Optical Lock-in Detection (OLID) and OLID-FRET Imaging Microscopy

Abstract

The success of imaging fluorescent proteins within living cells is limited by the presence of background signals - while some improvements to image contrast can be achieved through background subtraction, these benefits diminish when the background signal varies in time and space. Further this approach does not allow imaging below the background signal, a regime of interest for single molecule biology. We have developed an optical lock-in detection (OLID) approach and associated signal analysis that involve modulating the fluorescence emission of the probe through deterministic, optical control of its fluorescent and non-fluorescent states, and subsequently applying a lock-in detection method to isolate the modulated signal of interest from non-modulated background signals. The lock-in detection method involves pixel-by-pixel based cross-correlation analysis that maps the correlation between the total fluorescence emission within single pixels of an image detected over several cycles of optical switching, and a reference waveform that represents the defined response of the switch probe to the switching cycle. The fundamentally new approach to imaging, which is similar in principle to radar, allows for the selective detection of emission from optical switch probes even in the presence of a larger population of conventional fluorescent probes. We have developed new synthetic and genetically-encoded optical switches for OLID and OLID-FRET imaging in living cells and tissues including nitrospirobenzopyran and naphthozaxine based probes as well as fusion proteins of Dronpa and a new optically switchable mcherry. OLID imaging using these probes is remarkably efficient at imaging specific structures and proteins in high and time varying background signal environments such as in living cells, in culture, in brain slices and in live Xenopus embryos and zebrafish larvae.