58
ENDOGENOUS FLUORESCENCE
All of the preceding discussion applies to exogenous fluorochromes. The problem of
quantitation of endogenous fluorescence has not been seriously addressed. There are, however,
a few possible approaches which should be mentioned here. First is the use of fluorescence
parameters to obtain information about the physical state, local environment, or binding of
ligands to the fluorochrome.
Many fluorochromes change their properties with changes in their environment. This can be
exploited in a quantitative sense if it is possible to calibrate the change. Changes in
fluorescence intensity are most difficult to quantitate because the intensity is obviously a
function of both the amount of fluorochrome and its physical state. In the case of endogenous
fluorochromes, the amount is not easily controlled; in the case of exogenous fluorochromes,
the ability of the dye to penetrate the cell or be metabolized may dramatically alter its amount
in each cell.
More widely used is the measurement of fluorescence intensity ratios at two different
wavelengths, either of excitation or emission. These ratio measurements are independent of
the amount of fluorochrome and have been well exploited for measurements of calcium
concentration, ph, etc. in cells. The ratio can be calibrated either with free fluorochrome in
vitro or in the cells using various drugs to obtain equilibration between the cellular
compartments and the outside medium.
Another property of fluorochromes that changes with their environment is the fluorescence
lifetime. Direct measurement of this is difficult and cannot be done in flow cytometers.
However, it may be possible to indirectly measure the same phenomenon by using the phase
shift of emitted light when the fluorochrome is excited by a modulated excitation source. This
phase fluorescence is being explored in non-flow cytometric applications and there are a few
laboratories working on measuring phase shift in a flow cytometer. It is too early to tell if this
approach is technically feasible, but if it proves to be, it may be a very good method of
quantitating changes in endogenous fluorochromes.
REFERENCES
Flow Cytometry Standardization Forum Vol. 2, No. 1. Published by Flow Cytometry
Standards Corporation P.O. Box 12621, Research Triangle Park NC 27709. A. Shwartz
Ed.
ENDOGENOUS FLUORESCENCE
All of the preceding discussion applies to exogenous fluorochromes. The problem of
quantitation of endogenous fluorescence has not been seriously addressed. There are, however,
a few possible approaches which should be mentioned here. First is the use of fluorescence
parameters to obtain information about the physical state, local environment, or binding of
ligands to the fluorochrome.
Many fluorochromes change their properties with changes in their environment. This can be
exploited in a quantitative sense if it is possible to calibrate the change. Changes in
fluorescence intensity are most difficult to quantitate because the intensity is obviously a
function of both the amount of fluorochrome and its physical state. In the case of endogenous
fluorochromes, the amount is not easily controlled; in the case of exogenous fluorochromes,
the ability of the dye to penetrate the cell or be metabolized may dramatically alter its amount
in each cell.
More widely used is the measurement of fluorescence intensity ratios at two different
wavelengths, either of excitation or emission. These ratio measurements are independent of
the amount of fluorochrome and have been well exploited for measurements of calcium
concentration, ph, etc. in cells. The ratio can be calibrated either with free fluorochrome in
vitro or in the cells using various drugs to obtain equilibration between the cellular
compartments and the outside medium.
Another property of fluorochromes that changes with their environment is the fluorescence
lifetime. Direct measurement of this is difficult and cannot be done in flow cytometers.
However, it may be possible to indirectly measure the same phenomenon by using the phase
shift of emitted light when the fluorochrome is excited by a modulated excitation source. This
phase fluorescence is being explored in non-flow cytometric applications and there are a few
laboratories working on measuring phase shift in a flow cytometer. It is too early to tell if this
approach is technically feasible, but if it proves to be, it may be a very good method of
quantitating changes in endogenous fluorochromes.
REFERENCES
Flow Cytometry Standardization Forum Vol. 2, No. 1. Published by Flow Cytometry
Standards Corporation P.O. Box 12621, Research Triangle Park NC 27709. A. Shwartz
Ed.