Figure 1 : Simplified principle of a flow cytometer

To operate a flow cytometer requires a combination of:

Fluidic: To introduce and focus the cells.

Optics: A source of excitation and signal recovery,

Electronics: To convert the optical signals into proportional electronic signals and digitize them for analysis with a computer.

The cells must be suspended in order to be analyzed. The analysis of blood does not pose any problem as the cells are already in suspension. On the other hand, the cellular tissues must be dissociated and the aggregates eliminated in order to be analyzed.

Imagine having to count vehicles of different colors on a highway with many lanes (Figure 2). If the traffic is heavy and all the lanes are in use, it will be impossible to perform this task. To do this, you need to channel the vehicles into one lane and, in this way, you will be able to make an accurate analysis of the number of vehicles of each color.

In a flow cytometer the same procedure is used. The cells are brought to the center of the measuring nozzle and aligned one behind the other (by means of the hydrodynamic sample centering system) to be excited one by one with the light beam. The sheath liquid undergoes a progressive acceleration which causes the sample liquid to stretch and thus aligns the cells in the center of the jet (Figure 3).

Figure  3 : Hydrodynamic Focusing

When the starting sample is low in concentration, the user of a cytometer tends to increase the pressure on the sample in order to decrease the acquisition time. Unfortunately, this has the effect of decreasing the precision of the sample centering and leads to a scattering of the measurements since the cells will not necessarily pass through the focusing zone of the light source (Figure 4). This is easily visualized by passing calibration beads at different pressures, their peak widens as the pressure increases (increase in the coefficient of variation). This effect is particularly detrimental to cell cycle measurements where the sharpness of the peaks is a guarantee of the quality of the measurement. It is therefore preferable to increase the concentration of the sample rather than to increase the pressure on it. Of course, one must also take into account the information processing limits of the device. Similarly, by increasing the speed too much, we can find ourselves in a situation where several particles pass in front of the light source at the same time, compromising the reliability of the measurements.

The light excitation source must allow an illumination of the dyes at a wavelength close to their absorption maximum. It must be powerful, stable and requires a good focusing.
Two types of sources are currently used:
-Lasers (the most frequently used) which have a large number of advantages: power, stability, beam sharpness. Lasers have discontinuous emission spectra (Figure 5).

Figure 5 : main light sources used in flow cytometry

When there are several lasers in a cytometer, they can be arranged in 2 ways. Either they are all focused at the same place, they are then said to be collinear, or they are spatially shifted (the cell passes successively in front of each laser), they are then said to be staggered.
The interest of staggered lasers is that it allows, for fluorochromes with different excitation wavelengths but with equivalent emission wavelengths to be measured independently which is not possible with collinear lasers (Figure 6). Colinear lasers therefore lead to greater constraints in the choice of fluorochromes.

The different optical signals emitted by the cell must be focused, separated, and then routed to detection systems, photomultipliers or photodiodes. They are selected by different optical circuits, composed of alternating mirrors and filters.
A mirror is a reflecting surface. Depending on the treatment of its surface, we can obtain three types of mirrors: high-pass, low-pass and bandpass (Figure 7, 8). Moreover, the reflected wavelengths also vary with the angle formed by the incident ray and the surface of the filter.

Figure 7 : Different types of filters used by cytometry (R: Réflection,  : wavelength)


Figure 8 : Different types of filters used by cytometry

Figure 9 : Example of an optical path in a flow cytometer (©Becton Dickinson)

The scattered light provides information about the morphology and structure of the cell:

If light scattering is measured in the axis of the incident beam, the signal intensity can be correlated with cell size and viability.

At an angle of 90°, the measurement corresponds to the intracellular structure of the cell (cytoplasm refringence, morphology, nucleo-cytoplasmic ratio).

The simultaneous use of these two parameters makes it possible to distinguish, in peripheral blood for example, platelets, lymphocytes, monocytes and polynuclears (Figure 11).

Figure 11 :Use of double light scattering to distinguish the various blood subpopulations (FSC: small angle scattering, SSC: wide angle scattering).

This fluorescence can be spontaneous, but most often, it is brought to the cell by a fluorochrome. The fluorochrome absorbs the energy of the LASER and re-emits the absorbed energy by vibration and heat dissipation, emitting photons of a higher wavelength.
A fluorochrome is characterized by its absorption and emission light spectrum. The re-emitted light is always of a higher wavelength than the absorbed wavelength.
The characteristics of some fluorochromes used in CMF are summarized in Table 2 :

FLUOROCHROME	USE	Excitation Max*	Emission Max@
Hoechst 33342	ADN (A-T)	365	402
DAPI	ADN (A-T)	357	451
Chromomycine A3	ADN (G-C)	450	470
Iodure de Propidium	ADN	536	620
Acridine Orange	ARN/ADN	492	527 db/630 sbrin
Alexa 488	Protein labeling	498	520
FITC	Protein labeling	490	543
PE	Protein labeling	490/565	578
PerCP	Protein labeling	488	675
APC	Protein labeling	642	660
PE-Cy5.5	Protein labeling	490/565	693
PE-Cy7	Protein labeling	490/565	778
Brilliant Violet 510	Protein labeling	405	510
Brilliant Violet 650	Protein labeling	405	650
GFP	Gene Reporter	488	510
mCherry	Gene Reporter	585	610
dTomato	Gene Reporter	554	580
Rhodamine 123	mitochondrial potential	505	534
BCECF-AM	pH	440	530
SNARF-1	pH/traceur	488	580/630
CFSE	cell tracker	498	518
Cascade blue	cell tracker	399	423
Fura Red	Ca2+	450-500	660
Indo1-AM	Ca2+	331	405/480
Fluo-3	Ca2+	506	526

Table 2 : Characteristics of some fluorochromes used in cytometry. (*: excitation wavelenght, @: emission wavelenght).

There are
- Fluorochromes with a specific affinity for a cellular component: for example for the measurement of DNA, RNA, proteins, pH, calcium contained in the cell.
- Fluorochromes coupled to a specific ligand. This specificity can be achieved by coupling the fluorochrome to an antibody or a ligand specific to a cell component.
Be careful because the fluorochromes do not all have the same fluorescence yield and therefore will not have the same detection capabilities. To demonstrate this, it is 'sufficient' to couple the same antibody with different fluorochromes and to compare the intensity of the labelings on the same cells (Figure 12).

The optical signals are converted into electrical signals by the photomultipliers. These electrical signals have a value usually between 0 and 10 volts (analog signal). The computer can only process these data if they are in binary form (digital signal). The role of the analog-to-digital converter is to convert, as its name indicates, an analog signal (continuous value) into a digital signal (discontinuous value) that can be processed by the computer. That is to say, to transform a value between 0 and 10 volts into a binary value between 0 and 1023 for a 10-bit converter (2¹⁰) (Figure 13).The new machines now convert signals on several million channels.

Figure 13: Working principle of a converter

The numerical values from the converters are stored by the computer and presented on the cytometer screens in two forms:
1/monoparametric histograms where the x-axis represents the intensity of the analyzed signal and the y-axis the number of cells, the axes can be linear or logarithmic (Figure 14).


2/biparametric histograms or cytograms presenting two signals simultaneously (Figure 15).

Figure 14 : Presentation of flow cytometry results.