8th week of SIP - Flow Cytometry

sorry for this late entry...

Flow Cytometry
Flow cytometry is a technology that simultaneously measures and analyses physical or chemical characteristics of single particles, usually cells, delivered in a flowing stream to the laser beam. The properties measured include the particle's relative size, granularity and fluorescence intensity. The flow cytometer I'm currently using is known as FACSCalibur from Becton Dickson.

Fig 1: FACSCalibur. Taken from http://igc-wiki.igc.gulbenkian.pt/lib/exe/fetch.php?w=&h=&cache=cache&media=uic:flowcytometry:facscalibur.jpg


Mechanism of FACSCalibur
The cytometer consists of 3 main subsystems: fluidics, optics and electronics. The fluidics systems is responsible for the transportation of cells, from 0.2-150µm in size, in a sheath stream to the observation point where the laser intercepts the cells (refer to Fig 2).. Only 1 cell should move through the laser beam at a given moment. To achieve this, the sample is injected into a stream of sheath fluid within the flow chamber (flow cell). The flow of sheath fluid accelerates the cells and restricts them to the center of the sample core, a process known as hydrodynamic focusing. Priming of the instrument to remove air bubbles and debris in the fluidics system is necessary to ensure proper interception of cells at the laser beam.

Fig 2: Inside the flow cell. Taken from http://users.path.ox.ac.uk/~nrust/diag1.gif


In the optics system (refer to Fig 5), the laser will illuminate the cells in the sample stream and light signals are generated, which in turn will be processed by the light detectors. Forward scattered light (FSC), light scattered just off the axis of the incident laser beam in the forward direction, is detected by a photodiode. A photodiode is a mechanical obscuration in the path of the laser beam that prevents the laser light itself from reaching the FSC detector, thereby allowing the detection of the FSC. The FSC signal is proportional to the cell-surface area/size (refer to Fig 3). Therefore, the larger the cells, the more intense is the FSC signal. Side scattered light (SSC) is collected at approximately 90° to the laser beam by a collection lens and then redirected by a beam splitter to the SSC detector (photomultiplier tube). It is proportional to the cellular granularity (refer to Fig 3). Correlated measurements of FSC and SSC are useful in distinguishing the different cell populations in blood sample (refer to Fig 4).

Fig 3: FSC and SSC. Taken from http://www.uams.edu/flowcytometry/Images/FSC&SSC.jpg


Fig 4: Cell popultions based on FSC vs SSC. Taken from http://www.flowlab.ucalgary.ca/flowlab/images/FSC.gif


Emission of fluorescence by the fluorochromes conjugated to a monoclonal antibody is also collected by the same lens that collects the SSC. The fluorescence signals are directed to their respective detectors via a system of mirrors and optical filters. The first mirror that collects light encounters is a 560SP (short pass) mirror, which allows light shorter than 560nm to pass and reflects light longer than that to other optical filters. The light that passes through this mirror then reach a beam splitter that reflects 10% of the light to SSC detector and lets the remaining 90% pass, which will reach the FL1 detector for short wavelength (green) fluorescence. A 530/30 bandpass filter is placed in front of FL1 detector to allow only light of wavelength 530 ± 30nm to reach the detector, eg FITC. The bandpass filter serves to optimise the specificity of the detector. Light reflected by 560SP mirror will reach a 640LP (long pass) dichroic, a beam splitter. Wavelengths shorter than 640nm are reflected to the FL2 detector for medium wavelength (yellow/orange) fluorescence, and longer wavelengths are passed and will eventually reach the FL3 detector through a 670LP filter. The amount of fluorescent signal detected is proportional to the number of fluorochrome molecules on the particle.

PS: Sorry guys.. it's kinda hard to digest at first, but hope u'll understand.. From Fig 5, you'll notice a half mirror placed just after the 640LP filter rite? dun risk asking me its purpose.. it's more confusing.. It just basically for FL4 detector.. wad's FL4 detector purpose? also pls dun take risk.. just pretend it's not there.. thanx..

Fig 5 (below): Optics System in FACSCalibur. Taken from http://www.sickkids.ca/fcf/images/FACSlayout.gif


Fig 6 (below): Examples of fluorochromes detected by different detectors. Taken from http://images.google.com.sg/url?q=http://facs.scripps.edu/images/facscolors.jpg&usg=AFQjCNGHeSQIyJ8hHvjT2spGEVX5e-ZWRg


All the signals detected are converted into electronic signals by the electronic system, and then processed by the computer. Analysis of the cell population can be displayed in several formats. The format I'm using is dot plot, which provides a two-parameter display of data. Each dot represents 1 or more events. Gating is required to restrict the analysis to only the cell population of interest. In Fig 7, the lymphocytes population is gated (in red colour) on the left graph, and therefore the resulting display on the right would only reflect the fluorescent properties of only the lymphocytes. A quadrant marker is used to divide the two parameter plots into four sections to distinguish populations that are considered -ve, single +ve or double +ve. The lower-left (LL) quadrant display events that are -ve for both parameters. The upper-left (UL) quadrant display events that are +ve for the y-axis parameter (CD4) and the lower-right (LR) quadrant are events that are +ve for the x-axis parameter (CD8). The upper-right (UR) quadrant display events that are positive for both parameters (CD4/CD8), or double +ve. Gated events are expressed in the form of percentage.

Fig 7: Dot plot with a gate emcompassing the lymphocyte population. Taken from http://www.bio.umass.edu/micro/immunology/facs542/hdots.gif


If you're still reading, thanx so much for ur time! distinction for ur patience! tried to minimise the information load.. but it still seems like... haix.. took a month to understand this flow cytometer.. I should say its inventor must be real free and nth else better to do.. but nevertheless, it's a veri much useful instrument anyway (ability to perform 10 000 events, shorter turnaround time).. Should you have any qn.. (erm.. can try not to ask?) ask simple qn will do.. and not too many pls.. thanx so much!

Joan
TG01