Flow cytometry - Week 6

Question 1

what is the definition of flow cytometry

Answer 1

rapid measurement of the optical & fluorescent properties of a large number of particles.

Question 2

what does the flow cytometry instrument consist of

Answer 2

• The Fluidics system draws cells into the machine and channels them into a single file.
• it also contains an optical system, lasers, a white light source, and the lenses that focus the laser beams, there are also lenses that are similar to microscopic slides that focus the information that is coming from the sample as the light hits it and turns it into a recognisable image.
• there is also a computer system, which drives the process and/or collects the data so you could see what’s going on in real-time.
• it also has an electrical system which takes the light signal that bounced off cells and it turns that into something that can be recognised and processed electronically.
  how an image is made electronically is the light signal is converted into a voltage which goes through processes and turns the signal into a recognisable image.
• also, every cytometer has a waste pot, flow cytometer is an analytical method so once the sample is used it will go into the waste pot.
• there is a sheath buffer and other various cleaning agents used to prevent contamination from the previous sample which is done through a cleaning process the machine goes through after each use

Question 3

how were cytometry machines then vs now

Answer 3

the first machines used were very large but as technology advances, machines get more sophisticated and do more things and they also get physically smaller thus they have a smaller footprint

Question 4

what actually happens when your sample is taken up into the machine

Answer 4

Step 1: The sample is taken up into the machine through the sample injection port, it’s then loaded into the central core/ sample hopper.
Step 2: Inside your sample is the sample field, which is called the central core. The fluid is either under very low pressure due to a liquid handling pump or because of gravity so the sample is shaken down through the hopper until it forms a single file of cells into an ordered straight line. You don’t want 2,3 or 4 cells which are clumped up and go through the interrogation point you want a single line of cells going through.
Step 3: How the cells are kept in a straight line is by the use of outer sheath fluid which is on either side of the hopper. it is pumped down at relatively low pressure and it surrounds the column of cells and keeps them in an ordered straight line due to the forces between the fluid and cells. This is called hydrodynamic focusing which allows one cell to pass through the laser beam at a time.

Question 5

what happens when the flow rate of the central core and sheath fluid is fast or slow

Answer 5

The flow rate of the central core of cells and sheath fluid can be adjusted.
If the flow rate is slow, the cell passes the light beam one at a time and spends a relatively long amount of time in that light bream (in reality it’s less than a second, but you still get a lot of information about the cell) and you get high-resolution imaging.
The slower the cells go through the laser beam, the higher the resolution, thus the more information you get.

If you speed up the flow rate and have a fast flow rate and the cells go through the light beam at a relatively fast speed ( for example the cells go through the laser beam for a picosecond), this results in a low resolution and thus you don’t much information about that cell.

Question 6

how can some sample be lost in the flow cytometer

Answer 6

The machine used to make the hopper vibrates which means the sides of the hopper are not completely smooth and because the outside of a cell is made from a protein which is very sticky, it results in the cells sticking to the side of the hopper which can’t be unstruck by hydrodynamic focussing, thus it causes you to lose some of your sample.

The hopper is what your sample goes through.

Question 7

why must the samples be clean

Answer 7

the samples must be free of debris. In early processing debris like dead cells or broken cells is removed so the image that is taken is of all intact cells.

Question 8

how are samples cleaned or maintained clean

Answer 8

In the preparation of the sample dead cells are removed.
A way of maintaining a clean sample is by not getting air bubbles in the system. if you get air bubbles in the system it can distract the hydrodynamic focussing as the stream of cells starts wobbling from side to side and thus cause it to miss the laser beam.
One way to prevent bubbles is by using debubbling agents.

Question 9

what happens when your cell passes the interrogation point in the optical system

Answer 9

Step 1: As the cell passes through the interrogation point a laser beam is produced.
Step 2: The cells flow down the laser beam, one at a time, and as the light hits each cell it gets scattered.
Step 3: The vast majority of the light will continue through the detectors. the detectors are placed all through the surroundings to pick up the beam of all the scattered light. There is a whole semicircle of detectors which catch the light that has been refracted or deflected from that cell.
Step 4: The light then gets fed through to the electronics, the optical system often gets bounced around corners using mirrors because we want to keep things in a box, to keep the light in a manageable size we let the light go through corners
Step 5: Then it goes off to the electronics where the light is changed into a voltage.

Question 10

how can light goes through or around a cell

Answer 10

When the light beam goes straight and misses the cell, it can go around the cell to the detector that is on the other side, or the light beam goes into the cell and the organelles (which are the nucleus, mitochondria, lysosomes, ER, etc) which can cause the light to bounce off them.

Question 11

what is the forward scatter of light and what does it measure

Answer 11

The light that goes around the cell is called forward scatter (FSC). The light carriers on the same direction as the incident laser beam meaning it is carrying on in a straight line.
Forward scatter measures how big the cell is, it measures cell size. The wider the beam of the scattered light that is apart the bigger the cell is.

Question 12

what is the forward scatter of light and what does it measure

Answer 12

Some light is scattered sideways, and rarely some light is even scattered backwards, usually it’s scattered sideways and is called side scatter (SSC), and this measures the cell granularity, so the complexity of the cell.
The more light that is scattered sideways at an angle the more organelles are present in that cell, so it gives an idea of the complexity of the cell.

Question 13

how to remember what forward scatter and side scatter is

Answer 13

A way to remember it:
- Forward scatter (FSC), F for fatness measures the fatness (size) of the cell
- Side scatter (SSC), S measures the spottiness of the cell, how many spots the cell has got in it (how complex it is)

Question 14

how is emitted light from fluorophores measured

Answer 14

Emitted light from fluorophores registered by its corresponding detector.

Answer 15

Emitted light reflected off/passes through mirrors & filters before reaching photodetectors. Photodetectors also known as photo multiplying tubes detect refracted and reflected light.

What the photodetector does is it takes the photon of the light and turns it into an electric current.
photomultiplier tubes generate electric current proportional to the number of photons detected.

Question 16

why is a electric current amplified and how

Answer 16

The electric current can be amplified (because it’s milliamps) so it’s meaningful for the electronics to deal with. Electric current is amplified by a linear or logarithmic amplifier.

Question 17

why does an analogue-to-digital converter do

Answer 17

An analogue signal can be turned into a digital signal by an analogue-to-digital converter which turns it into something the computer can understand and then the signal goes into software for analysis and then the analysis is shown on a computer screen.

Question 18

what are channels

Answer 18

Each detector will detect a particular wavelength of light (e.g. Red, yellow, green or blue light), these are called channels.
a series of detectors for every wavelength of light are called channels.

Question 19

how are results made in flow cytometry

Answer 19

Once the computer has processed the light/ photon report from the detector into some kind of electric current it then turns into a digital signal which is the result.

Question 20

what do the results show

Answer 20

A scatter plot/ several scatter plots are what you see on the computer screen, it is a graph, and it shows forward scatter (FSC) on the X-axis and side scatter (SSC) on the Y-axis.
each one of the dots on the graph is a cell shown on a scatter plot.
The graph measures the fatness (forward scatter)/size of the cells against the side scatter (SSC)/ complexity of the cell.

Question 21

how do you know where the different types of cells are located on the scatter graph

Answer 21

often you are given calibration plots from the manufacturer to show you where different types of cells are gonna be reported on your scatter plot.

Question 22

what is the flow cytometry results/graph of blood look like

Answer 22

the graph shows a cell of a whole blood sample divided up into their constituents. Blood is made up of granulocytes, monocytes, lymphocytes, and red blood cells. the graph shows that:
- Granulocytes are quite high up on the forward scatter axis which shows you that they are big cells, they also are quite high up on the side scatter (Y-axis) which shows you that they are very complex cells.
- Monocytes are quite high up on the forward scatter axis as well which tells you that they are big cells but they are lower on the side scatter axis which tells you that they are less complex.
- Lymphocytes are around the middle of the forward scatter axis which tells you they are medium-sized and they are right down on the side scatter axis meaning they are very simple cells.
-Red blood cells are very tiny in comparison to the other cells
-There will also always be debris which is dead cells.

This is a very simple example of how flow cytometry can break down for you what’s in your sample, and what type of cells are present in your sample.

Question 23

how do you know how many cells there are in your sample

Answer 23

From the graph/scatter plot, you can quantitative your sample, you can draw a circle around a section on your graph (this is called gate) and you can tell the software to count the cells in that section. So you can count how many granulocytes, monocytes, lymphocytes and red blood cell is in your sample.

Question 24

what happens to your sample after the analysis

Answer 24

The sample after analysis goes to waste, so it’s not a preparative method but it can tell you analytically what is going on in your sample.

Question 25

why can the same laser be used to detect 2 different fluorephores

Answer 25

Different fluorophores can have the same excitation wavelength but distinct emission wavelengths. Therefore the same laser can be used to detect two or more fluorescent biomarkers.
The principle is certain fluorophores can be excited at the same wavelength, so electrons can jump up at the same wavelength (make those same jumps) but when they jump back down, they can give off different wavelengths of light. So you can excite in a sample 2 different fluorophores with one laser.

Question 26

what is an example of the same laser being used to detect 2 different fluorophores

Answer 26

phycoerythrin (PE) and fluorescein (FITC) are excited by the same wavelength of light but when the electrons calm down they give off different wavelengths of light, in this case, phycoerythrin is red and FITC is green.

Question 27

how is fluorophore flow cytometry carried out

Answer 27

Step 1: in a population of cells primary and then secondary antibody is added to it.
CD42a ( a surface label) is labelled with phycoerythrin (phycoerythrin is a red stain) by using immunocytochemistry. CD41( a surface label) is labelled with FITC (FITC is a green stain).
phycoerythrin and FITC are primary and secondary antibodies.
Step 2: The cell sample with the 2 fluorescent biomarkers is then put through a flow cytometer.
Results are shown on the calibration plot from the manufacturer.

Question 28

what do the results/graph show of fluorophore flow cytometry and how can you count how many cells you have

Answer 28

the results shows the following:
- Cells that have got both CD42a and CD41 on their surface will have been stained for both of them and they will show up on the top left corner of the scatter plot.
-Cells that have got only CD48a on their surface, will be shown in the top right corner of the scatter plot.
-Cells that have only CD41 on their cell surface, will be on the bottom left corner of the scatter plot
-If you have cells that don’t present either of those two cell surface molecules, there will be one on the bottom right corner of the scatter plot.

you can get the software to count the number of dots presented in any of the corners you are interested in because each dot is a cell.

Question 29

what is an example of where fluorophore flow cytometry is used

Answer 29

Fluorophore flow cytometry is used in a branch of immunology called immunophenotyping, where samples of white blood cells are taken from healthy people to find out how many healthy people have either CD48a present or CD41 present or both present.

Question 30

what happens when you stain your cells with a DNA intercalating agent

Answer 30

If you strain your cells with DNA intercalating agent e.g propidium iodide, essentially what happens is the molecule will insert itself into the 2 strands of the DNA and stays there thus called an intercalating agent.
DNA intercalating agent is a molecule that will sharpen the fluorescent light.

The more intercalating agent you see, the more DNA in the nucleus of that cell, as nuclei vary in different cells, there are some cells that are almost all nuclei thus some cells will have more intercalating agents than other cells.

Question 31

why do the nuclei vary in cells

Answer 31

nuclei vary in different cells, there are some cells that are almost all nuclei.

-Most cells are in G0 of the cell cycle stage because they are living life or they enter the cell cycle. When they enter the cell cycle from G0 they go into G1, here in humans they still have 46 chromosomes (23 pairs).
- in the cell cycle the cell then enters the S phase, those 46 chromosomes are copied and you get 96 chromosomes.
-The cell then moves onto G2, where those chromosomes are checked, and the DNA is checked to see if it is error-free, if not it is then repaired. Here the cells still have 96 (46 pairs) chromosomes.
-Then the cell is ready to go into mitosis, where it split apart into 2 daughter cells. here the cell has 46 (23 pairs) chromosomes each.
-Then the 2 daughter cells go into G0 or into the cell cycle. Here the cells have 46 (23 pairs) chromosomes as well.

Thus you can tell an approximation of what stage the cell is in the cell cycle by the amount of DNA it has in its nucleus/ or it contains.
A cell with a very big nucleus js probably existing the S phase or its in G2, in a human cell, here it has 96 chromosomes.
A cell that has a smaller nucleus, is probably in G1 or entering the S phase or is in G0.

Thus every cell has a different amount of DNA/nuclei in their cell as every cell is at a different stage in the cell cycle.

Question 32

what does cell cycle analysis from a scatter plot or histogram show

Answer 32

You can see the DNA and cell cycle analysis either in a scatter plot or in a histogram.

The X-axis on the graph shows the propidium iodide strain from 0 to 6 x 10^6, it’s unitless, 0 means there is no dye and as you go across the axis the more dye there is in the sample.
The Y-axis shows the number of cells, (the count of cells) in the sample.

The flow cytometer turns its analysis into a graph:
-In the graph the red section shows that there are a lot of cells with a little amount of DNA which is the G0/G1 phase, most cells spend most of their life in the G0 phase and some are in G1.
-The yellow section/ S phase of the graph represents is cells at different stages in the S phase, the cells at around 2 x 6^10 Propidium iodide have just entered the S phase, where they still have there 46 chromosomes, in the S phase the chromosome duplicate so by the time the cells exit the S phase they have 96 chromosomes.
-You also have a relatively small number of cells present in the G2/M phase, here the cell checks the DNA for imperfections and then the cell goes into mitosis. It is very rare to find a cell during mitosis as its hard to detect because mitosis usually is only taking place in the cell for 20 minutes.

Question 33

What is FACS and how are cells separated

Answer 33

Fluorescence-Activated Cell Sorting (FACS)
FACS separates a mixed sample of cells into different subpopulations.
how this happens is
FACS separates cells according to fluorescent labelling.
This method isn’t perfect but you will get about 95%-98% purity in this process.

how does the separation process using the labels occur:
Step 1: you have a piezoelectric crystal that physically causes vibration and that vibration causes the stream of liquid to be broken up so instead of having a stream of liquid come out, it comes out as single drops of liquid.
The concept of piezoelectric crystal is the vibration cause the breakdown of the liquid so it can come out as single drops, each droplet of liquid contains one cell.

Step 2: An electric charge is applied to the droplet depending on the fluorescent marker expressed by the cell in the droplet. Each fluorescence marker will respond differently to an electric charge

Step 3: The cells that you have marked and labelled e.g. with the green and red labels will respond as the following:
-The green label is negatively charged so when the cells come out as drops of liquid, the green labelled cells go towards the positive deflection plate
-conversely, the red-stained cells will have a positive charge and thus go towards the negative deflection plate.
- If you have cells with no stains on them, they go straight to waste.

This is called electrostatic deflection and it will use the electrical differences to attract the cells to different collection tubes. This is one way you can break the cells up into different populations.

Question 34

what is imagestream

Answer 34

Another subtype of a flow cytometer is called imagestream
It is an imaging flow cytometer, which is the main body of the instrument, this flow cytometry is run by a data acquisition computer.

Question 35

what does an imagestream show

Answer 35

What the imagestream does is instead of showing each cell as a dot, you get a photograph.
Imagestream is very fast as it takes a photograph of 5000 cells per second. In comparison on a microscope slide you see about 200 cells per slide.

Because a photograph is taken of the cell, you can look at all sorts of different things. For example:
- the main thing you can use imagestream is to determine where the protein is in the cell, is it in the cytoplasm or nucleus or cell surface?
Proteins often have different functions depending on where they are in the cell, with imagestream because you have taken a photograph if you have labelled your protein you can tell if the protein is in the cytoplasm or nucleus, because the location of the protein indicates its function.

You can also do many other things with imagestream like:
-apoptosis/ measure cell death
- if you take an image of seawater you can look for fluorescence creatures
- cell morphology, what the cell looks like, how cells interact with each other

Question 36

what are the different types of images that can be taken from an imagestream

Answer 36

You can take 3 images of the cell as they pass the interrogation point, which are the following:
-the cell in white light
-you can also see the same cell with Alexa fluor 48A antibody labelling for a protein, called gamma H2AX
-you can also see the Drac5 stain of the same cell that shows the nucleus

imagestream also gives you histogram plots as well as images of the cell.