Fluorescence refers to the phenomenon of photoluminescence. When a certain room temperature substance is irradiated by incident light of a certain wavelength, after absorbing the light energy, the electrons in the molecule reach a high energy level, enter the excited state, and immediately de-excite and return to the original state, and the excess energy is The form of light that radiates out, i.e. emits light with a longer wavelength than the incident light (usually in the visible wavelength band). Once the incident light is stopped, the luminescence phenomenon disappears immediately. That is, when an object absorbs the energy of short-wave light, it can emit light of longer wavelengths than it originally absorbed. Substances or molecules with this property are called fluorescein or fluorescent dyes.
When the laser beam is orthogonal to the cell, two fluorescent signals are generally generated. One is that the cell itself emits a weak fluorescent signal under laser irradiation, which is called cell autofluorescence; Quantitative analysis provides insight into the presence and quantification of the cellular parameter under study.
(1) Significance of the fluorescent signal
Fluorescent signals can reflect the biological properties of different cells. For example, monoclonal antibodies labeled with fluorescent dyes specifically bind to antigens, receptors or membrane glycoproteins on the cell surface, and emit fluorescence of a certain wavelength under the excitation of laser light. The intensity of the fluorescent signal reflects the relative quantity of membrane antigens, receptors or glycoproteins. By collecting and analyzing the fluorescent signals, the analysis of cell subsets and functions can be realized. The DNA is stained with DNA dyes, and the dyes are combined with the DNA chains in a certain way. Under the excitation of the laser, the dyes emit fluorescence, and by measuring the intensity of the fluorescence, the relative DNA content can be obtained, and then the cell cycle phase can be determined. analyze. The use of specific fluorescent dyes can also be used to measure cellular calcium ion concentration, intracellular pH, and cell membrane potential.
(2) Selection of fluorescent dyes
There are a variety of fluorescent dyes that can be used, and because of their different molecular structures, their fluorescence excitation and emission spectra are also different. When choosing the fluorescein labeled with the dye or monoclonal antibody, the wavelength of the laser light source configured by the instrument, that is, the excitation spectrum of the dye, must be considered; the excitation light wavelength of the instrument laser is as close as possible to the excitation spectrum peak of the fluorescent dye; in addition, the fluorescent dye must also be considered The emission color of the dye, that is, the emission spectrum of the dye, needs to be selected by a detector with a suitable wavelength band to detect the corresponding fluorescence signal.
Since the current flow cytometer is equipped with multiple fluorescence signal detectors, when the instrument detects multiple fluorescence simultaneously, each fluorescence detector only allows a fluorescence signal of a specified wavelength to enter and be detected, so the user must select Appropriate fluorescent signal receivers can receive the best signal. It is also necessary to pay attention to the use of fluorescent dyes with the same wavelength of laser light, and their emission wavelengths are different, so that they can be received by the detector of the corresponding wavelength band to achieve the purpose of simultaneous detection.
The choice of detector is based on knowing the emission spectrum of the fluorochrome. Taking three-color FCM as an example, if the fluorescence spectrum peak falls in the green range (wavelength 515-545nm), the first fluorescence detector is selected; if the spectral value falls in the orange-red range (564-606nm), the second fluorescence detector is selected; If the fluorescence spectral values fall in the deep red range (wavelength 650 nm), a third fluorescence detector is selected. At present, the laser that is often configured in desktop FCM is 488nm, and the commonly available dyes are PI, PE, FITC, PERCP, CY5, etc.
(3) Fluorescent-labeled antibody combination
When performing multicolor analysis by flow cytometry, if you want to get ideal analysis results, you need to choose the fluorescence matching of the antibody. Factors that are often considered are the following.
1. Fluorescence intensity of fluorescein
The ability of a particular antibody to differentiate between negative and positive results depends on which fluorescein the antibody is labeled with. Each fluorescein has different photon release ability and different relative fluorescence intensity. Generally, the staining index is used to compare the light signal intensity of different fluorescent labels. The staining index is the ratio of the difference between the positive signal and the negative signal to the width of the negative peak distribution, and it is used to judge the ability of the fluorescent dye to distinguish weakly positive expression. The figure below shows that the same monoclonal antibody was labeled with 8 different fluorescein, and different staining results were obtained. We need to find a fluorescent dye with a higher staining index to label the weak signal.
It can be seen that for a specific monoclonal antibody, due to the use of different fluorescein labels, the S/N ratio (signal-to-noise ratio) of negative nuclei-positive cells can differ by 4-6 times. Generally speaking, the order of fluorescence signal from strong to weak is: PE>APC>PE-CY5>PERCP-CY5.5>FITC>PERCP.
When selecting fluorescently labeled antibodies, the following factors need to be considered:
Fluorescein Labeling Efficiency: The amount of fluorescein labeled on the antibody (F/P) value also affects the relative fluorescence intensity. Each antibody can be labeled with several FITC or PERCP molecules (usually 2-9), while the amount of APC and PE labeled is about one fluorescent molecule per antibody. FITC is a small molecule compound, while PE, PERCP and APC are fluorescent proteins with larger molecular weight. Limited by the chemical properties of fluorescent labels, IGM-type antibodies are usually only labeled with small molecules of fluorescein, such as FITC, TEXAS RED, CY3 and CY5.
Antigen density for antibody detection: Highly expressed antigens can be detected with almost any fluorescein-labeled antibody, while lower-expressed antibodies need to be detected with fluorescein-labeled antibodies with a higher S/N ratio, so as to effectively distinguish positive cells Population and Negative Cell Purpose.
Cellular Autofluorescence: The level of autofluorescence varies for each cell population, although cells with high fluorescence intensity can be observed, but autofluorescence decreases rapidly in the high wavelength range (>600nm). When detecting cells with high levels of autofluorescence, better S/N ratios can be obtained by using fluorescent dyes with longer emission wavelengths such as APC. If it is a cell with a low level of autofluorescence, then the use of excitation light with a longer wavelength has little effect on the phenomenon of improving the difference between positive and negative. FITC-labeled antibodies can be used.
2. Nonspecific binding
Some fluorescently labeled antibodies exhibit low levels of nonspecific binding, resulting in increased fluorescence levels in negative cells. This nonspecific binding is usually caused by two factors:
Isotype controls for monoclonal antibodies: some IGG-type isotype controls are more susceptible to FC receptor binding by certain cell types
Fluorescein used: Sometimes CY3, CY5, CY5.5, and Texas Red directly labeled antibodies, and some tandom-conjugated antibodies, have enhanced binding to certain cell subsets. For CY5, studies have shown that this is mainly due to the weak interaction of the dye with low-affinity FC receptors; PE-CY5-labeled antibodies have a similar effect. In addition, in some cases (such as the analysis of monocytes with anti-HLA-DG PE/anti-monocyte PerCP-CY5.5), this feature can also be used to intentionally increase the amount of CY dye in the labeled antibody, This ensures that monocytes can be detected regardless of the difference in the level of CD14 expression of each monocyte.
In conclusion, fluorescent antibody-labeled fluorescent dyes should be carefully selected when performing multicolor analysis by flow cytometry. The main influencing factors are: selection of appropriate fluorescent antibodies (laser power and wavelength) according to different models; relative density of antigen expression of stained cells (brighter fluorescent dyes should be selected for antigens with low expression density); FC on negative cells receptor status. In addition, when doing multi-color analysis, it is necessary to select fluorescent dyes with less spectral overlap between fluorescence waves as possible for combination, and at the same time, correct adjustment and compensation are required.
3. Adjustment of Fluorescence Compensation
When cells carry two or more fluoresceins (such as PE and FITC) and are excited by laser to emit more than two different wavelengths of fluorescence, theoretically, the filter can be selected so that each fluorescence can only be detected by the corresponding detector, while Another fluorescence will not be detected. However, since the various fluorescent dyes currently used have broad emission spectrum properties, although their respective emission peaks are different, the emission spectrum ranges overlap to a certain extent, so a small amount of another fluorescent signal that does not need to be detected will also be detected. It is detected by this photomultiplier tube, so each photomultiplier tube actually detects the sum of two kinds of fluorescence, but each is dominated by a certain kind of fluorescence. As can be seen from the figure, the emission spectrum of fluorescein has a certain range, and a very small part of its emission signal is bound to enter another detection. The shade in the figure is the range of the detector’s detection spectrum. The FITC detector will detect a small amount of PE spectrum, while the PE spectrum detector will detect more FITC spectrum.
Since the spectral overlap accounts for a certain proportion of the detected signal, the method of fluorescence compensation is to subtract a part of the received signal (that is, the spectral overlap) in the other detector from the received fluorescence signal, so that the other A detector signal The signal detected in this detector is consistent with the negative background signal, so that the signal output in the photomultiplier tube really only represents the signal designated for detection, without the interference of the fluorescent signal of another wavelength. Using a standard known single-positive sample, the compensation value of the fluorescence signal can be reasonably set. The degree of compensation can be determined by the instrumental conditions under which the dual fluorescence parameters are measured simultaneously. When compensating, first measure the fluorescence (FL1) of a dye. At this time, in addition to the signal output of the photomultiplier tube that should receive the fluorescence, such as the FL1 detector, the other photomultiplier tube FL2 detector also often has a weak output. Adjust the compensation Then, measure another dye (FL2), and adjust the compensator FL1-%FL2 to make the average fluorescence intensity output by the FL1 detector match the negative signal. Negative signals are consistent. Such repeated adjustment, the FL1 and FL2 detectors can be fully compensated.
When doing multi-color analysis, each fluorescein single-positive sample must be used for detection, and the compensation with other fluorescence channels must be adjusted to ensure the accuracy of multi-color analysis results. Due to the complexity of fluorescence compensation for multicolor analysis, many analysis software allow offline compensation, which provides great convenience for the operator.
It is important to note that compensation is related to the specific fluorescein combination for a specific experiment, specific instrument setting. After the compensation is set, if the voltage of the PMT, the wavelength of the laser, the filter system, etc. changes, the fluorescence compensation value will change, and the compensation needs to be re-adjusted.