Single-molecule fluorescence spectroscopy and imaging

Observability of individual fluorescent molecules under a microscope has many applications in biology.1, 2 In particular, by grafting fluorophores to biomolecules, they can be tracked in real time in live cells for monitoring of their conformational changes and interactions with other molecules. In other words, singlemolecule imaging allows scientists to read the textbook of life pretty much as they will end up writing it, i.e., as a series of molecular events involving one or a few partners engaged in a spatiotemporal ballet. Observing single molecules is challenging because very few photons are emitted and even fewer are detected. There is, therefore, a strong incentive to improve detector performance to increase their temporal resolution and throughput. Single-molecule observations are usually performed in either wide-field or confocal geometry, using electron-multiplying CCD cameras or single-photon point detectors, respectively.3 Wide-field imaging with a camera allows looking at many molecules that are immobilized on a surface or in live cells, but this is limited by the camera’s noise and finite frame rate. Confocal microscopy, on the other hand, only illuminates a single microscopic spot in themolecular sample. Light emitted from this spot is collected by a point detector such as a single-photon-counting avalanche diode (SPAD). This approach has excellent temporal resolution but suffers from very low throughput. We have collaboratively explored several strategies to take advantage of the best of both worlds, i.e., the wide-field imaging capability and large parallelism of cameras and the high temporal resolution and noise-free detection of single-photon-counting detectors. In collaboration with the group of Oswald Siegmund (Space Sciences Laboratory of the University of California at Berkeley), we developed a photon-counting camera (see Figure 1) based on a large-area photocathode combinedwith amicrochannel plate to amplify each photo-electron.4 A position-sensitive Figure 1. Operational principle of our photon-counting camera. A fluorescence photon (with energy h ) is collected by the imaging optics and interacts with the photocathode, creating a photo-electron (e ). This photo-electron is amplified by a microchannel-plate stack, which thus generates an electron cloud. Measurement of the delay between the charge pulse at the back of the MCP and the laser pulse provides the nanotime information ( ). A position-sensitive anode determines the position (X, Y) of the electron cloud. A clock provides coarse timing information (T). The four coordinates are asynchronously sent to a computer for storage and processing.