ABSTRACT
Quantitative fluorescence microscopy can be performed using either transmitted or epi-illumination optical arrangements. In transmitted illumination, the excitation light is delivered from the bottom of the specimen, passing through the specimen and reaching the detector. This optical arrangement has been found to minimize reabsorption and provide better correlation between fluorescence intensity and fluorophore concentration. Quantitative fluorescence microscopy can also be performed using either photomultiplier tubes or cameras as detectors. After the droplets sink and come to rest (without flattening) on a siliconized coverslip, their diameter can be measured using an ocular micrometer, and their fluorescence measured with the same optics used to measure the real specimen. Droplet volume is then calculated from the droplet diameter. Because fluorescence is linearly related to excitation intensity fluorescence is a very sensitive and accurate way to measure concentration.
