Principles of Flow Cytometry & Instrumentation Flashcards

What is the main principle behind flow cytometry?

Answer: Light scattering and fluorescence

Flow cytometry is a technique used to analyze the physical and chemical characteristics of cells or particles in a fluid stream. Its main principle relies on illuminating individual cells with a laser beam and detecting two key signals: light scattering (indicating size and granularity) and fluorescence. Fluorescence is emitted by fluorochrome-labeled components, allowing for the identification and quantification of specific cellular markers.

What role does the laser play in flow cytometry?

Answer: Excites fluorophores for detection

In flow cytometry, the laser serves as the light source that illuminates cells as they pass through the interrogation point. Its primary role is to excite fluorophores, which are fluorescent dyes or proteins attached to specific cellular components. When excited by the laser, these fluorophores emit light at specific wavelengths, which is then detected and used to identify and quantify specific cellular markers.

Which component directs cells into single file?

Answer: Hydrodynamic focusing

Hydrodynamic focusing is a critical component in flow cytometry that ensures cells pass through the laser beam one at a time. A sheath fluid surrounds the sample stream, creating a narrow core where cells are aligned into a single file. This precise alignment is essential for accurate and consistent measurement of each individual cell's light scattering and fluorescence properties, preventing multiple cells from being analyzed simultaneously.

What is forward scatter (FSC) an indicator of?

Answer: Cell size

Forward scatter (FSC) in flow cytometry measures the amount of light that is scattered in the forward direction, along the path of the laser beam. This signal is primarily proportional to the cell's surface area and volume. Therefore, FSC is an excellent indicator of relative cell size, with larger cells typically producing higher forward scatter signals.

What does side scatter (SSC) measure?

Answer: Cell complexity/granularity

Side scatter (SSC) in flow cytometry measures the amount of light that is scattered at a 90-degree angle from the laser beam. This signal is primarily indicative of the internal complexity or granularity of a cell. Cells with more internal structures, such as granules or a complex nucleus, will produce higher side scatter signals, allowing for differentiation based on internal cellular characteristics.

Which detectors are used to measure fluorescence?

Answer: Photomultiplier tubes

Photomultiplier tubes (PMTs) are highly sensitive detectors used in flow cytometry to measure the weak fluorescent light emitted by excited fluorophores. PMTs convert photons into electrical signals, which are then amplified and processed to quantify the intensity of fluorescence. This allows for the detection and differentiation of various cell populations based on their specific fluorescent markers, providing crucial data for analysis.

What is the function of optical filters?

Answer: Isolate light wavelengths

Optical filters are essential components in flow cytometers, designed to transmit specific wavelengths of light while blocking others. This function allows the instrument to isolate the distinct fluorescent signals emitted by different dyes. By precisely separating these light wavelengths, filters ensure that each detector measures only the intended fluorochrome's emission, enabling accurate multi-color analysis.

Why is fluidics important in flow cytometry?

Answer: Aligns cells in flow

Fluidics is crucial in flow cytometry because it creates a hydrodynamic focusing effect, aligning cells into a single file. This process uses sheath fluid to narrow the sample stream, ensuring that each cell passes individually through the laser interrogation point. This precise alignment is fundamental for accurate single-cell analysis, preventing multiple cells from being measured simultaneously and ensuring reliable data acquisition.

Which type of light is typically used in cytometry lasers?

Answer: Visible spectrum lasers (e.g., blue, red)

Flow cytometers primarily utilize lasers that emit light in the visible spectrum, such as blue (488 nm) and red (633 nm) lasers, though violet and UV lasers are also common. These specific wavelengths are chosen because they efficiently excite the fluorochromes commonly used to label cells. Upon excitation, these fluorochromes emit fluorescent light, which is then detected to characterize the cells.