From its introduction in 1967 by Ploem (1), reflected light fluorescence microscopy, commonly called "epi-fluorescence," has enjoyed wide acceptance. Its optical path is relatively simple: full-spectrum light passing through an excitation filter is reflected by the dichromatic mirror into the objective lens to illuminate the sample; the excited sample emits fluorescent light, which is re-collected by the objective lens and passed through the emission filter to the camera. The recent development of biosensors based on genetically encoded variants of green fluorescent protein (GFP), coupled with advances in digital, multi-modes, epi-fluorescence microscopy, has introduced new powerful tools for observing protein dynamics and proteinprotein interactions at high spatial and temporal resolution within living cells. However, there are some disadvantages inherent in epifluorescence microscopy: a) mechanical switching of filter cubes to view different color fluorescence can cause misalignment of images; b) multi-pass filter cubes can eliminate the misalignment problem, but may attenuate the emission light; and c) the epi-fluorescent light source cannot be used in combination with transmitted light techniques such as Phase or differential interference contrast (DIC) microscopy.
展开▼
