level: Flow Cytometry
Questions and Answers List
Flashcards on case and lecture on Flow Cytometry
level questions: Flow Cytometry
| Question | Answer |
|---|---|
| what is the principle of flow cytometry? and what is forward and side scatter? | flow cytometry is a technique that analyses single cells or particles while they flow past lasers, while they are suspended in a buffer solution. the cells can be analysed for light scatter and fluorescence. light scatter is measured in two different directions: forward scatter (FSC) which indicates the relative size of the cell and the side scatter (SSC) which is the scatter at 90 degrees and indicates the internal complexity or granularity of the cell. |
| what are the different components of the flow cytometer and what are their functions? | fluidics: directing the liquid containing the cells to the light source. optics: focuses the light source on the cells and transmitting the scatter or fluorescence to the electronics. electronics: detecting the signal and converting it into digital data. |
| what are the characteristics of the fluidics component of the flow cytometer? | the fluidics are needed to direct the liquid containg the cells to the light source. it consists of the sheath fluid, which flows next to the sample fluid and makes sure that the sample cells flow in a single stream (because the sample pressure is bigger than the sheath fluid pressure). when the flow rate is high, the analysis is done faster, but this risks that the cells clump together and that you do not measure one cell at a time. high flow rates are used for qualitative measurements such as immunophenotyping. low flow rates are used for applications that need higher resolutions ( e.g. DNA content analysis). |
| what are characteristics of the optics component of the flow cytometer? | the optics focuses the light on the cells and transmits the scatter or fluorescence to the electrics. lenses are used to shape and focus the laser beam, which produces energized electrons in high energy states. when these electrons fall back, photons are produced and these photons deflect around the cell. the optics include a series of filters/dichroic mirrors that transmit part of the light, depending on the type of filter; - bandpass filters transmit a specific range of wavelengths. - short pass filters transmit wavelengths that are equal to or shorter than a specific wavelength. - long pass filters transmit wavelengths that are equal to or longer than a specific wavelength. |
| what are the characteristics of the electronics component of the flow cytometer? | the electronics detect the signal and convert it into digital data, which is analysed by the computer system. the light signals are converted to voltages by photodetectors, which are usually either; - photodiodes (PDs), which detect stronger light signals that are generated by FSC, they are very efficient. - photomultiplier tubes (PMTs), which detect weaker signals that are generated by SSC and fluorescence, because they are more sensitive. |
| what data is generated after flow cytometry? | a voltage over time curve is generated. the area of the curve is an indicator of the signal that is generated. the height of the curve is an indicator of the intensity of the signal and the width of the curve tells you something about the time it takes for the cell to pass through the laser (bigger width, more time). the data can be plotted linearly or logarithmically. the linear scale is often used when you analyse FSC and SSC and the log scale is used to analyse fluorescent scatter. when the data are log transformed, the resolution is increased and different peaks can be detected more easily. data can be presented in a histogram, dot plots (each dot represents a cell), contour plots and density plots (higher densities will have a different colour). |
| what is gating? | gating is the sequential identification and refinement of a subpopulation of cells, which is done by using a panel of molecules (markers) that are visualised by fluorescence; it is the selective identification of subpopulations of cells, done manually. a gate is a numerical or graphical boundary that contains only a specific subset of cells; e.g. cells with only the CD14+ surface marker. |
| what are different types of gating? | 1. flow stability gating: elimination of cells that are affected by clogging or other instrument-related issues. increases sensitivty of the experiment. 2. pulse geometry gating: elimation of doublet cells/clumped cells (as indicated by their disproportionate width). 3. forward and side scatter gating: identifiy the cells based on their size (FSC) and complexity (SSC) and remove any debris or cells that are not of interest (often cells in the bottom left of the graph). 4. subsetting gating: elimation of many unwanted cells, maintaining only cells that express the marker of interest. viability dyes are often used to eliminate dead cells. 5. backgating: done at the end of gating analysis, it allows for the visualisation of cells in a certain gate at a higher level and it determines whether any cells are being missed by previous gating strategies. |
| how does fluorescence work in flow cytometry? | when a fluorophore is coupled to the cells of interest, it absorbs the light from the laser. this light is emitted through emission of photons at a higher wavelength than the incoming wavelength (this is because some energy is lost to heat and vibration). the detector is placed in such a way that it detects the emission wavelength at its emission peak. the most widely used fluorophores for labelling antibodies are FITC, phycoerythrin (PE) and allophycocyanin (APC). when mutliple fluorophores are used, there will be some spectral overlap (overlap between the emission spectra). this can be corrected for by substrating a fraction of one fluorophore from another, called compensation. |
| what are applications of flow cytometry? | - immunophenotyping: simultaneously analysing mixed populations of immune cells for multiple parameters, makes use of fluorophore-conjugated antibodies that target specific surface markers on immune cells. - proliferation analysis: cells are stained with proliferation dye. when cells proliferate, the dye will be subdued. when cells do not proliferate, cells will remain bright with dye. - apoptosis: stain cells with dye that can only enter when cell membrane integrity is lost and thereby indicate apoptotic cells. - intracellular cytokine production: stain the cells with antibody-coupled fluorophore and permeabilize the cells so that antibodies can bind to intracellular cytokines. - FASC: cell sorting by giving cells in droplets different charges and attracking (or repelling) the droplets with deflection plates and thereby sorting them in different tubes. |
| what are pros and cons of flow cytometry? | pros: simple sample preparation, can analyse heterogenous subpopulations of cells and tissues, fast, detailed and large amount of data generated, accurate. cons: overwhelming amount of information, more expensive than alternatives (ELISA), necessary analytical measurements have to be done (e.g. correcting for fluorscence spillover, compensation), experience needed. |
| what is spectral flow cytometry? | spectral flow cytometry allows you to not only measure the emission peak of the fluorophore that is used, but the entire spectrum. this allows you to differentiate between fluorophores that have similar emission peaks. with this technique, a spectral signature can be created for each fluorophore (with the wavelength on the "x-axis" and the intensity on the "y-axis"). you can use spectral unmixing to separate the different spectral signatures of the fluorophores that you use. because of this you can use overlapping fluorophores and substract the autofluorescence signals. |