Fluorescent Dyes:
Introduction - Quantifying calcium - Definitions&References - Protocols&Examples
 

Protocols & Examples

Microscope & Setup

Most experiments can be performed in a wide-field fluorescence microscope, though some experiments have particular requirements that might conditionate our choice. In general, any fluorescent microscope with the required excitation/emission lasers/filters can be adjusted for measuring intracellular calcium, but advantages and restrictions of each equip have to be considered for experimental setup.

Confocal microscopes can record series of thin xy planes to build xyz stacks and render subcellular resolution. Also measurements in xz plane can be performed. These measurements can be interesting for studying calcium distribution along z axis, which cannot be performed with wide-field fluorescence microscopes, and the use of a confocal microscope renders higher spatial resolution. Confocal microscopes can mimic wide-field fluorescence microscopes: opening the pinhole renders thicker sections, similar to those images obtained with CCD cameras, and minimizes the laser intensity required for acquisition of thinner sections. High pinhole values (around 4 or 5 times the optimum value) render good results with HeLa cells using a 63x/1.32 objective (this value changes depending on cell thickness and microscope settings).

Newer confocals microscopes are faster than older ones, so time resolution is usually more than enough. If standard adjustments are not enough for our experiment, some parameters can be modified to increase speed. Adjusting scanning mode (bi-directional), scanning speed (higher values), resolution (it is not always necessary a high resolution image to quantify calcium!), etc. usually renders enough temporal resolution for most experiments.

Studying very fast changes in [Ca2+], as those observed in some cell lines, can be achieved by using an xt scanning mode. It consists on scanning just a line of the sample (x axis) while using the minimum dwell time. If xyt image are important, using a confocal microscope with a Nipkow disk system or a fast-scanning model allows capturing fast cell events without loosing xy information.

 

xzt scanning mode gives a different point of view of the studied cell, so it can render interesting information about calcium concentration changes in z axis.

Not all fluorescent calcium indicators can be measured in a standard confocal microscope. An UV laser is not usually installed (many confocal microscopes only have visible lasers), so indicators requiring UV excitation are not suitable for those microscopes. Also ratiometric methods with an excitation spectral shift require to be excited at two close wavelengths (e.g. 335 and 363 nm for Fura-2) in a very short time (but not simultaneously), which makes many confocal microscopes not suitable for this kind of measurement.

Fluorescence microscopes do not have z resolution, but this is not a major problem for many calcium imaging experiments. Different brands and models of microscope offer different settings, so choose a fluorescent calcium indicator that suits yours (and also your experiment!).

Temporal resolution depends on the microscope, but tricks like those detailed for confocal microscopes can also be used (note that fluorescence microscopes normally use a CCD camera to record images).

Some laboratories have adapted their systems to improve their performance in a particular experiment (see a description of Monck's Pulsed Laser Imaging System for studying kinetics of Ca2+ gradients or visit Monck's Lab web page) and suppliers have also developed high-speed filter wheels, fast shutters, specific software, etc. in order to satisfy the needs of most demanding experiments, including those with ratiometric methods.

 

 

Fluorescent Proteins

 

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