In correlative CFT-SXT imaging the cryopreserved specimen is imaged in a cryogenic high-numerical aperture light microscope prior to being cryo-transferred to the SXM for acquisition of SXT data. Landscape markers, clearly visible in data from both modalities, are used to accurately overlay one data set onto the other to produce a 3D multi-modal reconstruction. In such analyses, labeled-molecules are localized precisely within high-resolution reconstructions of the cell.
The workflow followed to collect and reconstruct a CFT data set. Cells containing fluorescently labeled molecules are pipetted into a thin-walled glass capillary and cryo-immobilized. The capillary is either cryo-transferred to a Dewar for storage or directly to the CLM where it is mounted in the cryo-rotation stage. Through-focus data are collected at a number of different angular rotations around a central axis (typically every 10° over a total angular range of 180°). A deconvolved image is calculated for through-focus series, followed by a tomographic reconstruction of the entire series to generate a 3D fluorescence image.
This combination of data from two complementary modalities produces significantly greater insights than is possible using SXT alone. We confidently predict this correlative method will become an important new tool for elucidating connections between molecular events and cellular phenotype. Correlated CFT-SXT can be applied to virtually any type of cell, presenting enormous imaging opportunities to the cell bio-research community. For this manuscript we will use the eukaryotic nucleus as a working example, since imaging and quantifying this organelle in its near-native state previously proved to be very challenging using other modalities. Using CFT-SXT the nucleus can be fully segmented, based on the degree of chromatin compaction together with guidance from localize fluorescent molecules. Information on the topology and packaging of the genome in the nucleus is invaluable to the generation of mathematical models that describe, for example, the movement of transcription factors and other molecules within the packed nuclear space. The below manuscript describes recent work using CFT-SXT to visualize and quantify the inactive X chromosome (download pdf version here).