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AMM > FACS > Flashcards

FACS Flashcards

1
Q

What is flow cytometry?

A

A laser-based lab test that can detect chemical and physical differences of cells or particles.

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2
Q

What is FACS?

A

Fluorescence Activated Cell Sorting

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3
Q

What are the two things FACS allows us to do with a sample?

A

Collect data on and sort our sample.

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4
Q

What are the three core systems of the Flow Cytometer

A
  1. Fluidics
  2. Optics
  3. Electronics
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5
Q

How are cells/particles carried to the laser intercept/light beam?

A

The fluidics system
Sheath fluids carry the cells/particles in a single file fashion.

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6
Q

What is the function of the fluidics system?

A

Fluidics system is responsible for transporting sample from the sample tube to the flow cell (and past the laser and detector).

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7
Q

What is the function of hydrodynamic focussing in the fluidics system?

A

Hydrodynamic focussing uses water to force the cells to travel at the same speed and to travel as single cells.

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8
Q

What makes up the fluidics system?

A

Flow cell
Sheath fluid
Droplet generator
Nozzle

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9
Q

What is the optics system of flow cytometry used for?

A

Responsible for illuminating cells and detecting the light signals they emit.

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10
Q

What are the components of the optics system of flow cytometry?

A
  • Lasers
  • Interrogation Point
  • Optical filters
  • Beam splitters
  • Lenses
  • Detectors
  • Mirrors
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11
Q

What does the optics system convert?

A

Emitted photons into an electrical signal, a photocurrent for the electrical system.

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12
Q

What does the laser from the optics system do?

A

Provide focused light beams that excite fluorescent molecules (fluorophores) within the cells as they pass through the interrogation point.

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13
Q

What are the main two components making up the electronics system?

A
  1. Photo diodes
  2. Photo Multiplier Tubes (PMTs)
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14
Q

What is the role of the electronics system?

A

Responsible for digitising and processing the photocurrent from the detector for analysis.

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15
Q

What are the three main parameters measured in FACS?

A
  1. Forward light scatter (FSC)
  2. Side light scatter (SSC)
  3. Fluorescence emission signals
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16
Q

How does forward light scatter separate cells?

A

Based on particle/cell size
More forward light suggests bigger cell.

17
Q

How does side scattered light separate cells?

A

Based on granularity and complexity of cells

18
Q

How do fluorescence emission signals separate cells?

A

Based on presence or absence of specific proteins

19
Q

How do fluorescence emission signals work?

A

Fluorophore (fluorescent dye) is used to label the protein of interest.

Cells pass through laser and dye absorbs light of a specific excitation wavelength.

Then emits light at a different emission wavelength

20
Q

Fluorescing cell possesses interrogation point and creates pulse of photon emission.

What is this detected by?

And what do they convert the light signal into?

A

Photon multiplier tubes
Convert light signal into a small electrical signal (a voltage pulse)

21
Q

Do the voltage pulse area and fluorescence intensity correlate?

A

Yes, the higher the fluorescence intensity, the higher the pulse area.

22
Q

What is the purpose of channel numbers?

A

To help categorise and display different fluorescence levels.

23
Q

How can we boost the intensity of and detect weaker signals?

A

Increasing the voltage

24
Q

Why do we put cells in a cell suspension during sample preparation/

A

To avoid clogging the system with clumps.

25
Q

What is flow cytometry’s greatest advantage?

A

Speed
Can sort 1000s of cells in seconds

26
Q

What happens at the point of interrogation?

A

Cells pass through in a single stream.
Laser interacts with cells.
Light scatter and fluorescence signals are generated here.

27
Q

What captures the scattered light?

A

Collection lenses

28
Q

What do beamsplitters and filters in the optical system do?

A

Filters are used to select specific wavelengths.
Beamsplitters can direct light paths to different detectors.

29
Q

How is flow cytometry data usually represented?

A

Via histograms or dot plots

30
Q

What are isotope controls?

A

Primary antibodies lacking specificity to the target but match the class and type of the primary antibody used in the application.

Negative control to help differentiate non-specific background signal from specific antibody signal.

31
Q
A

AMM (18 decks)

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