Measurement in the confocal microscope.
All measurements require that the microscope must be aligned as accurately as possible, and the gain (or PMT voltage) and black level must be set to avoid any overflow or underflow. Measuring surface profiles and relative depths is straightforward and can be carried out to a higher accuracy than the depth resolution of the microscopes, even though the actual images may look poor. Measuring the thickness of objects which are labeled throughout is less accurate. Length and 2D area measurements are common image analysis problems and easily carried out with image analysis software. Volume measurements are conceptually equally simple but require manual techniques or 3D analysis software. 3D surface area measurements require specialist software, or can be carried out with stereological techniques. Fluorescence intensity measurements require careful calibration. For ratiometric measurements filters and/or laser lines should be chosen to optimize the response and calibration should be done in conditions as close as possible to the experimental ones. FLIM allows exploration of the chemical environment, and multiple labelling even where spectra overlap. When the hardware is available it is also usually the method of choice for measuring FRET, which can measure molecular interactions in the nanometer range. Without FLIM hardware, either intensity measurements with correction for bleed-through and cross talk or acceptor bleaching are the most popular methods of measuring FRET.
Australian Centre for Microscopy & Microanalysis, University of Sydney, Sydney, NSW, Australia.
Guy
G
Cox
D002138:Calibration; D005453:Fluorescence; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D018613:Microscopy, Confocal / Q000379:methods*; D010316:Particle Size; D012984:Software