Written by Tim Bushnell, Ph.D.
The importance of the optical system of your flow cytometer was established in Part I of this series, noting in particular the benefits of foundational knowledge to gain a comprehensive view of your data, as well as troubleshooting throughout your experiment.
In Part I, the role of lasers was broken down. This follow-up article investigates the lenses, mirrors, and filters in your flow cytometer.
To review these three elements…
- Lasers illuminate the stream with coherent, focused light of specific wavelength (energy) and power. This illumination facilitates the generation of fluorescence signals from cells labeled with fluorophores and light scatter signal from redirected laser light.
- Lenses focus laser light and collect light scatter and fluorescence optical signal and direct this signal to the optical detection path.
- Mirrors are responsible for directing light through the detection path and partitioning it so that fluorescence and scattered light are directed to the appropriate detectors.
- Filters, placed in front of detectors, function to restrict the light that is introduced to the PMT detectors so that each detector can be dedicated to measure fluorescence from a specific set of fluorophores.
As the lasers interact with particles and cells at the observation point or the interrogation point, scattered and fluorescence light is generated. In order to measure this light, the cytometer needs to collect as much of it as possible. This is the job of lenses.
Howard Shapiro phrases this duty nicely when he says “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 other regions a very small distance away.”
In other words, good lenses allow us to collect the light we’re interested in (scattered and fluorescence light) while avoiding irrelevant light (e.g. errant laser light).
The optical collection system of a cytometer must accomplish two goals. First, it must gather as much light as possible from the interrogation point. Second, it must collimate that light so that all rays propagate parallel to each other and can travel through the collection path without diverging. The lenses on a cytometer are designed to do these two things and do them well.
The collection lens system, which usually consist of multiple lenses, is placed directly in front of the interrogation point. Collimating lenses may be positioned some distance away from the collection lens, depending on the optical design of the cytometer.
The collection lens dedicated to the detection of both fluorescence and side scatter signal is typically positioned 90° relative to the angle at which the laser beam interacts with the stream.
Forward scatter signal, on the other hand, is collected at 180° relative to the angle that the laser hits the stream (the FSC path is in front of the laser, looking at it straight-on).
Figure 4 below illustrates a generalized configuration of the optics at the interrogation point, as seen from above, looking down at the cytometer.
One prominent feature of the forward scatter detection system is the obscuration bar. This device prevents laser light from hitting the forward scatter detector. Because of its position, the forward scatter collection paths “look” directly at the laser beam. If there wasn’t any device that blocked non-scattered laser light, any relevant forward scatter signal that found its way to the FSC detector would be entirely drowned out by laser light.
The obscuration bar is a horizontal piece of metal that blocks laser light but allows scattered light to pass over it and into the detector.
Figure 5 below shows how the forward scatter obscuration bar interacts with laser light.
Many cytometers use optical fibers to direct collected light to the fluorescence and side scatter detection system. In these types of systems, the output of the collection lens is focused on the ends of fibers, which are routed to the detection path. This can be very advantageous in overall cytometer design.
Detection paths can be integrated in spaces in the instrument that they would not be able to otherwise thanks to the flexible path that fibers offer.
In some systems, the lens and the fibers are directly coupled using optical gel which may minimize light loss due to refraction.
As light passes through different types of mediums (water, quartz, and air), it bends at the media interfaces. The degree to which this occurs depends on the difference in refractive index between the two mediums: the greater the difference, the more refraction occurs. By coupling the lens, which is typically glass or quartz, to material with a similar refractive index, like gel, there may be less loss as light transitions between the mediums.
The downside of gels is that they can crack and uncouple the lens from the fibers, which will prevent most collected light from entering the fibers and require a service engineer to repair.
Some cytometers use optical fibers to deliver lasers to the interrogation point. This strategy also provides a space-saving benefit in terms of where the lasers can be positions in the instrument. However, a downside to this approach is that there can be significant power loss between the laser output and the interrogation point as laser light travels through the fiber.
Additionally, fibers are not compatible with higher energy light, especially UV wavelengths, which can degrade the material of the fiber over time and require frequent replacement.
Once light has been collected and collimated from the interrogation point, it must be partitioned by wavelength so that each detector can be dedicated to the measurement of a specific spectral band.
Again, Shapiro phrases this very elegantly: “Optical filters (and mirrors) provide spectral resolution, allowing discrimination between scattered, fluorescent, and background light.”
Mirrors generally direct and partition light through the detection path while filters are placed directly in front of each detector to ultimately determine the band or wavelength range of light that interacts with that detector.
Dichroic mirrors are pieces of glass that are coated on one side with a material that allows light above or below a certain wavelength to pass through while reflecting the rest. Placed at 45° relative to the direction of incident or oncoming light, dichroic mirrors come in longpass and shortpass flavors. A 600 LP (longpass) mirror, for example, reflects light shorter than 600 nm while allowing light longer than 600 nm to pass through. A 600 SP (shortpass) would do the opposite.
The activity of a dichroic mirror is best illustrated using a graph of percent transmission (how much gets through) as a function of wavelength.
Figure 6 below is a transmission graph from the product information of a 590 LP mirror manufactured by Chroma Technologies, one of the primary manufacturers of optical filters and mirrors used in flow cytometry.
At wavelengths below 600 nm, the transmission of light drops off precipitously using this 590 LP mirror. At 590 nm, transmission is 50% and continues to drop quickly. All of the non-transmitted light is reflected.
Dichroic mirrors are positioned in the optical detection path so that the coated surface faces the incident beam of light. You may be wondering what the effect would be if the mirror were installed backwards, so that the uncoated side were facing the direction of oncoming light. Interestingly, it probably wouldn’t have much effect at all.
The light that passes through the mirror would not be affected. However, the reflected light may bend slightly by the time it is reflected. If the filter is installed “backwards”, the incident light would travel through glass twice — once to reach the coating and once after it is reflected from the coating —which may result in some refraction.
In practice, this usually has little impact on fluorescence measurements. Certainly, installing dichroics backwards isn’t recommended but it is interesting that the effect of doing so is not as severe as it may seem.
Filters are pieces of glass coated on both sides that allow light of a certain collection, or band, of wavelengths to pass through while absorbing or interfering with photons of other wavelengths.
These come in bandpass, longpass, and shortpass flavors. Bandpass filters are the ones that are most commonly used in flow cytometry. Positioned in front of the detectors, these components determine what collection of wavelengths, and ultimately which fluorophores, will be measured by each detector. Bandpass filters are named based on the center and width of the band of light that will pass through.
For example, a 525/50 filter allows light to pass that is of a range of wavelengths of 500-550 nm (525 +/- 50 nm). Note that the entire band’s width is 50 nm — the range is not 525+/-50 nm but is 525+/-25 nm (25 nm on either side of the center wavelength).
Figure 7 below illustrates the transmission curve, from Chroma Technologies, of a 525/50 bandpass filter.
As shown, the transmission of this filter drops almost asymptotically at 500 nm and at 550 nm. This particular filter, given its transmission band, is ideal for measuring fluorescence of FITC, GFP, or any other fluorophore with similar emission spectra.
Finally, Figure 8 integrates both dichroic mirrors and bandpass filters to illustrate how they cooperate in a detection path. The arrows represent the direction of light as it passes through the path.
One final comment about filters. While they are, for the most part, very good at letting relevant light pass and keeping out irrelevant light, there are certain circumstances when the wrong kind of light — especially laser light — can sneak past the guards and sabotage detection.
This is most typically a problem in the “PE” channel measured off the 561 nm laser.
This channel’s bandpass’ center is usually centered around 575-590 nm and its longer (wavelength) edge can be precariously close to 561 nm. There is some variability in filters as well, that result in laser light leakage.
Finally, filters are only able to block up to a certain point. If enough light — say, high-powered laser light — is directed on them, a certain proportion of that light will pass through. The effect of all these extraneous photons can be severe.
Excessive background light in a detector can cause a drastic loss in sensitivity. If measuring 8-peak beads under conditions of high optical background, you will see both the dim peaks much higher on the scale than they would be otherwise, as well as merging of peaks.
Knowing this can be useful for troubleshooting. If you are having trouble resolving a population in a channel, especially one close to a laser line, it may be worth investigating a laser light leakage issue into that channel. 8-peak beads can be a useful first-line diagnostic tool in this regard.
This article outlined some of the major components of the optical systems used in flow cytometry. While it is certainly possible to explore these topics in much more depth, what is presented here should provide enough insight to understand what happens before a signal is produced from the PMT detectors. Additionally, this article may also equip you with a knowledge toolkit that can help troubleshoot problems you may encounter when performing your next cytometry experiment.
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My other passions include grilling, wine tasting, and real food. To be honest, my biggest passion is flow cytometry, which is something that Carol and I share. My personal mission is to make flow cytometry education accessible, relevant, and fun. I’ve had a long history in the field starting all the way back in graduate school.