The EMCCD camera is operating as a direct replacement of our old ICCD (intensified CCD) camera for normal microbeam operations and we are exploring a reduction of the UV illumination of the fluorescent stain (Hoechst 33342) that binds to the DNA in the cell nuclei and is used for target identification. We are working with our biological microbeam users in the further reduction of the amount of nuclear stain used – which can have deleterious effects on its own. Requiring fewer fluorescence photons to be generated in the sample for target detection allows the reduction of the amount of stain and the intensity of the UV illumination required to perform targeted irradiations.
The EMCCD camera has given us an opportunity to reduce potentially confounding factors of UV exposure and stain toxicity in our sensitive experiments, such as bystander effects or genomic instabilities, where very slight effects can have huge ramifications on the experimental results.
The EMCCD technology allows the multiplicative gain of the image pixels directly at the readout of the CCD sensor that is mounted in a vacuum housing and cooled to -70 C by a Peltier cooler, reducing the thermal electrical noise in each pixel to less than 1 electron per read. This allows a single photon to generate a large enough signal to be read as part of an image, making extreme low-light imaging possible on a single sensor without a coupled intensifier. The EMCCD camera technology also allows us to acquire enough signal from the sample in our present setup to target our samples in 1 image frame, where previously several frames had to be aggregated, thus increasing the speed and reliability of the sample targeting.
