Correlative microscopy combines different imaging techniques to provide a comprehensive understanding of a sample. This method allows researchers to observe the same area of a cell or tissue using multiple modalities, such as light and electron microscopy, and then combine the resulting data. This integration offers insights a single technique cannot provide.
Why Combine Microscopy Techniques?
Individual microscopy techniques possess inherent trade-offs, limiting the complete understanding of complex biological and material systems. Light microscopy, for instance, offers the advantage of imaging living samples and providing a broad contextual view of cells and tissues. However, its resolution is limited by the wavelength of visible light, typically around 0.2 micrometers, making it challenging to discern fine sub-cellular structures like mitochondria or ribosomes.
Electron microscopy provides high resolution, enabling visualization of structures at the nanometer scale, like organelles and individual molecules. This detailed view has limitations. Samples usually require fixation, dehydration, and often heavy metal coating, preventing direct observation of living processes. Sample preparation can also be time-consuming.
Additionally, electron microscopes offer a narrow field of view, making it difficult to locate specific events. Correlative microscopy addresses these limitations by leveraging complementary strengths, allowing researchers to first identify areas with light microscopy and then examine them in detail with electron microscopy.
The Process of Correlative Microscopy
Performing correlative microscopy involves a workflow to ensure that the exact same region of interest is imaged by different techniques. The process often begins with preparing the sample, which might involve marking specific areas with fiducial markers—tiny, identifiable reference points visible under both light and electron microscopes. For biological samples, cells might be grown on specialized grids or treated to preserve both their fluorescence and ultrastructure.
Following preparation, initial imaging is typically performed using a light microscope, such as a fluorescence microscope, to locate and capture images of specific cells, organelles, or dynamic events of interest. This step provides functional information and a broader context.
After light microscopy, the sample is carefully transferred to an electron microscope. This transfer requires precise stage control to ensure the previously identified area is relocated accurately.
The electron microscope then images the identical region, providing high-resolution ultrastructural details. For cryo-correlative light and electron microscopy (cryo-CLEM), both imaging steps are performed under cryogenic conditions, which helps preserve the sample in a near-native state and reduces damage.
Finally, images from both microscopes are computationally aligned and overlaid using specialized software. This creates a unified view, combining functional insights from light microscopy with detailed structural information from electron microscopy.
Unlocking Biological Insights
Correlative microscopy has advanced our understanding of biological systems by allowing scientists to bridge the gap between cellular function and ultrastructure. One notable application is in studying how viruses infect cells, where light microscopy can track the initial interaction and entry, while electron microscopy reveals the precise structural changes within the host cell and the virus’s location at a nanometer scale. This combined view offers a detailed understanding of the infection mechanism.
The technique has also been instrumental in dissecting complex cellular processes like cell division and migration. Researchers can use fluorescent labels to observe the dynamics of the mitotic spindle during cell division or track the movement of adhesion molecules during cell migration using light microscopy. Subsequently, electron microscopy provides high-resolution images of the underlying cytoskeletal arrangements and organelle interactions that drive these processes, offering insights into the mechanics at play.
Correlative microscopy also aids in understanding disease mechanisms, such as how bacteria interact with host tissues. It precisely localizes pathogens within host cells and observes resulting cellular damage.
Beyond Biology: Other Scientific Discoveries
The utility of correlative microscopy extends far beyond biological research, demonstrating its versatility across various scientific disciplines. In materials science, this approach is applied to analyze defects in newly developed materials, such as those found in CoNi-based superalloys, by combining techniques like transmission electron microscopy (TEM) with 3D atom probe microscopy. This allows researchers to understand how defects and deformations form and affect material properties.
Correlative microscopy also plays a role in characterizing composite structures and understanding material degradation mechanisms. Researchers can use light microscopy for an initial overview of a material’s morphology and then employ electron microscopy for detailed nanoscale imaging of its composition and structure.
In nanotechnology, it is used to characterize nanoparticles, determining their size, shape, and composition, and observing their interactions with their environment. This is particularly relevant for applications like drug delivery systems.