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Multicolour Analysis
There are occasions where it is important to know if antigens co-exist on the same cells and in these cases multicolour analysis can help. The ability to simultaneously measure multiple parameters on a cell by cell basis is probably the most powerful aspect of analytical flow cytometry. Once more than two colours are being used, the importance of the relevant controls, dye combinations, electronic compensation and optical set-up becomes much more important, and before embarking on such experiments it is vital that you discuss it with a member of the FACS Lab.
It is also important to be aware that all fluorochromes will have a range of fluorescence emission and because of the optical filters used in flow cytometers, there will be overlap between the emission spectra once more than one fluorochrome is being used. In these cases, there will need to be some form of compensation for this spectral overlap whether it is provided by hardware (as in the Labs Becton Dickinson machines) or software (as on the MoFlo). A good introduction to fluorescence compensation is provided by Mario Roederer.
The optical filters found in most flow cytometers can be of several types and knowledge of the ones that are used or that can be used will aid in the design and the feasibility of multicolour experiments.
Long Pass filters allow light above a specified wavelength to pass through. The usual nomenclature is for example, 600lp, where light above 600nm passes through.
Short Pass filters allow light below a specified wavelength to pass through, e.g. 500sp.
Band Pass filters allow light between two wavelengths to pass through. The usual designation is for example 530/30bp where the centre of the transmitted light is at 530nm and the band is 30nm ie light 15nm either side of the centre is transmitted (515-545nm in this example).
Dichroic Filters or Mirrors are also used and these are specialised long or short pass filters. A 560lp dichroic for example will allow light above 560nm to be transmitted but will reflect all light below 560nm in a specific direction.
The filters available in the Lab's cytometers is as follows:
- FACScan
- We can analyse up to three colours on the FACScan. The collection optics
are fixed and allow us to collect green (530/30), orange (585/42) and
red (650lp) fluorescence. As with any of the Laboratory's cytometers any
combination of fluorochromes that emit in these wavelengths may be used.
- FACSCalibur
- We can analyse up to four colours on the FACSCalibur. Three of these
are from the 488nm laser and are at similar wavelengths to the FACScan
(the red fluorescence on a Calibur is collected above 670nm). The fourth
colour is excited by the red diode laser and emitted fluorescence is collected
through a 661/16 filter.
- BD LSR
- The latest Becton Dickinson cytometer is more versatile than the FACS
Calibur. We can measure either 6 fluorochromes: either three from the
488nm laser, two from the UV and one from the 635nm laser, or four
from the 488nm laser and two from the UV. The ability to change the optical
filters means a greater range of fluorochromes may be used on the LSR.
The filters we have are: 380lp, 400/40, 424/44, 500/11, 510/20, 530/28,
572/26, 610/20, 660/13,670lp and 682/13.
- Laser Scanning Cytometer
- In this slide-based cytometer, scanning of the slide is done sequentially
by two lasers. Four fluorochromes may be measured from the 488nm laser
and two from the 635nm laser. The filter sets we have are: 530/30, 580/30,
625/28 and 650lp.
- FACS Vantage and MoFlo
- Both of these machines are more configurable than the benchtops. The
FACS Vantage will allow three 488nm excited fluorochromes and either
two UV-excited fluorochromes, or two 635nm-excited fluorochromes,
or one UV- and one 635nm-excited fluorochromes.
The MoFlo can measure 7 colours simultaneously - the current set-up allows
any combination of 7 from four colours off the 488nm laser, two from the
UV and two from the HeNe.
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