All biological specimens, from organelles to bacteria, somatic cell layers to tissue, and model organisms, are three-dimensional. A greater understanding of the structure-function relationship in cells and tissues is now within reach through high-resolution correlation of chemical markers and structural components in three dimensions.
Advances have been made in 3D spatial localization of fluorescent probes with optical sectioning techniques and provide the ability to specifically label proteins, structures, and/or organelles. However, when you image these targets within the sample, light emanates from above and below the focal plane. This effect causes a decrease in both resolution and quality in the resulting image. Optical sectioning helps to achieve 3D, high-contrast images from fluorescent samples. Selective excitation refers to exciting only the fluorophores in the desired focal plane. One way to accomplish this is with multiphoton microscopy, which uses a pulsed laser that allows signal to only come from the raster-scanned focal spot; this method is especially useful for deep-tissue imaging. A new form of selective excitation can be achieved using light sheet microscopy, also referred to as selective plane illumination microscopy (SPIM).
Field-emission scanning electron microscopy (FESEM) is capable of imaging biological microstructures at nanometer scale resolution. In order to generate 3D images using scanning electron microscopy, the samples are often embedded in resin and then physically sliced. One approach is to collect each section in a serial ribbon on tape, glass slide, or slotted grid and image each section, to build up a 3D tomogram of the entire sliced volume. This technique allows each section to be documented and revisited with both the optical and electron microscope and even allows for samples to be modified with different probes, highlighting different structures.
Alternatively, although a destructive technique, thin layers are removed from the “face” of the block containing the embedded sample. The block face is imaged with the FESEM after removal of each thin layer, like removing a page out of a phonebook to read the next page, thereby creating a sequential series of electron microscopy images that can be recombined into a final, 3D reconstruction. Block-face images have significantly lower distortions associated with material removal than the compression seen in the cutting direction of thin sections, thus the final 3D EM reconstructions from this approach align more closely with the 3D light microscopy volume image.
With the block-face imaging technique, there are two primary methods used to remove the thin slices of material, either integrating an ultramicrotome directly into the electron microscope chamber or using a focused ion beam (FIB) to mill away the specimen. While the ultramicrotome approach is a faster method of removal, the FIB is capable of much finer slice thicknesses, thereby improving the z-resolution in the final 3D reconstruction.
Three dimensional EM reconstructions have traditionally been very labor intensive, comprising mostly of manual collection of the thin sections and manual montaging of large area sequential images. By automating EM instrumentation using the ZEISS Atlas 3D and 3View packages, researchers can create high resolution 3D tomograms (3D images) which produce massive amounts of information, previously too difficult or time consuming to collect.
Overlaying these 3D light microscopy and 3D EM images, known as correlative light and electron microscopy (CLEM) is shedding new light on the complex world of biological interactions. This data is generating new understandings of biological systems, from cell vulnerabilities to virus attack, to cancer cell migration mechanisms.
Filed Under: Drug Discovery
