Ultra-sensitivity, speed and resolution: Optimizing low-light microscopy with the back-illuminated electron multiplying CCD

摘要

The back-illuminated Electron Multiplying Charge Coupled Device (EMCCD) camera is having a profound influence on the field of low-light dynamic cellular microscopy, combining highest possible photon collection efficiency with the ability to virtually eliminate the readout noise detection limit. We report here the use of this camera, in 512x512 frame-transfer chip format at 10 MHz pixel readout speed, in optimising a demanding ultra low-light intracellular calcium flux microscopy set-up. The arrangement employed includes a spinning confocal Nipkow disk, which whilst facilitating the need to both generate images at very rapid frame rates and minimize background photons, yields very weak signals. The challenge for the camera lies not just in detecting as many of these scarce photons as possible, but also in operating at a frame rate that meets the temporal resolution requirements of many low-light microscopy approaches, a particular demand of smooth muscle calcium flux microscopy. Results presented illustrate both the significant sensitivity improvement offered by this revolutionary technology over the previous standard in ultra low light CCD detection, the GenIII+ ICCD, and also portray the advanced temporal and spatial resolution capabilities of the EMCCD.