Do you feel there is any decent method for measuring volume of an intracellular object (eg: mitochondria, nucleoli, etc) using confocal microscopy and 3D rendering, or is a good estimate the best technology can currently provide?
It's tough, because confocal does not offer isotropic resolution. X/Y are very accurate, but Z is almost half the resolution. I can imagine that using fluorescent microspheres of known diameter within the sample, or in parallel, you can do an even closer mathematical determination. Also, depending on the chromatic aberration of your lenses, you may need a microsphere of appropriate color for each wavelength you are imaging.
Hi! Jordan´s suggestion on using microspheres for calibration purpose is great. Such microspheres of defined diameter and with different fluorescent labels are commercially available, e.g. from Molecular Probes (look for fluorescent beads). Please consider, that any fixation of your object may cause shrinkage and the measured (calculated) volume may be inaccurate. This should be no problem when performing life cell imaging in an appropriate buffer. For volume determinations, some imaging systems (e.g. ZEN by Zeiss) can be equipped with specific software modules, which address the problem of isotropy, which Jordan has mentioned. Good luck!
It's tough and time consuming, but it's possible. Imaris software is the best in 3D rendering. The image quality is the key, good staining, tight Z-stacks, and optimized deconvulotion, all will make the 3D rendering and the volume measuring easier.
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