What Is A Flow Cytometry Laser And How Flow Cytomtery Optics Function

The optical system of a flow cytometer consists of an elegant coordination of many components that function concordantly and synchronously to generate the signals that we need to measure in order to shed light on the biology at hand.

Understanding the optical system of a flow cytometer may seem unnecessary for performing a typical experiment, but the more you know about your instrument, the better you will be at understanding nuanced aspects of your data, as well as troubleshooting any potential issues that may arise during an experiment.

A cytometer’s optical system can be broken down into two major parts: A) lasers, and B) lenses, mirrors, and filters.

Lasers and some lenses comprise the excitation optics that generate optical signal, while other lenses, mirrors, and filters form the emission optics which collect optical signal. A brief description of the roles of each of these components is listed below, followed by more detailed descriptions.

Part I of this article will focus on lasers. Look for Part II for the remaining discussion on lenses, mirrors and filters.

• 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.

Before diving deeper into the optical components, it is worth discussing some fundamental concepts about electromagnetic radiation, or light.  While we typically think of light as something that is visible, the electromagnetic spectrum spans a very large range, of which visible light is only a small portion.

Figure 1 below from NASA illustrates the limited range occupied by visible light in the entire spectrum.

Electromagnetic radiation is characterized by its wavelength. Wavelength is inversely proportional to energy, so the longer the wavelength of light, the lower the energy.

Electromagnetic radiation with very short wavelengths, like gamma (~10 picometers) or X-rays (0.01 – 10 nanometers), has very high energy — high enough to break covalent bonds and wreak havoc on biological systems. Those with longer wavelengths, like microwaves (1 mm – 1 m) and radio waves (whose wavelengths can be in the kilometer range), are lower in energy.

Flow cytometry is primarily concerned with the visible spectrum, which occupies a portion of the spectrum in about the middle, with wavelengths of about 380 – 700 nanometers or so.

The spectral range that is utilized in flow cytometry is actually a bit wider than the true visible spectrum, typically between ~350 nm to ~800 nm. The wavelength of visible light determines its “color”. Ultraviolet light, the highest energy light used in flow cytometry of wavelengths below about 400 nm, is not visible. The “cytometric spectrum” can be very roughly conceptualized as follows:

• ultraviolet light occupies the mid-to-high 300 nm range
• violet light occupies the low 400 nm range of the spectrum
• blue light occupies the mid-to-high 400 nm range
• green light occupies the low 500 nm range
• yellow light occupies the mid 500 range
• orange light occupies the high 500 nm range
• red light occupies the range above about 600 nm
• light above ~700 nm is not visible.

When it comes to your flow cytometer’s lasers, there are 4 factors that you should understand. These 4 factors are…

1. Coherence.

In order to measure fluorescence from labeled cells, a light source is necessary to produce this fluorescence. Light can be generated in several ways, but the most effective way for the conditions and configurations of flow cytometry is by utilizing the laser.

Lasers, whose name is actually an acronym (Light Amplification by Stimulated Emission of Radiation), are especially suited for flow cytometry for two primary reasons…

First, laser light is coherent. Second, laser output is of a very narrow energy range — the wavelength of light can be specified with high precision.

Coherence is the best thing about lasers as far as flow cytometry is concerned. In technical language, this means that all of the light that is emitted by the laser, according to Shapiro, is “in phase with and propagating in the same direction.”

In practical terms, this means that all of the photonic power of a laser can be directed and focused onto a very small spot. Unlike microscopy, particles flowing through a cytometer spend a very short amount of time in the illumination spot. Given a stream velocity of 20 meters per second, a beam spot of 20 micrometers, and a 15 micrometer cell, the cell is illuminated for only 0.015 microseconds. That’s not a whole lot of time.

To maximize the likelihood that sufficient fluorescence events are produced by a labeled cell, and in order to best measure that fluorescence, it is necessary to bombard that cell with as many photons as possible.

The coherence property of lasers ensures that the photon density at the illumination point is high enough to allow us to precisely measure the fluorescence necessary to glean useful biological information.

In contrast, other kinds of light sources, such as arc lamps or LEDs, which are commonly used in fluorescence microscopy, are not coherent. Their light output travels in all directions from the origin, and specialized optics are required to gather this light and direct it onto a measurement point.

Another benefit of lasers is that they can be designed to produce light of a very narrow spectral, or wavelength, range.

Unlike a typical fluorescent or arc lamp, which output white light, or a wide spectral band of photons, laser output can be tuned, depending on the construction and materials of the laser, to produce a particular color, or wavelength, of light. Laser lines commonly used in flow cytometry are: 355 nm, 375 nm, 405 nm, 488 nm, 530 nm, 561 nm, and 640 nm.

2. Spontaneous and stimulated emissions.

The way that lasers work is interesting. The laser consists of material, called the lasing medium, typically through which electrical energy is pumped. This causes electrons in the medium to be excited, or transition to higher energy states. When the electrons fall back to lower energy states, a photon is generated.

The emission of these initial photons results from spontaneous emission — they are not in-phase or polarized and are not necessarily of the same energy (wavelength), as reported by Shapiro. However, the incredible thing, which Einstein showed, is that when electrons of a molecule or atom are excited to a higher energy state, the presence of a photon nearby with a particular energy will increase the probability that the excited molecule will EMIT a photon with the same energy.

This is called, in contrast to spontaneous emission, stimulated emission, and is the logic behind the word “stimulated” in the acronym “laser”.

In other words, photons have a “mob mentality.” If one if doing something (i.e. propagating in a certain direction with a certain wavelength), other photons like to follow-suit and do the same.

By equipping cytometers with multiple lasers, each outputting a specific wavelength, a spatially-separated system can be constructed in which fluorophores are illuminated and excited at distinct points on the stream.

In this kind of system, each laser is focused on its own spot, or interrogation point, on the stream, so only fluorophores with excitation spectra in the range of the laser’s wavelength will be excited at each interrogation point.

By constructing a system like this, it is possible to simultaneously measure and differentiate fluorophores which have very similar emission spectra but different excitation like, for example, PE-Cy7 and APC-Cy7.

Both of these fluorophores emit photons at the same wavelength (Cy7’s emission spectrum, whose maximum is in the high 700 nm range). However, PE-Cy7’s excitation spectrum is largely restricted to the 488 nm or 561 nm laser lines, while APC-Cy7 excitation spectrum is largely restricted to the 640 nm laser.

Spatially-separated systems can differentiate between these two fluorophores because each beam spot, or interrogation point, is associated with its own dedicated collection path. In other words, fluorescence signal from 561 nm excitation is routed exclusively into the 561 nm collection path, while fluorescence signal from the 640 nm collection path is routed exclusively into its own, separate path.

The simultaneous detection of both of these fluorophores would not be possible if PE-Cy7 and APC-Cy7 were excited at the same point in time and space.

This is only possible when the illumination source of a single interrogation point consists of a very narrow range of wavelengths.

3. Colinear systems.

In colinear systems, on the other hand, multiple lasers are focused on the same spot. This can be a cost-effective and convenient way to accommodate two excitation lines when two separate beam spots (interrogation points) are not practical or possible.

However, these systems carry the caveat of the inability to simultaneously measure two fluorophores with very similar emission spectra, as described above.

For example, it is very challenging to measure and distinguish Brilliant Violet 786 from APC-Cy7 simultaneously using a 405-640 nm collinear system. Both Brilliant Violet 786 and APC-Cy7 fluorescence over largely the same range of wavelengths but excite at very different wavelengths (~405 nm for Brilliant Violet 786 and ~640 for APC-Cy7).

Since both of these dyes will be excited at the interrogation point in a collinear system and then be collected into the same optical path, both will be measured by the same PMT. In contrast, Brilliant Violet 786 and APC-Cy7 can be measured simultaneously on a spatially-separated system.

4. Lasing mediums.

There are a few different kinds of lasers, with respect to the lasing medium, and while a detailed discussion of this aspect is beyond the scope of this article, most lasers in current cytometers are solid-state lasers.

In these kinds of lasers, the lasing medium is a solid, as opposed to a gas or plasma. Lasers these days are much smaller, have lower power requirements, and do not require the amount of warm up time that they used to in the past.

The evolution of laser design is one of the reasons that cytometers and sorters have gotten so much smaller in the last decade. The era of the “benchtop” cytometer has in large part been facilitated by the development of smaller lasers without sacrificing output power.

In a flow cytometer, lasers must be shaped by the excitation optics before they reach the interrogation point and interact with cells. This shape is typically elliptical which results in a Gaussian energy profile. In other words, the photons are most “dense” in the middle of the beam and taper off towards the edges.

Figure 2 below illustrates this property of the elliptical laser shape.

Because energy is densest in the center of beam, cells must flow through this portion of the spot in order to generate most fluorescence. When flow rates are high, the core stream through which cells flow widens, which results in more variation in cells’ positions in the beam.

Cells located towards the edge of the beam will be exposed to fewer photons and generate less fluorescence, while cells that flow through the center of beam will be exposed to more photons and generate more fluorescence.

Figure 3 below illustrates how flow rate can affect cells’ positions in the laser beam.

Finally, another helpful property of lasers is that their output, or power (in milliwatts), can also be specified. Laser power, which is essentially a measurement of how many photons are output per unit time, is usually adjustable on a cytometer but sometimes not.

Typical powers range from 20 mW to 100 mW, although some cytometers are equipped with very high-powered and considerably dangerous lasers of up to several hundred mW. Although not always apparent in practice, the more power the better (when it comes to lasers).

The more photons that a fluorophore sees, the higher the chance that that fluorophore will fluoresce and the higher the chance that a useful biological measurement can be made.

Regarding power and safety, with the exception of UV lasers (~355 nm), lasers on cytometers in the typical range of powers are not terribly dangerous, as long as care is taken to not look directly into the beam. That being said, leave laser alignment to your service engineers unless you’ve had formal laser safety training. Any laser can be dangerous in the right context.

For further reading, check out the following: Shapiro, H.M. Practical Flow Cytometry. New York: John Wiley & Sons, 2005.

Part I of a look into your cytometer’s optics focused on the dynamics of the lasers and their application in flow cytometry. Understanding the coherence property of lasers and how they impact fluorescence, along with principals of emissions, use of colinear systems and lansing mediums helps you understand the intricacy of the equipment you’re using while providing you with the opportunity to troubleshoot during your experiments.

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