: Employ recent advances in technology, and if needed, invent new ones
  6. Team work: Success requires a cross-functional team, including expert researchers and engineers

The outcome of this design philosophy is the C6 Flow Cytometer System: a low-cost, full-featured instrument that is easy-to-use, is compatible with standard protocols, produces results that are comparable with existing instruments, and requires minimal maintenance.

Key innovations

All flow cytometers are composed of three major, interdependent sub-systems: fluidics, optics, and electronics. Focusing on the interfaces between the sub-systems, Accuri’s team designed all components to optimise overall system performance, including creating intuitive, user-friendly software. This resulted in several interlocking innovations that together surpass anything on the market today.

Innovation #1: Reliable, high-performance fluidics system

Conventional flow cytometers typically use either syringe pumps or air-over-water pressurised fluidics systems. Historically, these systems have been selected because they can provide smooth, non-pulsatile flow of the sample, which is essential to achieve high performance. But these approaches to fluidics have negatives; they add cost, complexity and size to instruments, and require regular maintenance, a time-consuming nuisance.

Early in product development, the Accuri team looked for an alternative to conventional fluidics designs. A non-pressurized approach would negate the need for pressure systems or expensive syringe pumps and reduce the size and cost of the instrument. Peristaltic pumps were appealing. They are compact, inexpensive, low maintenance and offer direct drive fluid flow. The challenge was how to produce a smooth pulse-free flow from an inherently pulsatile pump.

The solution came from an in-house innovation on how to regulate the fluid flow. Novel pulse dampeners, combined with a sophisticated microprocessor controlled dynamic feedback system, were invented to produce a sensitive, zero pulse flow that was user adjustable. The C6 Flow Cytometer fluidics system starts up automatically with the instrument’s power switch and produces consistent, robust control of the velocity of both the sheath and sample fluids to within extremely tight tolerances (<0.01%).

Accuri’s patented pulse dampeners and pressure sensors allow the use of inexpensive peristaltic pumps to provide a non-pressurised, zero pulsation, push/pull system with sophisticated microprocessor controlled dynamic feedback.

The C6 Flow Cytometer fluidics system employs two peristaltic pumps in a “push/pull” configuration. One pump pushes sheath fluid into the flow cell, the other pulls the combined sample and sheath fluid from the flow cell to the waste tank. The differential between the two pump pressures creates suction, which draws up the sample. The push/pull design allows for independent control of both the sheath and sample flow speeds, making it possible to precisely control the sample core diameter. This enables the user to quickly fine-tune the fluidics for each individual sample, if so desired.

Peristaltic pumps are ‘direct drive’ pumps. One benefit of the direct drive design is that it makes it possible to meter the sample fluid uptake. As a result the C6 Flow Cytometer can automatically calculate the events per microliter for each run, providing counts and concentration. Another benefit of the direct-drive approach is that it makes it easy to remove clogs, such as a clump of cells, which might occur during sampling. By design most clogs will occur in the Sample Injection Probe (SIP), not in the interior fluidics lines. Clogs can be pushed out of the flow cell with a brief burst of sheath fluid followed by a high-volume flush.

Because the C6 Flow Cytometer does not rely on pressurization, it is very tolerant of types and brands of sample tubes. Several types of tubes can be used, including any brand of 12x75mm tubes and most capless microcentrifuge tubes, making it easier to use in the lab. A cracked tube is no longer a source of user frustration.

The instrument uses four non-pressurized fluid tanks: sheath, waste, cleaner, and decontamination solution. Each of these tanks is equipped with durable sensors that automatically alert users when the tanks need to be filled or emptied. Sample is drawn by siphon through the SIP into the flow cell, where the sample fluid is focused using the hydrodynamic force applied by the sheath flow. Although hydrodynamic focusing is the conventional approach to sample presentation, the Accuri flow cell is not. It’s housing contains a fused quartz capillary, replacing the more common and expensive all-quartz designs.

C6 fluidics tanks

To address researchers’ desire for a low maintenance system, the C6 Flow Cytometer is programmed for automatic decontamination of the fluidics system. Pressing the power switch to turn off the instrument triggers the C6 Flow Cytometer to automatically flush the fluidics lines with decontamination and cleaning solution. When next powered on, the C6 Flow Cytometer automatically flushes and primes the fluidics lines with sheath fluid prior to reporting that it is ready for the first sample.

The high performance C6 Flow Cytometer fluidics system:

  1. Powers on with a single switch,
  2. Self-cleans and alerts when fluids need attention,
  3. Allows the use of a range of sample tube configurations,
  4. Meters sample fluid uptake and automatically calculates the events per microliter,
  5. Allows the user to independently adjust speed of the sample uptake,
  6. Allows the user to adjust the core stream diameter for a variety of cell sizes, and
  7. Uses fewer components, contributing to a small footprint and lower cost.

Innovation#2: Inexpensive, robust optics

Most conventional “high-end” flow cytometers contain large optic tables composed of a complex array of dozens of photomultiplier tubes, lenses, filters, beam splitters or mirrors, each aligned in a precise manner to get optimum results. These dense optic tables are expensive to build and are very sensitive to jarring or bumping, which results in misalignment that requires service.

The Accuri team had two optic system design goals: 1) decrease complexity to lower costs and 2) increase stability to lower service requirements. To achieve these goals the optic table was designed with the shortest possible light path. Novel configurations of the optics subsystems were needed to make this possible.

First, the Accuri system employs two solid-state lasers (488nm and 640nm) that are arranged to be co-linear, but modulated so that they are not co-temporal.

Next the lenses and filters were simplified. Fixed alignment, multi-section focusing and gross collection lenses replace the conventional flow cytometer lens and mirror assemblies, increasing signal strength while reducing user alignment issues and assembly complexity. Interference filters are easily accessed and can be swapped by the user.

Finally, a direct light detection scheme was employed. Photomultiplier modules are clustered in a pie-shaped design around the flow cell to maximise light collection and reduce alignment issues. Focused on the same spot, these detectors sample data only when the desired laser combination is exciting the sample. This arrangement makes it possible to assign any of the 4 fluorescent detectors to read from either of the lasers. A 24-bit analog to digital (A-D) converter is used to convert the signals from the photomultiplier tubes, creating a totally digital system.

Clustered in a ‘pie’ design around the flow cell, the C6 Flow Cytometer’s photomultiplier tubes maximise light collection and reduce alignment issues.

The reliable C6 Flow Cytometer optics system:

  1. Is robust and withstands jostling; the C6 Flow Cytometer is easy to transport,
  2. Allows the user to easily swap out interference filters, and
  3. Uses fewer components, contributing to a small footprint and lower cost.

Innovation #3 Sophisticated, simplified electronics

The digital signal processing (DSP) systems in conventional flow cytometers typically max out at 16 to 18 bits, limited in part by the analog to digital converter (ADC) card. DSP and ADC determine an instrument’s dynamic range; the <4 decades of dynamic range in conventional cytometers (and even 5 decades in the most advanced digital systems) is less than the range of signals in some biological experiments.

Limited dynamic range has been managed through the use of voltage and amplifier gain controls on the photomultiplier tube (PMT) detectors. To observe faint signals the voltage is set high; to detect bright signals the voltage is set low. Although effective, there are two major drawbacks to the use of voltage and gain: 1) bright and faint signals cannot be resolved simultaneously, and 2) the user must carefully set the voltage in real-time or the desired data may not be detected. The complexity of managing dynamic range with control settings contributes greatly to the steep learning curve for novice flow cytometrists, as they must learn the “art” of setting voltage and amplification gains.

Electronics are the nerve system of a flow cytometer. They tie the other systems together and can make the instrument either complex or simple to operate. The Accuri team focused on building electronics that would facilitate use of the instrument, with increased dynamic range as a key objective. They utilized state-of-the-art 24 bit ADC chips and advanced electronic design, including sophisticated electronic filtering. The result was an instrument with 6 full decades of dynamic range, a first in flow cytometry history.

The C6 Flow Cytometer simultaneously collects 16 million channels, displayed as 6 full decades, of digital data. This means it has the ability to look at both faint and bright signals simultaneously and with great resolution. This unparalleled dynamic range obviates the need for adjustable voltage settings on the photomultiplier tube detectors. Eliminating the need for voltage control speeds data collection, and, for the first time, makes it possible to separate data collection from data analysis. Data can be collected and analyzed later, without concern over lost data due to improper PMT settings. The learning curve is greatly reduced because a common source of user error, the “art” of setting voltage and amp gains, has been eliminated.

As part of the effort to simplify the electronics system, the Accuri team redesigned the instrument/computer interface. The communication pathways in conventional flow cytometers are typically proprietary, requiring a dedicated interface card installed in the computer that operates the cytometer. In contrast, the C6 Flow Cytometer is USB Plug-n-Play. This approach is user friendly, familiar, and helps reduce complexity, cost and size.

The sophisticated C6 Flow Cytometer electronics system:

  1. Provides six full decades of dynamic range,
  2. Obviates the need to learn the “art” of adjusting voltage and gain settings,
  3. Is USB Plug-n-Play on a standard PC or laptop, and
  4. Uses fewer components, contributing to a small footprint and lower cost.

Innovation #4: Intuitive software

Most flow cytometry software is difficult to navigate and time consuming to learn, especially the software that drives the instrument. The complexity of the user interface is a common complaint among current users and limits the growth of the technology. Manufacturers offer training classes to compensate for the complexity of their software design, but classes do little to reduce the barriers to entry and can overwhelm users, especially novices.

Accuri listed ease-of-use as a top priority from the start of product development. Because the user interface is critical to the user’s experience CFlow software was designed from the ground up to be intuitive and easy-to-use. To achieve this goal, Accuri partnered with Menlo Innovations (, a well-recognized software development firm with an established track record in developing user-friendly software. Menlo Innovations employs “anthropological” methods, which include extensive observation and interview of users by design specialists to ensure a user-focused design process and a user-friendly interface.

The user interface was built under the direction of one of Accuri’s life scientists, in consultation with many outside researchers with a wide variety of experiences in flow cytometry. Features such as the tabbed architecture and “just where you need it” utility placement greatly speed learning and facilitate use. As a result CFlow software is intuitive to the user. User testing shows that new users become fluent with CFlow in less than 30 minutes assisted only by a simple, pictorial three-page Quick Start Guide.

The CFlow interface.

CFlow plots.

The intuitive CFlow software:

  1. Can be used on any standard PC or laptop with 2 GB of RAM,
  2. Is intuitive and does not require training classes,
  3. Meets the needs of both novice and proficient users,
  4. Eliminates layers of complexity, decreasing the learning curve,
  5. Uses the FCS 3.0 file format, making it compatible with other software, and
  6. Allows drag and drop of plot images to MS Office and copy and paste statistics to spreadsheet programs.

The result: A powerful, affordable, easy to use, and compact flow cytometer

The C6 Flow Cytometer System offers an entirely new vision for the role of flow cytometry in the life sciences. Designed from the ground up to be compact and easy to use, the high capability C6 Flow Cytometer can fit in any lab for ready access to all researchers, experts and novices alike.

Manufactured in the USA and selling for an affordable price, Accuri’s product meets the company’s goal of making flow cytometers accessible to a wider range of life scientists. The C6 Flow Cytometer System gives life scientists access to a reasonably priced, powerful tool that they can quickly and easily use to accelerate their research goals.

1: The C6 and optional CSampler. The added automation allows you to prepare your samples and then analyze them in the same plate, eliminating manual transfers from plate to tubes. It’s compatible with both regular and deep-well 96-well plates, as well as 12 x 75 mm tubes.
2. C6 instrument dimensions.

3: A reverse view that shows the back of the C6 and how easily it connects to the fluid tanks.


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