Flow cytometry, a powerful technique in cell biology and medical diagnostics, intricately combines fluidics, optics, and electronics to unpack the complexity of cellular composition.
In this article, we delve into the optics of flow cytometry, exploring the critical role played by optical devices in the precise counting and comparison of cells.
Unpacking a Flow Cytometer
Flow cytometer technology can be broken down into a few basic sections:
- Fluidics: the flow of cell material through the instrument
- Optics: provide the equipment necessary to count/compare cells and provide samples
- Electronics: in charge of processing the samples and returning data for analysis
The place where they all meet is called the interrogation point. The interrogation point is composed of one or more lasers and 2 or more photodetectors.Forward Scatter vs. Side Scatter.
Forward Scatter vs. Side Scatter
A photodetector on the opposite end of the laser will measure forward scatter (FSC), while a photodetector perpendicular to the laser measures what is called side scatter (SSC).
In most flow cytometers, forward scatter measures the size of a cell by the amount of light scattered when a cell passes through the beam.
Side scatter measures the internal complexity of a cell as light is refracted by the cell at different wavelengths, and thus scattered to the side. Side Scatter can also detect cells marked with fluorescent markers.
[RELATED READING: HOW FLOW CYTOMETRY TECHNOLOGY IS USED TODAY]
Flow Cytometer Design Variations
There are several designs for flow cytometers that arrange the interrogation point according to the instrument’s intended use. Some are simple, composed of a single light source and two sensors, one for forward scatter and one for side scatter.
More complex designs exist that use multiple light sources and measure a wider spectrum of wavelengths for side scatter.
More complex designs can often be broken down into two main variations: co-linear and parallel. Co-linear designs divide SSC signals using dichroic mirrors in a linear arrangement. Parallel designs place multiple laser diodes and SSC sensors in line with each other, making more than one interrogation point.
About These Measurements
The data collected by these scatter patterns sort the cells later in the process.
For example, lymphocytes will have a different FSC and SSC profile compared to granulocytes or monocytes. If a high lymphocyte count is discovered, it could indicate an infection, cancer, or other health condition that should be tended to immediately.
As such, it is important for the optics and electronics to be in precise alignment so that an accurate cell count is achieved. A false positive or false negative is something nobody wants.
Choose MISUMI for Precision and Accuracy
Specialized MISUMI products are used to ensure precise alignment of optical and electronic components within flow cytometer equipment. MISUMI offers the following products:
Smart Laser Autocollimators, prisms, and mirrors are integral components that contribute to the precision and reliability of these advanced instruments.
