Fluorescent Dyes - Choosing a dye
There are many commercially available calcium indicators, so it is important
to choose the right one for each experiment. Some facts to be considered
are listed below:
- Range of calcium concentration. In general, quantification
is possible if calcium concentration is close to Kd value.
As a rule of thumb, [Ca2+] has to be between the following
values during the experiment:
0,1 · Kd
< [Ca2+] < 10 · Kd
- Instruments. Not
only epifluorescence microscopes are suitable for ion imaging, but
also confocal microscopes can be used and offer advantages. Illumination
source and detection filter/settings have to be suitable for the fluorochrome:
the optimum λexcitation for the fluorochrome should
be very similar to our excitation light source. Very fast excitation/emission
changes are required sometimes to follow fast biological processes.
- Loading. Many indicators are available as acetoxymethylester
(AM), but some of them have to be microinjected or electroporated
inside the cell. These compounds require a special equipment and training.
- Compartmentalization. Some dyes tend to accumulate
inside intracellular reservoirs (endoplasmic reticulum, mitochondria,
vesicles, etc.). This behaviour depends not only on the dye used,
but also on the loading conditions and cell type. There is not a general
rule, but sometimes it can be overcome it the loading protocol is
methods. Though non-ratiometric indicators are easier to measure and
quantify, ratiometric indicators are more insensitive to bleaching,
concentration differences, optical path, etc. giving robust measurements.
- Quantum efficiency/photobleaching. A high quantum
efficiency gives a stronger fluorescence signal, allowing to work
with lower laser intensities. It is important to use low excitation
intensities in order to minimize photobleaching. If UV light is used,
higher intensities can induce phototoxicity.