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Flow cytometer
Flow cytometry is a technique for obtaining various information from cells by measuring the scattered light and fluorescence that are generated when the cells are irradiated with laser light. A instrument specifically designed for flow cytometry is referred to as a flow cytometer.
Flow cytometers rely on fluorescent labeling with specific antibodies to detect markers both inside and outside the cells, This allows quantitative evaluation of cell populations based on multiple parameters such as cell type, active state and cell cycle status, etc.
How a flow cytometer works
A flow cytometer consists of 3 main components: a fluidics, an optics and an electronics. Each plays a different role.
- Fluidics: Consists of tubes and glass cells through which cells and sheath fluid flow, and requires high accuracy both in the cell flow amount and speed.
- Optics: Consists of light sources for excitation of cells, and detectors for detecting fluorescence and scattered light from cells, lenses and filters. Each device must have high stability and sensitivity to achieve highly accurate cell analysis.
- Electronics: Receives electrical signals output from the optical system, processes the data, and analyzes the measurement results.
Basic principle
When a suspension of cells labeled with a fluorescent dye is poured into a thin tube, the cells move through the tube at certain intervals.
The cells are then irradiated with laser beams. The fluorescence emitted from the fluorescent substance and the light scattered by the cells are detected and monitored by the photodetectors.
1. Target cells are fluorescently labeled.
2. Cells are poured into flow path system and move through tube at fixed intervals.
3. Cells are irradiated with laser beams.
4. Light is detected via the optical system.
Technology trend: Multicolor analysis
To obtain even more information that has not been available up to now in flow cytometry, it is necessary to stain cells with multiple types of markers. In measurements that precisely distinguish between various combinations of markers; multicolor analysis utilizing lasers and detectors for different wavelengths plays an active role.
Conventional fluorescence detection measurements are usually limited to the wavelength range where fluorescence intensity is high. However, recent advances in measurement and analysis techniques allow us to obtain fluorescence information across the entire spectrum. This improves the accuracy of multicolor analysis and raises the bar in cell analysis to a more advanced level.
Fluorescence spillover and compensation
During multicolor analysis, since multiple fluorescent dyes are used in the measurement, fluorescence from different wavelengths might overlap among the measurement items, causing interference due to light leaking into channels not originally intended for measurement. The technique to correct this problem is called compensation. To apply compensation, the fluorescence overlap between the target fluorochromes must be predicted and subtracted from the signal. The accuracy of this compensation processing relies to a large extent on the sensitivity characteristics of detectors in the flow cytometer.
Wavelength spectra in conventional flow cytometry
Conventional flow cytometry cannot measure as many items at one time compared to multicolor analysis, but there is less overlap between wavelength spectra, which makes the measurement simpler to perform.
Wavelength spectra in multicolor analysis
Multicolor analysis requires complex and exacting measurements due to the large number of items to measure at one time, which leads to overlaps between measurement spectra.
Supporting multicolor analysis
Multicolor analysis in flow cytometry now requires versatile detectors with higher performance.
To obtain a large volume of accurate data, the detection wavelength range must be extended to the near-infrared region This means that the detectors need to have higher sensitivity in the near infrared region.
To meet these demands, Hamamatsu is working hard to improve the performance of its detectors used in multicolor analysis.
As one example, we are constantly improving the performance of photomultiplier tube modules utilizing crystalline materials in the photocathode and have achieved both higher sensitivity and extended near-infrared sensitivity compared to conventional photomultiplier tubes utilizing alkali materials in the photocathode.
We offer a lineup of photomultiplier tubes having multiple detection channels in a single unit and also provide easy-to-use modules with functions well-suited for multicolor analysis.
Photomultiplier tube quantum efficiency comparison
Related Products
Devices for flow path system
These are glass cells designed to install into flow path systems. Their high dimensional accuracy contributes to precise control of flow speed and flow rate.
This power supply provides the required voltage to cell sorters. Its highly regulated voltage output contributes to highly accurate cell sorting.
Devices for optical system
We provide photomultiplier tube modules with higher sensitivity extending to the near-infrared region to meet needs stemming from recent advances in multicolor analysis.
Users can select from various models such as those with a thermoelectric cooler according to their particular application.
APD modules are high sensitivity, fast response photodetector modules with a built-in Si APD (silicon avalanche photodiode). APD features internal signal multiplication (about several hundred times) called avalanche multiplication and so is ideal for light measurements requiring high sensitivity and fast response.
MPPC modules are photon counting modules with built-in MPPCs. They incorporate an MPPC, I/V converter, and power supply, making them easy to use.
The MPPC features high gain, high photon detection efficiency, and wide dynamic range, so it can obtain high S/N characteristics even for high-speed, weak signals.
We provide TDI cameras with features optimized for imaging flow cytometry. Flexible customization and options are also available.
Photodiodes that can detect everything from ultraviolet light to visible light with extremely high quantum efficiency. CAN package, surface mount type, etc. are available.
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