Brainscape's Knowledge GenomeTM

Browse over 1 million classes created by top students, professors, publishers, and experts.


See full index

Clinical Immunology > Lecture 4 - Flow Cytometry > Flashcards

Lecture 4 - Flow Cytometry Flashcards

1
Q

What is flow cytometry?

A

Use of focused light (lasers) to interrogate cells delivered by a fluidics system

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
2
Q

Why do we use flow cytometry?

A
  • Allows isolation of specific cell sub-populations
  • Essential for diagnostics and research
  • Analyses individual cells
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
3
Q

Parameters that the flow cytometer takes into account

A
  • Size
  • Granularity
  • Surface molecules
  • Cytoplasmic & nuclear molecules
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
4
Q

FACS

A
  • Fluorescence Activated Cell Sorting

- Discovered by Lou Herzenberg in 1972

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
5
Q

Components of flow cytometer

A
  • Fluidics
  • Optics
  • Electronics
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
6
Q

Fluidics

A
  • Cells in suspension
  • Flow single-file
  • Focuses the cells for ‘interrogation’
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
7
Q

Optics

A
  • Generates light signals
  • Scatter light and emit fluorescence
  • Light collected & filtered
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
8
Q

Electronics

A
  • Processes optical signals
  • Converts them to proportional digital values
  • Stored on a computer
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
9
Q

How fluidics works

A
  • For accurate measurements cells must:
  • be measured one at a time
  • travel single-file through a stream at the point of laser interrogation
  • Accomplished by injecting sample into sheath
    fluid as it passes through a small (50-300µm) orifice
  • When conditions right sample fluid :
    • flows in central core
    • does not mix with sheath fluid
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
10
Q

Components of fluidics system

A

Sheath flow:

sheath tank&raquo_space; sheath filter&raquo_space; sheath interior reservoir&raquo_space; bubble filter

Sample flow:

test tube&raquo_space; sample injection tube (SIT)

Sheath flow and sample flow merged together:

Flow cell&raquo_space; interrogation point (laser works here)&raquo_space; waste interior reservoir&raquo_space; waste tank

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
11
Q

How optics works

A

Consist of:

  1. Lasers (BD Canto II can have 3 lasers)
  2. Fiber optic cables = carry beams to steering prisms
  3. Steering prisms = direct laser beams to the fluid stream

Collection optics = Direct emitted light that will be processed as useful data

Collection optics consists of:

  1. Fiber optic cables = direct emitted light to appropriate emission block
  2. Filters = direct signals in emission block to appropriate detectors
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
12
Q

Detectors (in optics)

A
  • Light must be converted from photons into volts to be measured
  • Use photodiodes for forward scatter
  • Use photomultiplier tubes (PMTs) for fluorescence and side scatter
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
13
Q

Photomultiplier Tube

A
  • Amplifies signal for detection
  • Voltage applied to the dynodes changes the parameter/setup
  • Increases in log scale
  • Voltage applied also linked to compensation setup
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
14
Q

Summary of fluidics

A
  • Sample = single cells = test tube
  • Liquid drawn up & pumped into flow chamber
  • Cells flow through flow chamber - one at a time
    500-2000 cells per second
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
15
Q

Summary of optics

A
  • Laser beam of bright light hits cells
  • Light bounced off each cell = information about the cell
  • Fluorochromes absorb light & emit specific color
  • Filters - send emitted light to color detectors
  • Light detector: Processes light signals & sends information to the computer
  • Color detectors: Collect different colors of light emitted by fluorochromes
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
16
Q

Summary of electronics

A

Computer – data from the light detector and the color detectors

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
17
Q

Forward scatter channel = size

A
  • Majority of photons pass through stream unobstructed
  • Some photons contact cell membranes & diverge from their path
  • Light scattered in forward direction
  • Detector in line with laser path (opposite side of stream)
  • ”Scattered” light collected in Forward Scatter channel (FSC)
  • Proportional to cell size: Bigger cell = more light scattered = higher detected signal
18
Q

Side scatter channel = complexity

A
  • Cells translucent
  • Many photons pass through cytoplasm
  • Photon strikes organelle = photon reflected > angle than generated by FSC
  • light scattered to side (perpendicular to axis laser light is traveling)
  • Detected in the side scatter channel by Side Scatter (SSC) detector
  • SSC proportional to cell complexity: More organelles = more light scatter = higher detected signal
19
Q

FACS: size & granularity

A

Light scatter:

  • Differentiation of size = FSC
  • Differentiation of complexity (granules) = SSC
  • Each dot = one event = one cell
20
Q

Fluorescent labelling

A

Absorption of light:

•causes an electron in the fluorescent compound to be raised to a higher energy
- Level = excitation

The excited electron:

  • quickly decays to its ground state
  • emits the excess energy as a photon of light
  • this transition of energy is called fluorescence
  • Each fluorescent compound has its own emission wavelength
  • Fluorescein isothiocyanate (FITC) emits light at ~530nm when excited by a 488nm (blue) laser
21
Q

Fluorescence

A
  • Some molecules absorb light energy&raquo_space; higher energy state = excited state
  • The energy of the excited state, “decays” or decreases&raquo_space; emission of light energy
  • This process is called fluorescence
  • To “fluoresce” means to emit light via this process
22
Q

Fluorophore in ground state

A
  • A fluorophore is a molecule that is capable of fluorescing
  • In its ground state, it is in a low-energy, stable configuration&raquo_space; does not fluoresce
  • ground state = low energy
23
Q

Absorption of light

A

• When light from an external source hits the fluorophore&raquo_space; absorbs the light energy

  • happens in ground state (low energy)
24
Q

Excitation

A
  • If the energy absorbed is sufficient&raquo_space; higher-energy state, called an excited state.
  • This process is known as “excitation”
25
Q

Energy loss (after excitation)

A

• Fluorophores are unstable at high-energy configurations» lowest-energy excited state, which is semi-stable

26
Q

Emission

A

• Fluorophores then rearrange from semi-stable excited state&raquo_space; ground state
» excess energy emitted as light

• Light energy emitted is of a longer wavelength than light energy absorbed, due to energy lost during the transient excited lifetime

  • energy drops down to almost ground state
27
Q

Visible light spectrum

A

• Fluorophores absorb a range of wavelengths of light energy, and also emit a range of wavelengths

• Within these ranges are the excitation maximum and the emission maximum
» absorbed and emitted light are different colors on the visible spectrum

28
Q

Visible light spectrum (ranges and sections)

A

400nm: short wavelength, high frequency
700nm: long wavelength, low frequency

Violet: 400 - 455 nm
Blue: 455 - 492 nm
Green: 492 - 577 nm
Yellow: 577 - 597 nm
Orange: 597 - 620 nm
Red: 620 - 700 nm
29
Q

Alexafluor graphs

A
  • point of excitation is at the wavelength written in the title of the graph, and point of emission is slightly afterwards
30
Q

Fluorescent labeling

A

Cells pass through stream

  • Laser light excites fluorescent tag
  • Emit photons of light at higher wavelength
  • Fluorescence emitted by each fluorochrome
  • Detected in a wavelength-specific detector
  • Recorded as voltages for analysis
31
Q

The importance of controls (flow cytometry)

A

Must have unstained cells

  • check for autofluorescence
  • set up negative area

Must have single stained cells

  • check for spillover
  • set up compensations
  • set up positive regions
  • after this, multiple stained cells can be run
32
Q

Gating

A

Stained with monoclonal antibodies against:

  • CD45 labelled with FITC
  • CD3 labelled with PE-Cy5 (gated on lymphocytes)
  • CD4 labelled with PE
33
Q

What properties of a cell or particle can be measured by a flow cytometer?

A
  • Size
  • Granularity
  • Surface molecules
  • Cytoplasmic & nuclear molecules
34
Q

What light source is used in most flow cytometers?

A
  • focused light (lasers)
  • LASER = Light Amplification by Stimulated Emission of Radiation
  • fluorescent light
35
Q

What are the 3 main systems in a flow cytometer?

A
  • Fluidics
  • Optics
  • Electronics
36
Q

Light emitted from a particle is collected by:

A

.

37
Q

Fluorescent compounds have defined absorption and _____________ spectra

A

emission

38
Q

Spectral spillover is corrected by using:

A

.

39
Q

(T/F) Particles must be in single-cell suspension before flow cytometric analysis

A

True

40
Q

(T/F) A dot plot can be used to display two parameters.

A

True

41
Q

(T/F) A gate can be used to restrict the analysis to a specific population within the sample

A

True

Clinical Immunology (15 decks)

Brainscape helps you reach your goals faster, through stronger study habits.
© 2024 Bold Learning Solutions. Terms and Conditions