SCYM Principles of Flow Cytometry & Instrumentation

Question 1

What is the main principle behind flow cytometry?

Accepted Answer

Light scattering and fluorescence

Answer

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.

Question 2

What role does the laser play in flow cytometry?

Accepted Answer

Excites fluorophores for detection

Answer

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.

Question 3

Which component directs cells into single file?

Accepted Answer

Hydrodynamic focusing

Answer

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.

Question 4

What is forward scatter (FSC) an indicator of?

Accepted Answer

Cell size

Answer

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.

Question 5

What does side scatter (SSC) measure?

Accepted Answer

Cell complexity/granularity

Answer

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.

(SCYM) Specialist in Cytometry Practice Test

(SCYM) Specialist in Cytometry Practice Test

SCYM Principles of Flow Cytometry & Instrumentation