Detecting and measuring the chemical and physical characteristics of cell populations or particles is an important part of life sciences research. This article will discuss the spectral flow cytometer, an instrument that researchers use to carry out this task in laboratories worldwide.
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Flow cytometry – an overview
Flow cytometry is a technique that analyses thousands of cells rapidly, with the data processed by a computer. Samples of cells contained in a fluid medium that has been labeled with fluorescent markers are injected into the flow cytometer. Each individual cell is illuminated by a laser, causing the markers to fluoresce. The scattered light is then recorded and analyzed.
There are a number of uses for flow cytometry in research (the technique is used in clinical trials, clinical practice, and routine research.) These include:
- Cell sorting
- Cell counting
- Biomarker detection
- Diagnosis of health disorders
- Detecting microorganisms
- Determining the function and characteristics of cells.
There are many different types of flow cytometry used in research. Spectral flow cytometry differs from traditional flow cytometry in terms of instrumentation and capability, though it is still based on the same fundamental principles.
Spectral flow cytometry
A traditional flow cytometer uses photomultiplier tubes to detect emitted photons after they have been directed through mirrors by the instrument’s optics. Single fluorochromes are detected by the equipment. In spectral flow cytometry, conversely, the emission spectra of all fluorescent molecules present in the sample are detected by multiple detectors or channels.
The fluorescent spectrum of each molecule is recognized and recorded. The spectra are separated using a process called spectral unmixing to provide in-depth analysis of the data and provide information on the cell populations themselves.
Principles of the spectral flow cytometer
The ability to measure multiple fluorescent spectra makes a spectral flow cytometer more flexible than a traditional flow cytometer. The equipment has capabilities that far exceed conventional flow cytometry methods.
The core functions that the instrument must have include:
- The means to spatially separate light based on wavelength.
- A multichannel detection system that simultaneously measures every fluorescent signal at different wavelengths individually.
- Powerful enough data processing power to perform real-time spectral unmixing.
They are capable of multiparametric analysis of cell population samples at the single-cell level. This is enabled by using either hydrodynamic focusing or a microfluidics chip. Single cells are aligned in a flow chamber or cell prior to being passed through a laser. Cellular properties and the fluorescence characteristics present are determined by the excitation of individual events by the lasers used.
In the spectral flow cytometer, a spectrograph and a multichannel detector (usually a charge-coupled device or CCD) are used in place of the traditional optics, mirrors, and photomultiplier tubes of a conventional flow cytometer. By replacing the optical designs of conventional flow cytometry with light dispersion elements such as prisms and diffraction gratings, spectral detection is made possible. Each dispersion element disperses light in a different manner.
Advantages of spectral flow cytometers over conventional flow cytometers
One of the main problems with traditional flow cytometry is its limitations in multiparameter analysis. The more parameters involved in the detection, the more complex the hardware needs to be. Detectors, optical filters, and other components need to be added to the instruments. For a 30-parameter instrument, there need to be 32 independent detectors and 50-60+ additional optical components. And this is just the detection part of the equipment.
This increases the cost of the equipment almost to prohibitive levels – a 50 parameter flow cytometer can cost around $1 million. Add to this the fact that the equipment is relatively outdated, and it can be seen that traditional flow cytometry is not necessarily advantageous for current biological research.
One of the main advantages of a spectral flow cytometer, therefore, is the ability to provide multiparameter analysis of individual cells at a fraction of the cost and complexity of conventional flow cytometers. This is advantageous, especially if a project has budget concerns.
Another advantage of spectral flow cytometry is the ability to measure the whole fluorescence spectrum for each individual fluorophore in the sample. This allows for improved resolution of overlapping fluorophores based on the spectral shape which would otherwise not be possible with conventional flow cytometry. Additionally, cellular autofluorescence can be used as a parameter, which is not possible otherwise.
In a conventional flow cytometer, there is an issue with fluorescence spillover from nearby fluorochromes. To minimize this and resolve multiple signals, much of the emission spectrum is disregarded. This means that a huge amount of potentially useful data is not captured by the spectrometer. Spectral flow cytometers get past this issue due to their ability to capture, resolve, and analyze the entire emission spectrum.
Spectral flow cytometry is a relatively new technology. Though there are some teething problems with this cytometry technique, it is distinctly advantageous compared to conventional flow cytometry, a field that is around 50 years old. Spectral flow cytometry is an exciting field that will no doubt contribute to future developments in many fields of biological research.