Read this article to learn about the principles of fluorescence.
Fluorochromes and Light:
Fluorochromes are essentially dyes, which accept light energy (e.g., from a laser) at a given wavelength and re-emit it at a longer wavelength. These two processes are called excitation and emission.
The process of emission follows extremely rapidly, commonly in the order of nanoseconds, and is known as fluorescence. Before considering the different types of fluorochrome available for flow cytometry, it is necessary to understand the principles of light absorbance and emission.
Light is a form of electromagnetic energy that travels in waves. These waves have both frequency and length, the latter of which determines the colour of light. The light that can be visualized by the human eye represents a narrow wavelength band (380-700 nm) between ultraviolet (UV) and intra red (1R) radiation (Fig. 15.5).
Sunlight, for example, contains UV and IR light that, although invisible to the eve, can still be felt as warmth on the skin and measured scientifically using photo detectors. The visible spectrum can further be subdivided according to colour, often remembered by the mnemonic ‘ROY G BY’ standing for red, orange, yellow, green, blue and violet. Red light is at the longer wavelength end (lower energy) and violet light at the shorter wavelength end (higher energy).
Stokes Shift:
When light is absorbed by a fluorochrome, its electrons become excited and move from a resting state (1) to a maximal energy level called the ‘excited electronic singlet state’ (2). The amount of energy required will differ for each fluorochrome and is depicted in Fig. 15.6 as Fexcitation.
This slate only lasts tor 1-10 nanoseconds because the fluorochrome undergoes internal conformational change and, in doing so, releases some of the absorbed energy as heat. The electrons subsequently fall to a lower, more stable, energy level called the ‘relaxed electronic singlet state’ (3). As electrons steadily move back from here to their ground state they release the remaining energy (Eemission) as fluorescence (4).
As Eemission contains less energy than was originally put into the fluorochrome it appears as a different colour of light to Eexcitation. Therefore, the emission wave-length of any fluorochrome will always be longer than its excitation wavelength.
The difference between Eexcitation and Eemission is called Stokes Shift and this wavelength value essentially determines how good a fluorochrome is for fluorescence studies. After all, it is imperative that the light produced by emission can be distinguished from the light used for excitation. This difference is easier to detect when fluorescent molecules have a large Stokes Shift.
Maximal Absorbance and Maximal Emission:
The wavelength of excitation is critical to the total photons of light the fluorochrome will absorb. FITC (fluorescein isothiocyanate), for example, will absorb light within the range 400-550 nm but the closer the wavelength is to 490 nm (its peak or maximum), the greater the absorbance is. In turn, the more photons absorbed, the more intense the fluorescence emission will be.
These optimal conditions are termed maximal absorbance and maximal emission wavelengths. Maximal absorbance usually defines the laser spectral line that is used for excitation. In the case of FITC, its maximum tails within the blue spectrum. Therefore, the blue Argon-ion laser is commonly used for this fluorochrome, as it excites at 488 nm, close to FITC’s absorbance peak of 490 nm.
FITC emits fluorescence over the range 475-700 nm peaking at 525 nm, which falls in the green spectrum. If filters are used to screen out all light other than that measured at the maximum via channel A (see Figure 15.8), FITC will appear green. Hence, ‘fluorescence colour’ usually refers to the colour of light a fluorochrome emits at its highest stable excited state.
However, if FITC fluorescence is detected only via channel B (see Fig. 15.8), it will appear orange and be much weaker in intensity. How the flow cytometer is set up to measure fluorescence will ultimately determine the colour of a fluorochrome.
Why Use a Fluorescent Probe?
The purpose of a fluorescent probe, such as a fluorochrome-conjugated antibody, is to directly target an epitope of interest and to allow its biological and biochemical properties to be measured more easily by the flow cytometer.
Fluorescent probes are useful in a wide range of applications including identifying and quantifying distinct populations of cells, cell surface receptors or intracellular organelles; cell sorting; immunophenotyping; calcium influx experiments; determining nucleic acid content; measuring enzyme activity, and for apoptosis studies. By changing the excitation light and using more than one fluorochrome, it is possible to analyze several parameters of the sample at any one time. This forms the basis of multi-colour fluorescence studies.
Which Fluorochromes are Useful for Flow Cytometry?
There are dozens of fluorescent molecules (fluorochromes) with a potential application in flow cytometry. Instead, some of the most useful fluorochromes for surface or intracellular epitope detection are described in table 15.1, including the very latest in fluorescent probe technology—tandem dyes. There is enough variation in the two tables to cover most researchers’ needs.
Single dyes:
Some of these single dyes, e.g., FITC have been in use for the past 30 years but are now facing competition from alternatives like Alexa Fluor® dyes, which offer the user greater photo stability and increased fluorescence.
Tandem dyes:
In a tandem dye, a small fluorochrome takes a ‘piggy-back’ ride on another larger fluorochrome. When the first dye is excited and reaches its maximal singlet state, all its energy transfers to the second dye (an acceptor molecule), located in close proximity. This activates the second fluorochrome, which then produces the fluorescence emission.
The process is called FRET (fluorescence resonance energy transfer). It is a clever way to achieve higher Stokes Shifts and, therefore, increases the number of colours that can be analyzed from a single laser wavelength. The majority of tandem dyes have been manufactured for the standard 488 nm laser, which is found in most flow cytometers.
Tandem dyes are very useful for multicolor fluorescence studies especially in combination with single dyes. For example, Alexa Fluor® 488, Phycoerythrin, PerCP-Cy5.5 and PE-Cy7 can all be excited at 488 nm, but will produce green, yellow, purple and infrared emissions respectively, which can be measured using separate detectors.
Fluorescence Compensation:
One consideration to be aware of when performing multicolor fluorescence studies is the possibility of spectral overlap. When two or more fluorochromes are used during a single experiment there is a chance that their emission profiles will coincide, making measurement of the true fluorescence emitted by each difficult. This can be avoided by using fluorochromes at very different ends of the spectrum, e.g., Alexa Fluor® 405 and Phycoerythrin; however, this is not always practical.
Instead, a process called fluorescence compensation is applied during data analysis, which calculates how much interference (as a %) a fluorochrome will have in a channel that was not assigned specifically to measure it. Figure 8 helps to explain the concept.
The graphs show the emission profiles of two imaginary fluorochromes ‘A’ and ‘B’ which are being detected in FL-1 and FL-2 channels respectively. Because the emission profiles are so close together, a portion of fluorochrome A spill over into FL-2 (red shade) and conversely, some of fluorochrome B reaches FL-1 (dark blue shade).
To calculate how much compensation needs to be applied to the data set if both dyes are used simultaneously, some control readings must first be taken. Fluorochrome A should be run through the flow cytometer on its own and the % of its total emission that is detectable in FL-2 (spillover determined. The procedure should be repeated with fluorochrome B, except that this time FL-1 is spillover.
Suppose the results are:
This means that when the two fluorochromes are used for a dual-colour experiment, the true reading for fluorochrome A in FL-1 = (total fluorescence measured in FL-1) minus (5% of fluorochrome B’s total fluorescence)
Similarly, the true reading for fluorochrome B in FL-2 = (total fluorescence measured in FL-2 minus (17% of fluorochrome A’s total fluorescence) Fortunately, modern flow cytometry analytical software applies fluorescence compensation mathematics automatically, which simplifies matters considerably.
