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Flow Cytometers

How Flow Cytometers Work & How We Save You Time & Money

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Flow cytometry diagram

Flow cytometry is a widely used method for measuring cellular properties as they move through a fluid stream past an optical system comprising an excitation light source (laser), lenses, mirrors, and lastly, filters, which are placed in front of detectors.

Biotech diagram

The cellular characteristics measured include the expression of cell surface and intracellular molecules, various cell types in heterogeneous cell populations, purity of isolated subpopulations, and single-cell size and volume. The resulting emitted photons are collected and become the data that identifies characteristics about the cells in question. It is a powerful technique commonly used in the life sciences.

This technique is also often used for high-throughput measurement of antigens on live cells. There is no better option in this case, as the technique utilizes fluorescent markers on antibodies or other proteins in order to quantifiably detect changes in protein expression via excitation of the various fluorescent markers. Antibodies allow scientists to detect a specific antigen, making them useful tools for protein characterization on the surface of live cells.

The antibodies can be directly labeled with a fluorescent marker—such as FITC (fluorescein isothiocyanate)—or can be visualized by binding to a secondary antibody that is labeled. This allows flow cytometry to analyze or sort cells based on more than one color, each representing a different antigen that is bound by a different antibody.

Over the past 30 years, single-cell flow cytometry analysis has become an indispensable tool for many scientific researchers and clinicians working in the life sciences performing data analysis and, more specifically, cell analysis.

The evolving technology ensures its application in a number of fields, including cell biology, drug discovery, cancer research, neuroscience, stem cell research, pathology, immunology, hematology, plant biology, food science, and marine biology.

FCM Components, Data Visualization, Sample Handling, & More

An open hand with a DNA strand coming out of the palm

Flow cytometers, sometimes referred to as cell sorters (depending on whether cell analysis or cell sorting is being performed), are made up of three main components that are integral to collecting accurate flow cytometry data.

The fluidics system transports particles in a sample stream—with the help of flow cells—to the interrogation point and takes away the waste. Flow cells are sample cells designed to help liquid samples continuously flow through the beam path. This is especially useful for any samples that can be easily damaged by the excitation light source.

The optics system consists of a series of optical filters and mirrors to direct light of specific wavelengths emitted by cells or particles towards the detectors, amplifying the signal produce by single cells.

The electronics system then converts the signals from the detectors into electronic signals that can be processed by the computer. For some instruments equipped with a sorting feature, the electronics system is also capable of initiating sorting decisions to charge and deflect cells or particles. This is often referred to as fluorescence-activated cell sorting, or FACS.

The Fluidics System

The fluidics system within the analyzer aligns and transports particles in a fluid stream through the laser beam’s path for identification. When a sample in a solution is injected into the instrument, the individual cells are randomly distributed in a three-dimensional space. For accurate data collection, it is important that each specific cell passes through the laser beam on its own, with each cell passing through single file.

Most flow cytometers accomplish this by injecting the sample stream into a stream of faster flowing sheath fluid within the flow chamber, along with additional flow cells. The flow of the sheath fluid accelerates the particles and restricts them to the center of the sample core so that a single stream of particles is created. This process is known as hydrodynamic focusing.

The Optics System

After hydrodynamic focusing, each particle passes through one or more beams of light. The excitation by laser causes the fluorescent reagents bound to cells or particles to emit light, which is detected according to their specific wavelength range. Light scattering, through a forward scatter channel (FSC) or fluorescence emission provides information about the particle’s properties, and cell granularity or internal structural complexity is provided via a side scatter channel (SSC).

When using a combination of fluorescent dyes, reagents should have distinct emission spectra. For example, MACSQuant instruments will use various dichroic mirrors and optical filters to direct light of specific wavelengths to the according fluorescence detectors. This arrangement creates “fluorescence channels.” Various filters are used to control which wavelengths enter the fluorescence channels. These filters are referred to as long-pass, short-pass, and band-pass according to their optical properties.

  • Long-pass: blocks light up to a certain wavelength.
  • Short-pass: transmits light up to a certain wavelength.
  • Band-pass: transmits light within a defined wavelength range.

Fluorescent measurements taken at different wavelengths can provide quantitative and qualitative data about fluorochrome-labeled cell surface receptors or intracellular molecules, such as DNA and cytokines. The argon-ion laser is most commonly used in flow cytometry. This is because the 488 nm light that it emits excites more than one fluorochrome, while the laser and the arc lamp are the most popular light sources in modern flow cytometry.

The Electronics System

Once a cell or particle passes through the laser light, emitted side scatter and fluorescence signals are diverted to the photomultiplier tubes (PMTs) and a silicon photodiode collects the forward scatter signals.

All of the signals are routed to their photodetectors via a system of mirrors and optical filters. Optical filters control the specificity of detection by blocking certain wavelengths and transmitting others.

Visualizing Data

To visualize the data collected, values of each measured optical parameter are plotted in various ways. The plots used can be univariate or bivariate. In the case of flow cytometry, univariate plots are represented as histograms while bivariate plots are represented as dot plots.

Univariate plots (histograms) visualize one optical parameter, such as fluorescence or light scattering, against event number. For each event, each measured parameter’s signal intensity values are stored in a data file and, as the events accumulate, a curve is generated to illustrate the distribution of events according to their individual signal intensity.

Bivariate plots (dot plots), on the other hand, allow for the simultaneous visualization of two optical parameters on a single graph. As more events are plotted, ones that share similar intensity values accumulate in clusters. These clusters can often represent specific cell populations.

Sample Handling

Most cell sorter instrumentation can utilize sample volumes ranging from several microliters to hundreds of microliters. If you have a small volume of precious cells, consider using a microfluidics-based flow cytometry chip.

The development of microfluidic, lab-on-a-chip (LOC) technologies is one of the most innovative and cost-effective approaches toward the advancement of cytometry.

Automation

Automated sample introduction into the analyzer allows for walk-away cell viability assays to be performed, among other analytical experiments. The addition of automation also allows for a larger numbers of samples to be analyzed, using a variety of formats, from single test tubes to multi-well plates.

Single tubes are often contained within rectangular grid arrangements or carousel formats to match laboratory equipment used for sample preparation.

Immunophenotyping

Immunophenotyping is just one of the many uses of flow cytometry, although it may be the most common. It is used in the identification of markers on cells, particularly the immune system, and allows researchers and scientists to study the protein expressed by cells.

Cell identification is based on the markers or antigens present on a cell’s surface, nuclease, or cytoplasm, and helps identify the lineage of cells using antibodies that detect the markers on each cell (thus the “immuno-” prefix).

Immunophenotyping can be as simple as identifying a cell using a single marker or as complex as applying homing profile, activation states and cytokine release all in one panel. Because of this, experimental protocols often combine surface and intracellular staining. Immunphenotyping is routinely used in basic research as well as clinical applications to diagnose disease or to monitor and evaluate residual disease.

Some examples of immonphenotyping include differentiating between acute myeloid and lymphoid leukemia, as well as B and T cell lymphoid neoplasms, which includes chronic lymphocytic leukemia and lymphoma. This method is also used for reactive and neoplastic expansions of lymphocytes, predicting prognosis in lymphoma, and in the identification of lymphocyte subsets.

We Lease a Variety of FACS Machine Brands & Models

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Founder-Friendly Leases

Our lease agreements are founder-friendly and flexible, helping you preserve working capital, strengthen the cash flow of your business, and keep business credit lines open for expansions, staffing, and other crucial operational expenses and business development opportunities.

2-5 Year Lease Lengths

Leases range from 2 to 5 years. Length will depend on several factors, including how long you want to use the equipment, equipment type, and your company’s financial position. These are standard factors leasing companies consider and help us tailor a lease agreement to fit your needs.

Your Choice of Manufacturer

We don’t carry an inventory. This means you’re not limited to a specific set of manufacturers. Instead, you can pick the equipment that aligns with your business goals and preferences. We’ll work with the manufacturer of your choice to get the equipment in your facility as quickly as possible.

Maintenance & Repair Coverage

Bundle preventive maintenance and repair coverage with your lease agreement. You can spread those payments over time. Easily maintain your equipment, minimize the chances something will break down, repair instrumentation quickly, and simplify your payment processes.

End-of-Lease Options

At the end of your lease, you have multiple options. You can either renew the lease at a significantly lower price, purchase the machine outright based on the fair market value of the original pricing, or call it a day and we’ll come the pick up the equipment for you free of charge.

No Loan-Like Terms

Our leases do not include loan-like terms, which can be restrictive or harmful in certain situations. We do not require debt covenants, IP pledges, collateral,  or equity participation. Our goal is to maximize your flexibility. When you lease with us, you’re collaborating with a true business partner.

In-House Underwriting Process

Our underwriting is done in-house. You can expect quicker turnaround, allowing you respond to your equipment needs as they arise. We require less documentation than traditional lenders and financiers and can get the equipment you need in operation more quickly.

Popular Flow Cytometer Manufacturers

Agilent
BD Biosciences
Beckman Coulter
Bio-Rad
Cytek
Cytonome
Milliporesigma
Miltenyi Biotec
Sartorius
Sony
Sysmex
Thermo Fisher Scientific