Principles of Flow Cytometry & Instrumentation Flashcards

What is the primary function of the sheath fluid in a flow cytometer?

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.

Which optical component separates emitted light into different wavelength bands for simultaneous detection in flow cytometry?

Answer: Dichroic mirror

Dichroic mirrors (beam splitters) reflect certain wavelengths while transmitting others, routing light to appropriate detectors for multicolor analysis.

In flow cytometry, what does the term 'doublet discrimination' refer to?

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.

What is the consequence of increasing the flow rate in a flow cytometer beyond the optimal range?

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.

Which laser line is most commonly used to excite fluorescein isothiocyanate (FITC)?

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.

What is the purpose of a bandpass (BP) filter in a flow cytometer's optical system?

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.

In a flow cytometer, what does a photomultiplier tube (PMT) measure?

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.