http://www.med.cam.ac.uk/flowcytometry-core-facility/
BRC Phenotyping Hub and COMPENSATION TUTORIAL
CIMR Flow Core UNIT
http://intranet/flow/facs.html
Tutorial II: Optics, Spectra and Compensation
Choice of fluorochromes
When light is absorbed by a fluorochrome molecule, its electrons move from a ground state to an excited
state. The amount of energy required (Eexcitation) will differ for each fluorochrome. The fluorochrome then
undergoes internal conformational change causing dissipation of energy and releases some of the
absorbed energy as heat, and an intermediate state at a lower energy level is reached. The electrons move
back from there to their ground state they release the remaining energy (Eemission) as fluorescence.
According to Plank’s Law, the emission wavelength of any fluorochrome will always be longer than its
excitation wavelength (in case of single photon excitation, as the energy of emission is less than the energy
of excitation). The difference between Eexcitation and Eemission is called Stokes Shift. The emission light shall be
easily distinguished from the excitation, thus, the fluorochromes with large Stokes Shift are better for
fluorescence studies, especially for multicolour experiments.
It is important to select a fluorochrome with excitation maximum close to the one of
the laser lines of the instrument!
Excited Singlet
State
Heat
out
Eexcitation
Relaxed
Singlet State
Eemission
Light in
Light out
Ground State
E= hν=h(c/λ)
Eem<Eex
Plank’s law
λem>λex
Why do we need Compensation?
Every fluorescent molecule emits light with a particular spectrum unique to that molecule. These emission
spectra overlap, in some cases significantly. Thus, for simultaneous detection of emissions, whole spectra
must be considered rather than only emission maxima. Compensation is the process by which we correct
for the spectral "spillover". This correction can be performed in a digital or analogue way (by software or
hardware). They differ in the way they will process and affect raw data. While the hardware compensation
is based on subtraction of electronic pulses and the raw data are affected irreversibly; the software
compensation is corrective, and applies mathematical algorithms to the data without affecting them.
Tutor:
For local use: Anna Petrunkina, BRC Phenotyping Hub ©
Date:
http://www.med.cam.ac.uk/flowcytometry-core-facility/
BRC Phenotyping Hub and COMPENSATION TUTORIAL
CIMR Flow Core UNIT
http://intranet/flow/facs.html
Source of this figure: http://www.drmr.com/compensation/indexDetail.html
In this figure, excitation spectra
are shown in green, and
emission spectra in red. The
light blue line is the wavelength
of an argon ion laser (488 nm).
Each of the three dyes can be
excited at this wavelength.
However, when the dyes are
excited, each emits with a
characteristic spectrum:
• FITC: Exmax~ 520 nm,
• PE: Exmax~ 575, and
• PE-Cy5 Exmax~670.
Above are shown the spectra for fluorescein ("FITC"), phycoerythrin PE and PE-Cy5
• To measure these emission spectra simultaneously, and separate the signals, we choose optical
filters ("bandpass filters") which only transmit specific wavelengths of light.
- The bandwidth of the filters is represented by the grey shaded areas. In general, filters are chosen
which collect the emitted light near the emission maximum.
- For example, for detecting fluorescence of FITC, a BP 530/30 filter is used. The pass-band is
centred on 530 nm, and the width of the pass-band is 30 nm.
• It is nearly impossible to perform spectral separation with a filter selecting the fluorescence from
only one specific dye.
- Fluorescein/FITC has a significant emission in the region that we measure PE (575 nm).
- Whenever fluorescein is present, we will get signal both in FL1 (BP 530/30), and in FL2 (585/30).
- If a second fluorochrome (such as PE) is also present, it will contribute to the FL2 signal.
Correcting the amount of fluorescence detected in the FL2 for ‘fluorescein’–photons will allow to
account for specific PE fluorescence and vice versa.
BP
BP
530/30
580/30
FITC: Em max= 519
PE: Em max=575
FITC-Fluorescence in FL2
PE-Fluorescence
in FL1
520
540
560
580
600
λ
FL2-%FL1= corrected FL2
FL1-%FL2= corrected FL1
Tutor:
For local use: Anna Petrunkina, BRC Phenotyping Hub ©
Date:
http://www.med.cam.ac.uk/flowcytometry-core-facility/
BRC Phenotyping Hub and COMPENSATION TUTORIAL
CIMR Flow Core UNIT
http://intranet/flow/facs.html
Examples of correct & wrong compensation (Mario Roderer; http://www.drmr.com/compensation)
In this hypothetical experiment, cells were stained with FITC CD3 and with a PE isotype control,
and collected at different compensation values to correct for the FITC spillover into the PE channel
(panel 1 is uncompensated; 2-5 are for increasing compensation values).
X
V
X
X
Question #1: Which panel
represents proper
compensation?
Question #2: On what
basis did you make this
determination?
FITC CD3
Compensation: The protocol (Source: M. Roderer, modified by the FACS Core)
Five steps must be taken to assure proper compensation. These steps should be taken with every
experiment where the cell types differ, where the reagents differ, or where any instrument settings change daily compensation is thus probably not frequent enough! NEVER use compensation from others’ expts
(1) The compensation tube must consist of cells that are unstained as well as cells that are singly-stained
with the fluorescent probes. The stained (positive) cells must have the same autofluorescence (when they
are unstained) as do the unstained (negative) cells in the compensation tube (e.g., all are lymphocytes).
(2) The PMT voltages must be set high enough guarantee that the negative population is off the axis in
every channel. But they must be low enough to ensure 1) your positive population is within the detectable
range, and 2) spillover in the relevant channel is not too high to make the compensation impossible (100%).
(3) An analysis gate is set so that only cells with identical autofluorescence characteristics are viewed (e.g.,
a lymphocyte gate). An analysis gate is set to include all of the negative cells and all of the positive cells.
(4) The centers of the positive and negative cell populations are aligned by matching the median/mean
fluorescences. That is achieved by calculating average values (MFI). It is advisable to use rather median or
geometric means than arithmetic means, especially if normality of distributions cannot be assumed.
(5) Save or print out your compensation protocol. It is important for your records. You can also import your
compensation protocol next time and run all your controls (including unstained) through it. Remember: you
must always check all your single-stained samples pair-wise AND you must re-do your compensation if you
changed voltage
Tutor:
For local use: Anna Petrunkina, BRC Phenotyping Hub ©
Date: