Improving signal-to-noise ratio in flow cytometer optics: Key techniques

Flow cytometer SNR

This article will review flow cytometry to help readers understand the challenges faced when increasing flow cytometer SNR (signal-to-noise ratio). 

Quick review: What is flow cytometry?

Flow cytometry is an optical technique often used to reveal details about cells. These cells are tagged with fluorescent compounds and subsequently suspended in a liquid that flows through an ‘interrogation point’. Hydrodynamic focusing forces them to pass through in a ‘single file’ configuration, or one after the other.

During this stage, excitation light sources (usually one or more laser lines) douse the sample with light. The lasers may be configured in one of two ways: collinear (where the cells are exposed to several lasers at one time) or parallel (where laser lines are separated and each cell is processed one at a time).

A detector then collects the emission from the excited fluorophores in addition to the side and forward scatter, which is then converted to a photocurrent and recorded by the system.

Parallel and collinear configurations in flow cytometer optics

Parallel and collinear configurations in flow cytometer optics. Image Credit: Avantier Inc.

Forward scatter detection occurs when the lasers are directly pointed at a detector. In contrast, side scatter detection involves using a detector configured in a side placement, and the lenses are positioned to route the side-scattered light to the detector.

Understanding signal-to-noise ratio in flow cytometer optics

When improving the signal-to-noise ratio (SNR), the focus is not solely on enhancing fluorescence signals from tagged cells in a standard flow cytometer setup. The objective is to increase the significant signal relative to the amount of random errors, reduce background noise, and emphasize relevant data. The signal-to-noise ratio results from almost every part of the flow cytometry assembly, as well as from experiment apparatus, sampling techniques, and staining methods.

Key optical components that influence the SNR include:

  • Detectors (Photomultiplier tubes or photodiodes)
  • Dichroic Mirrors
  • Lasers
  • Lenses
  • Optical filters (short pass filters, long pass filters, bandpass filters)

Improve Flow Cytometer SNR involves looking at each of the optical components

Improve Flow Cytometer SNR involves looking at each of the optical components. Image Credit: Avantier Inc.

Lasers and flow cytometer SNR

When the flow cytometry configuration is being set up, a laser's appropriate wavelength specifications and power should first be determined. However, a few factors are typically overlooked. These factors can make a huge difference in the SNR.

Output power stability: How high are the time-dependent variations in the laser output power? Two values determine the strength of the laser power or potential lack thereof: peak-to-peak (which reveals sudden, high spikes or deep dips) and root mean square, or rms (which indicates an average of variations).

Both numbers can be regarded as noise readings, and reducing them is crucial for improving the SNR. For example, when measuring blood cells, ensuring that the RMS noise is below 0.1 is essential. Smaller particles will necessitate even lower noise levels, but a higher RMS noise is acceptable if you are studying phytoplankton because the signal will also be stronger.

Laser beam quality: The ability of the laser light to properly focus on a small spot is another key parameter that demands close attention. This is measured in terms of M2, where an optimal, diffraction-limited beam has an M2 of 1. When working with a laser with M2=3, the laser focus spot will be 2x greater in size than the spot of a perfect laser. Moreover, it will have a non-Gaussian intensity profile and likely create random hot spots. These issues increase and lower the signal power density, which can be tricky to navigate. 

Filters, mirrors, and lenses

A chain is only as strong as its weakest link, and even the most superior of lasers with optimal output power stability and laser beam quality will not come close to producing high SNR if subpar optics are being used.

Therefore, it is crucial that the bandpass filters, mirrors, and lenses used in a flow cytometry workflow are manufactured to the highest standards, and just as importantly, the right ones should be used. One key parameter is the range of wavelengths used for the filters. 

For example, suppose an application requires counting excitation from FITC and DY-505, with excitation maximums at 495 and 503 and emission maximums at 519 and 530; a laser beam with specific wavelengths of 500 nm would be able to excite both. Two bandpass filters with 525 x 25 nm could be placed in front of the two detectors, but this would produce inaccurate data and require double counting. Therefore, a 510 x 10nm bandpass filter and another at 532 x 10 nm would be the better choice. 

About Avantier Inc.

Avantier Inc. is an unparalleled leader in providing imaging systems solutions and optical solutions.

They offer advanced precision custom optical design, optical engineering, optical lens assembly, rapid optical proto-typing, image processing and manufacturing services.

Avantier is ISO 9001:2015, ISO 13485:2016, and ISO 14001:2015 certified.


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Last updated: Jun 21, 2024 at 2:44 AM

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