Correlative Light and Electron Microscopy (CLEM) combines the strengths of two distinct microscopic techniques. This method allows researchers to first observe dynamic processes within living cells using light microscopy, and then to examine the fine details of the same cells or specific regions of interest at a much higher resolution using electron microscopy. By bridging the observational gap, CLEM provides a more complete understanding of biological phenomena, linking cellular function with underlying structural organization.
The Need for Correlative Microscopy
Light microscopy offers unique advantages for studying biological systems. It allows researchers to image live cells, observing dynamic processes such as protein movement or organelle reorganization in real-time. Fluorescent tags can be attached to specific molecules or structures, making it possible to track their behavior and interactions within a living environment. However, light microscopy faces a fundamental limitation in its resolving power, known as the diffraction limit, which restricts its ability to distinguish objects closer than approximately 200 nanometers. This means that fine cellular ultrastructure, like individual proteins or the precise details of membranes, remains unresolved.
Electron microscopy, conversely, offers significantly higher resolution, often reaching down to 0.2 nanometers. This unparalleled resolution allows for the visualization of cellular ultrastructure, including the precise arrangement of organelles and intricate details of molecular complexes. Despite this exceptional detail, electron microscopy has its own constraints. Samples must be prepared through processes like fixation, dehydration, and embedding, which are incompatible with living cells. The electron beam and vacuum environment required for imaging are detrimental to live specimens, meaning dynamic processes cannot be directly observed. Furthermore, electron micrographs are typically black and white, and extensive sample preparation can sometimes introduce artifacts.
Principles of Correlative Microscopy
The core principle of correlative light and electron microscopy involves a precise workflow to ensure the same region of interest is analyzed by both techniques. The process typically begins with light microscopy, often using fluorescence microscopy, to identify a specific cellular event or structure. Researchers capture images or videos of this region, noting its position and dynamic behavior. This initial observation establishes the functional context or identifies rare events.
Following light microscopy, the sample undergoes specialized preparation for electron microscopy. This preparation aims to preserve both ultrastructural integrity for electron microscopy and the fluorescent signal from initial light microscopy observations. Fixation methods, such as chemical fixation using aldehydes, are carefully chosen to stabilize cellular components while minimizing damage to fluorophores. Subsequently, the sample is typically embedded in a resin that is transparent to light and stable under the electron beam.
A defining step in CLEM is the accurate alignment of images obtained from the two different microscopes. This is commonly achieved through fiducial markers, which are small objects visible in both light and electron micrographs. Examples include fluorescent beads that also contain electron-dense material, such as gold nanoparticles. These markers serve as reference points, allowing image registration software to precisely overlay the light and electron microscopy datasets. The software matches marker positions or recognizable structural features, transforming and rotating images until they are aligned, creating a combined view.
Different approaches exist within CLEM, adapted based on the specific research question and sample type. In “on-section” CLEM, light microscopy is performed directly on ultrathin sections of embedded tissue already prepared for electron microscopy. Alternatively, “pre-embedding” CLEM involves observing live cells with light microscopy, then fixing, embedding, and sectioning the cells, with careful tracking of the region of interest to ensure the same area is examined by electron microscopy.
Applications Across Science
Correlative light and electron microscopy has significantly advanced cell biology by allowing researchers to connect dynamic cellular processes with their underlying structural components. For instance, scientists can observe the intricate stages of endocytosis by tracking fluorescently labeled molecules with light microscopy. Following this, CLEM allows for high-resolution visualization of the precise membrane curvature, vesicle formation, and protein machinery involved at the electron microscopy level within the exact same internalized structure. Similarly, autophagy, where cells recycle damaged components, can be monitored live, with subsequent electron microscopy revealing the detailed ultrastructure of autophagosomes.
In neuroscience, CLEM offers significant opportunities to investigate the structural basis of neuronal function and disease. Researchers can use light microscopy to identify specific neurons or synapses exhibiting particular activity patterns, such as during synaptic plasticity. Subsequently, electron microscopy is employed to reveal nanoscale architectural changes within these identified synapses, such as alterations in the density of synaptic vesicles or the morphology of dendritic spines. This helps in understanding how functional changes observed with light microscopy are encoded in the brain’s fine structure, and how these structures are affected in neurodegenerative conditions.
CLEM also plays a role in pathology and disease research, enabling a deeper understanding of various ailments. Pathologists can use light microscopy to identify specific disease markers or abnormal cellular changes in tissue sections, such as cancerous cells or regions affected by infection. The correlative approach then allows for a detailed examination of the underlying ultrastructural alterations within these identified areas using electron microscopy. This can include visualizing damaged organelles, precisely localizing pathogens within host cells, or characterizing the formation of abnormal protein aggregates. This detailed view at the nanoscale helps in elucidating disease mechanisms and identifying potential targets for therapeutic interventions.