SCYM Principles of Flow Cytometry & Instrumentation 2 — Questions and Answers
Question 1: What is the primary function of the sheath fluid in a flow cytometer?
- To lyse red blood cells before analysis
- To hydrodynamically focus cells into a single-file stream (Correct answer)
- To stain cells with fluorescent dyes
- To cool the laser during operation
Correct answer: To hydrodynamically focus cells into a single-file stream
Sheath fluid surrounds the sample core and uses laminar flow to hydrodynamically focus cells into a narrow, single-file stream through the interrogation point.
Question 2: Which optical component separates emitted light into different wavelength bands for simultaneous detection in flow cytometry?
- Neutral density filter
- Dichroic mirror (Correct answer)
- Photomultiplier tube
- Collimating lens
Correct answer: Dichroic mirror
Dichroic mirrors (beam splitters) reflect certain wavelengths while transmitting others, routing light to appropriate detectors for multicolor analysis.
Question 3: In flow cytometry, what does the term 'doublet discrimination' refer to?
- Detecting two different fluorescent labels on the same cell
- Identifying and excluding two cells passing through the laser simultaneously (Correct answer)
- Measuring two scatter parameters at once
- Gating on two populations with similar scatter properties
Correct answer: Identifying and excluding two cells passing through the laser simultaneously
Doublet discrimination uses pulse width or pulse area vs. pulse height plots to identify and exclude events where two cells pass through the laser at the same time.
Question 4: What is the consequence of increasing the flow rate in a flow cytometer beyond the optimal range?
- Increased fluorescence signal intensity per cell
- Decreased coefficient of variation (CV) for peak measurements
- Reduced resolution due to cells passing the laser too quickly (Correct answer)
- Longer acquisition time for the same number of events
Correct answer: Reduced resolution due to cells passing the laser too quickly
Excessive flow rate causes cells to spend less time in the laser beam, reducing photon collection time and broadening peaks, which increases CV and reduces resolution.
Question 5: Which laser line is most commonly used to excite fluorescein isothiocyanate (FITC)?
- 355 nm UV laser
- 405 nm violet laser
- 488 nm blue laser (Correct answer)
- 633 nm red laser
Correct answer: 488 nm blue laser
FITC has a peak excitation at approximately 490 nm, making the 488 nm blue argon laser the optimal and most widely used excitation source for this dye.
Question 6: What is the purpose of a bandpass (BP) filter in a flow cytometer's optical system?
- To block all light below a specific wavelength
- To transmit only light within a defined wavelength range (Correct answer)
- To split the laser beam into two equal beams
- To amplify fluorescent signals before detection
Correct answer: To transmit only light within a defined wavelength range
A bandpass filter transmits only a specific range of wavelengths (e.g., 530/30 passes light from 515–545 nm) while blocking wavelengths outside that range.
Question 7: In a flow cytometer, what does a photomultiplier tube (PMT) measure?
- The speed of cells passing through the flow cell
- The size of the cell nucleus directly
- The intensity of light emitted or scattered by cells (Correct answer)
- The electrical impedance of individual cells
Correct answer: The intensity of light emitted or scattered by cells
PMTs convert photons of light (from fluorescence emission or scatter) into an amplified electrical signal proportional to light intensity.
SCYM Principles of Flow Cytometry & Instrumentation 2
What is the primary function of the sheath fluid in a flow cytometer?