How Lenses, Mirrors, and Filters Work In Your Flow ...

Written by Tim Bushnell, PhD

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This article explores the essential roles of lenses, mirrors, and filters in your flow cytometer.

1. Lenses

Lenses play a crucial role in the flow cytometer's optical system. When lasers interact with particles or cells at the interrogation point, they generate scattered and fluorescent light. To effectively capture this light, lenses are employed to maximize collection efficiency.

As Howard Shapiro aptly remarks, "The lenses provide spatial resolution, enabling us to collect a great deal of light coming from a very small region of space (i.e., the interrogation point) and relatively little of the light coming from nearby regions."

This ability to differentiate desirable light from irrelevant sources, like stray laser light, underscores the importance of high-quality lenses. The optical system of a cytometer has two fundamental objectives: gather as much light as possible from the interrogation point and collimate that light into parallel rays for efficient transmission.

Typically, a collection lens system, composed of multiple lenses, is strategically positioned near the interrogation point, with collimating lenses placed further away, tailored to the cytometer’s optical design.

To collect both fluorescence and side scatter signals, the collection lens is often set at a 90° angle to the laser beam's interaction path, while forward scatter signals are gathered directly at 180°.

This arrangement is further enhanced by the use of an obscuration bar, which prevents laser light from interfering with the forward scatter detector, ensuring that collected signals remain distinguishable.

In many cases, optical fibers are utilized to direct the collected light to the detection system, allowing integration of detection paths into the cytometer’s design while minimizing light loss.

2. Mirrors

Following the collection of light, mirrors are employed to partition this light by wavelength, enabling each detector to target specific spectral bands for measurement.

As expressed by Shapiro, "Optical filters (and mirrors) provide spectral resolution, allowing discrimination between scattered, fluorescent, and background light." Mirrors act as directors of light, while filters determine which wavelengths interact with the detectors.

Dichroic mirrors, specific types of mirrors, reflect certain wavelengths while allowing others to pass through. By placing these mirrors at a 45° angle to incoming light, they can effectively segregate data based on wavelength.

3. Filters

Filters are essential components that permit light of specific wavelengths to pass while absorbing others. Generally available as bandpass, longpass, and shortpass types, bandpass filters are predominant in flow cytometry, positioned before detectors to define the spectrum of measurable wavelengths.

For instance, a 525/50 bandpass filter allows the passage of light between 500-550 nm and is particularly effective for detecting common fluorophores such as FITC or GFP.

While filters work effectively to isolate relevant light, some circumstances may lead to interference from unwanted laser light, particularly in channels closely associated with laser wavelengths.

Excess background light can significantly diminish sensitivity in collected data, leading to the merging of peaks and indistinct measurements, indicating the necessity of troubleshooting potential laser light leakage.

This overview provides insight into the optical components vital for flow cytometry and equips you for addressing challenges in future cytometry experiments.

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