In the past decade, our lab has collected more than 26,000 tomograms of more than 200 different biological specimens. The wealth of information from our group and others is wasted without a way to curate the data. In response to this need, we developed the Caltech Tomography Database, a secure, searchable repository tailored to 3-D imaging datasets.
One major goal of our lab is to increase the throughput of ECT imaging. A decade ago, it could take all day to collect a few tomographic tilt-series. Now, we can automatically collect a tilt-series every 15 minutes, and we are currently developing even faster data collection. To facilitate high-throughput data processing, we developed an automatic processing pipeline to handle data as it comes off the microscope and perform initial analysis.
Image quality is increasing thanks to advances in cryo-EM technology. We have replaced traditional phosphor-charge-coupled devices with direct detectors capable of recording individual electron hits. We have implemented energy filters to block inelastically-scattered electrons, increasing resolution of thick biological samples. We plan to implement phase plates to boost contrast, allowing data collection with less radiation damage.
One of the main difficulties in resolving a structure by ECT is identifying it in the complicated environment of the cell. We were the first to use correlated light microscopy and ECT of a frozen cell to identify a subcellular object, and have since used it to identify several structures in vivo. Taking that a step further, we recently developed correlated cryogenic super-resolution light microscopy (“cryo-PALM”) and ECT. This allows us to pinpoint the location of a fluorescently-tagged protein of interest, which we can then image at high resolution by ECT.