OGB-5N loaded neuron. Image courtesy of Dr.Cho

Conventional microscopy with a CCD camera is the simplest approach to conduct functional imaging. When performing VSD imaging, the CCD camera needs to be fast enough to capture millisecond events. When performing Ca.I, image acquisition speed does not need to be as fast as VSD imaging. However, since calcium indicator fluorescence emission is not strong, it requires a sensitive and low noise camera, exceeding the capability of a conventional video camera. When performing Cal.I with wide-field epifluorescence microscopy and a moderate to high magnification objective lens, individual cellular responses may contain signals from cells in out of focus planes. One may also need to consider photo-toxicity when using UV light as an excitation source for calcium (or chloride) indicators. The image acquisition speed of conventional scanning confocal microscopy is limited to 1 to 10 Hz frame rates and usually it is not suitable for VSD imaging. However, it provides powerful, three dimensional resolution for Cal.I. The penetration depth of visible excitation laser light (usually utilized in confocal microscopy) may be limited to 50 to 80 microns from the surface and that may in turn restrict the structures one can image with this technique. Depending on experimental demands, an image acquisition rate of 10 Hz may not be sufficient. Various strategies can be implemented to overcome this temporal resolution issue, including (a) scanning a small area, (b) conducting line scans to further limit scan area, (c) specifying arbitrary line traces of varying configurations, (d) conducting point by point scanning, (e) use of a high speed scanning device (rather than a conventional galvonmeter) such as a resonant galvo-scanner or an accousto-optical deflector (AOD). Alternatively, multi-beam scanning devices such as a spinning disk or a swept-field confocal microscope can provide high image acquisition rates, as fast as 25 Hz to 2 kHz depending on the CCD camera in the system, and the amount of light released by the experimental preparation (Table 2). One of the advantages of two photon over confocal microscopy is that, since infrared light interacts less with the tissue, one can image fluorescence in deeper structures; easily 200 microns from the surface and up to 1 mm depths in some cases. Another reason why one may want to use two photon microscopy is to avoid toxicity associated with use of UV light for UV-excitable dyes such as fura-2 or MQAE. Image acquisition rates are limited to 1 to 10 Hz, as in the case of scanning confocal microscopy, and therefore two photon microscopy usually is not suitable for VSD imaging unless performing point scanning or line scanning. Multi-beam multi-photon microscopes are sufficiently fast to allow for conduct of Cal∙I or even VSD imaging.