FCM

Question 1

what is cytometry?

Answer 1

the measurement of the physical or chemical characteristics of cells or other biological particles at a single cell level

Question 2

What is fcm?

Answer 2

technology that allows analysis of multiple characteristics of particles (cells) as they flow through a beam of light

Question 3

what is the purpose of the fluidics sytem?

Answer 3

transports particles in a fluid stream past the lasers

Question 4

What is hydrodynamic focusing?

Answer 4

sample is injected into a central core which is enclosed by an outer sheath of faster flowing liquid, creating a drag effect on a narrowing core –> single file particles

Question 5

How does pressure influence the flow rate and width of the flow core?

Answer 5

higher sample pressure –> higher flow rate and greater width of flow core

Question 6

What are a couple of disadvantages of increasing the flow rate?

Answer 6

1. > 1 cell can pass through the flow cell at any given time
2. cells may pass through the laser beam off-centered (poor illumination) and off the lens focal plane (poor fluorescence collection)

Question 7

What is acoustic focusing?

Answer 7

acoustic waves applied to the sample prior to hydrodynamic focusing –> allows for very fast running speed with no loss in resolution or increases in coincidence (basically puts all the cells in a single file before the sheath –> faster processing)

Question 8

What two components make up the optics system?

Answer 8

1. lasers that illuminate particles
2. optical filters that direct light signals

Question 9

What is forwards scatter (FS)?

Answer 9

measurement of diffracted light –> tells you the relative size of the object

Question 10

What is side scatter (SS)?

Answer 10

measurement of the refracted and reflected light at an interface where there is a change of refractive index –> internal complexity of the object

Question 11

Describe the forward and side scatter of: neutrophils, monocytes, lymphocytes, and debris to one another

Answer 11

Side scatter: neutrophils > monocytes > lymphocytes > debris

Forward scatter: monocytes > neutrophils > lymphocytes > debris

Question 12

What are the three main forms of optical filters? Describe each

Answer 12

1. long pass = allow any longer wavelength through
2. short pass = allow any shorter wavelength through
3. band pass = allows wavelength of a certain range through

Question 13

What is the function of the detectors and electronics in FCM?

Answer 13

convert light signals to electronic information that can be processed by a computer

Question 14

What detects the light signals and what does it create?

Answer 14

detect = PMT (photomultiplier tube)

creates a pulse

Question 15

What are the three main kinds of FCM? Briefly describe each.

Answer 15

1. analyzers - standard (FSCm SSC, and fluorescence)
2. sorters - FACS
3. imaging cytometers - similar to an analyzer, but get fluorescent images of each particle