Flow cytometer

Question 1

What does a flow cytometer do?

Answer 1

It measures multiple PHYSICAL CHARACTERISTICS of a single cell at 500-4000cells/sec in a FLUID STREAM.

Question 2

What is required in a flow cytometer? components.

Answer 2

Fluidics, optics and electronics. Fluidics (sheath fluid) to deliver particles to the detector. filtered isotonic saline. Optics allow the visualisation of cells, which consists of an EXCITATION SOURCE (light/laser/fluorescence) and a DATA COLLECTION OPTIC (filters, mirrors, detectors: photodiodes/photo multiplier tubes PMT). Electronics convert optical signals into ELECTRICAL SIGNALS for analysis.

Question 3

How does fluorescence occur in a flow cytometer?

Answer 3

Fluorescence occurs when a molecule is EXCITED by a light photon of one wavelength and returns to the ground state BY EMITTING LIGHT OF A LONGER WAVELENGTH = fluorescence. The sample has to be stained with a fluorescent DYE or used with a FLUOROCHROME bound to an antibody, in proportional amounts with the sample so that the fluorescence intensity is proportional to the cell.

Question 4

What is an arc lamp? uncommon.

Answer 4

Glass envelope containing gas/vapour at HIGH PRESSURE.

Question 5

What is a PLASMA TUBE used as an excitation source for laser beams?

Answer 5

A plasma tube contains gas UNDER PRESSURE that will fluoresce when CURRENT is applied and the fluorescence is reflected along the tube.

Question 6

How do multiple lasers in a flow cytometer work?

Answer 6

One laser excites the MOLECULAR TAGS and the other excites the MICROSPHERE one at a time. The fluorescent intensity of the microsphere identifies the reaction and is later measured by the detector.

Question 7

What does a dichroic mirror do as a data collection optic?

Answer 7

A dichroic mirror allows light of a certain wavelength to be REFLECTED while the remaining wavelengths PASS THROUGH. It acts as a beam splitter.

Question 8

What are the 3 filters used in a flow cytometer?

Answer 8

The SHORTpass filter allows light BELOW a certain wavelength through. The BANDpass filter allows a RANGE of wavelengths through. The LONGpass filter allows light ABOVE a certain wavelength through.

Question 9

What are the advantages of using photodiode and photomultiplier tube (PMT) detectors in a flow cytometer?

Answer 9

The PHOTODIODE detector is great for the VISIBLE spectrum, but there’s no adjustable gain and requires cooling. The PMT CAN ADJUST GAIN for sensitivity, detects LIGHT, AMPLIFIES the signal to detect weak fluorescence. But PMT is not great for detecting >650nm.

Question 10

In a flow cytometer, what does the forward and side scatter detect?

Answer 10

The forward scatter detects SIZE and the side scatter detects GRANULARITY which are the internal structures of the cell. Dead cells swell and burst so the internal structures are leaked out and will have LOWER SIDE SCATTER.

Question 11

What does spectral overlap indicate in a flow cytometer?

Answer 11

Spectral overlap is the range where the 2 fluorochromes emit the same wavelengths.

Question 12

What is a frequency histogram used for a flow cytometry analysis?

Answer 12

A frequency histogram is a graphical representation of the NUMBER OF EVENTS for each PARAMETER analysed. Parameters such as size, granularity, fluorescence (dimmer/brighter).

Question 13

How does gating work in a flow cytometer?

Answer 13

Gating SELECTS A CELL POPULATION to analyse OTHER PARAMETERS.

Question 14

What parameters can be measured in a flow cytometer?

Answer 14

INTRINSIC parameters such as cell size, cytoplasmic granularity, and pigment content (haemoglobin). EXTRINSIC parameters where reagents are required, such as DNA & RNA content, DNA synthesis & degradation.

Question 15

What is “coincidence” in a flow cytometer? and how can it be prevented?

Answer 15

Coincidence is when more than 2 cells are detected in the time frame of a DROPLET FORMATION. Use ANTI-coincidence gating to prevent it as it creates a TIME WINDOW AROUND the particle of interest relating to droplet formation, if OTHER PARTICLES ARE DETECTED during this time window they are directed to WASTE.

Question 16

How does the multiplex flow cytometry (Luminex) work?

Answer 16

Multiplex flow cytometry allows multiple analyses of the physical characteristics of a cell in a fluid stream in one tube (Max. 100). Mention fluidics, optics, and electronics also. REAGENTS are bound to POLYSTYRENE MICROSPHERES that are dyed with RED & INFRA-RED FLUOROCHROME to allow the definition of 100 different microspheres. The SURFACE CHEMISTRY of the microspheres also allows binding with antibodies, peptides, antigens etc. for SPECIFIC BINDING WITH CELLS in the sample. The sample is then INCUBATED with the microspheres to facilitate binding, then WASHING is performed before adding PHYCOERYTHRIN (PE) REPORTER and then samples are analysed on the Luminex which has high throughput capabilities on the 96-well plate.

Question 17

What is cell sorting in a flow cytometer?

Answer 17

Cell sorting is when a flow cytometer separates out a cell population with a GATE into a NEW TUBE. It is done using the ELECTROSTATIC DEFLECTION of a STREAM OF AIR and MECHANICAL SORTING of the sample.

HYDRODYNAMIC FOCUSSING in a nozzle is VIBRATED by a transducer breaks the stream of air into DROPLETS. Then laser and signal processing is followed by a sort decision. There is an ELECTRONIC DELAY until the cell reaches the BREAK-OFF POINT where the stream is CHARGED then the CHARGED DROPLETS DEFLECT via ELECTROSTATIC FIELD from plates at high voltage. MECHANICAL COLLECTION is done by (tubes, wellplates, slides) to collect the sorted cells from the stream.

Question 18

What is droplet charging affected by during cell sorting in a flow cytometer?

Answer 18

Droplet charging is affected by SHEATH TEMPERATURE AND PRESSURE which can cause the CHARGING PULSE to be delivered to the WRONG droplet and CHARGE MORE THAN 1 droplet.

Question 19

What is phase gating in flow cytometry?

Answer 19

Phase gating determines if the cell is in the CENTRE OR OUTSIDE QUARTERS of the droplet window for droplet charging.