Dual Color STED Microscopy with Ultrafast Photon Counting

Abstract

To more precisely quantify association between cardiac proteins, we built a dual-channel super-resolution stimulation emission depletion (STED) microscope with an ultrafast photon-counting acquisition system. STED microscopy is unique among super-resolution technologies due to its capability of optical sectioning and fast imaging speed. A major limitation of STED is the aggravated photobleahching, to which fast scanning speed is an excellent solution. In order to keep up with fast scanning speed and to reduce counting nonlinearity, we invented a photon counting acquisition system based on ultra-fast analog-to-digital conversion (ADC), pushing the readout rate to the gigahertz range. Incorporated into a resonant scanning confocal microscope, this system can acquire images at 16,000 lines per second and 58,000 pixels per line. By adding a continuous wave (CW) depletion laser and photon arrival time control elements, the confocal microscope was converted to a time-gated STED microscope. A second STED channel was built with picosecond depletion pulses. The dual-channel STED microscope is able to achieve a resolution of ∼40 nm in biological samples which allows a more stringent analysis of colocalization in native systems.