Flow Cytometry Flashcards
Objective of fluidics subsystem
One cell moving past laser beam at a particular time.
Pressurized laminar flow (hydrodynamic focusing)
Injected through sheath fluid
Three subsystems of flow cytometry
Fluidics
Optics (excitation and collection)
Electronics
What does laser stand for
Light amplification by stimulated emission of radiation
Two main types of photodetectors
Photodiodes - strong signals
Photomultiplier tubes - more sensitive, small fluorescence
What is the role of optical filters
Determine specificity of detection
Long-,short- and band-pass
Flow cytometric measurements
Physical - relative size/ internal complexity
Chemical - relative fluorescent intensity
Forward scatter related to
Cell size
Side scatter related to
Cellular granularity and internal complexity
What is a fluorochrome
Organic molecule which accepts light energy (excitation) at a given wavelength and re-emits (emission) at a longer wavelength.
Critical aspects of staining for FC
Ab non-specificity (
Appropriate controls (ns binding - stain with 2ndry Ab only, background - don’t stain)
Ab titrations
Explain stokes shift
Ground state molecule is excited. Energy is given off in form of heat. Remaining energy is emitted at longer wavelength light.
Types of fluorochromes
Classic dyes Alexa Fluor / brilliant violet Pacific dyes Tandem dyes Quantum dots
How is light converted to data
Photodetection
Amplification
Signal processing (quantify)
What is used for dual and single parameter display
Dotplot and histogram
Why do we need compensation in FC
Emission at range of wavelengths. Emission spectra tail extends to longer wavelengths leading to SPECTRAL OVERLAP = detection in more than one detector
What is compensation
Removal of background signal in other channels. Calculates the amount of interference a fluorochrome will have in a channel.
What is the goal of compensation
For a given channel, the median signal of both positive and negative populations in other channels should be the same.
Practical considerations when running FC
Ensure PMT voltages are correct
Compensation before acquiring
Make singly stained controls
How does one select population of interest in FC
Gating
Name types of gating
Polygon
Quadrant
Histogram (interval)
What is a CFSE assay
Carboxyfluorescein succinimidyl ester
Diffuses into cells, cleaved by esterases to for membrane impermeable fluorescent dye. Cells given stimulus. As cells divide signal in each cell halves.
Components of cell sorter
Interrogation point Charging stream Droplet formation Electrical plates Waste collection Collection vessels
