Correlative Light and Electron Microscopy

Correlative Light and Electron Microscopy: Bridging the Gap in Multimodal Imaging

Correlative Light and Electron Microscopy (CLEM) has emerged as a powerful and indispensable tool in the field of biological and materials sciences. This technique integrates the strengths of both light microscopy (LM) and electron microscopy (EM), allowing researchers to obtain comprehensive, multiscale information about biological specimens and materials. In this article by Academic Block, we delve into the principles, applications, challenges, and recent advancements in Correlative Light and Electron Microscopy, shedding light on its pivotal role in unraveling the mysteries at the nanoscale.

Principles of Correlative Light and Electron Microscopy

CLEM involves the sequential or simultaneous acquisition of images using both light and electron microscopy techniques. The fundamental principle lies in combining the advantages of high spatial resolution offered by electron microscopy with the specific labeling and live-cell imaging capabilities of light microscopy. The integration of these two techniques enables researchers to correlate structural and functional information, ultimately providing a more comprehensive understanding of the specimen.

Workflow of Correlative Light and Electron Microscopy:

The CLEM workflow typically begins with live-cell or fluorescent imaging using light microscopy to capture dynamic processes in real-time. Subsequently, the specimen is fixed, embedded, and sectioned for electron microscopy analysis. The correlation of images is achieved through fiducial markers, unique structural features, or advanced software algorithms that align the datasets accurately.

Applications of Correlative Light and Electron Microscopy

Cellular Biology: In cellular biology, CLEM has proven invaluable in elucidating the spatial organization of cellular structures, organelles, and dynamic processes. By combining live-cell imaging with high-resolution electron microscopy, researchers can track cellular events from initiation to the ultrastructural level, providing unprecedented insights into cellular function.

Materials Science: In materials science, CLEM has found applications in studying nanomaterials, catalysts, and other advanced materials. The ability to correlate optical information with nanoscale structural details enables a deeper understanding of material properties, defects, and interfaces, fostering advancements in material design and development.

Neurobiology: CLEM has played a crucial role in neurobiology by allowing researchers to investigate the intricate details of neuronal connections, synapses, and subcellular structures. This has significant implications for understanding neurodegenerative diseases and developing targeted therapies.

Challenges and Solutions in Correlative Light and Electron Microscopy

Despite its numerous advantages, CLEM poses several challenges, including specimen preparation, imaging artifacts, and data correlation. Achieving accurate alignment between light and electron microscopy datasets remains a considerable hurdle. However, ongoing developments in sample preparation techniques, imaging modalities, and computational algorithms are addressing these challenges.

Sample Preparation: One of the critical challenges in CLEM is ensuring the compatibility of specimens for both light and electron microscopy. Sample preparation methods must preserve cellular structures, maintain fluorescence signals, and withstand the harsh conditions of electron microscopy. Recent advances in high-pressure freezing, cryo-fixation, and novel embedding resins contribute to improving the overall quality of specimens.

Image Registration: Accurate image registration is essential for successful correlation between light and electron microscopy images. Fiducial markers, which are visible in both imaging modalities, are commonly used for alignment. However, the development of advanced software algorithms, machine learning approaches, and automated image registration techniques is streamlining and enhancing the precision of this process.

Multimodal Imaging Platforms: To overcome the challenges associated with sequential imaging, multimodal imaging platforms that seamlessly integrate light and electron microscopy are gaining prominence. These platforms allow researchers to switch between imaging modalities without compromising sample integrity, enabling more efficient and reliable correlative studies.

Mathematical equations behind the Correlative Light and Electron Microscopy

Correlative Light and Electron Microscopy (CLEM) involves the integration of two distinct imaging modalities: light microscopy (LM) and electron microscopy (EM). While there are no specific mathematical equations that universally define CLEM, I can provide an overview of the fundamental concepts and mathematical considerations involved in the correlation process.

  1. Image Registration:

    • Image registration is a crucial step in CLEM, where the images obtained from light microscopy and electron microscopy need to be aligned accurately. This process is often performed using mathematical transformations.
    • The transformation can be represented by equations that describe translation, rotation, scaling, and deformation. For instance, a simple 2D transformation might involve the following equations:

      x′ = a⋅x + b⋅y + c ;

      y′ = d⋅x + e⋅y + fx′ ;

    where (x,y) are the coordinates in one image, and (x′,y′) are the transformed coordinates in the other image.

  2. Fiducial Marker-Based Alignment:

    • Fiducial markers are often used as reference points for image registration. The correlation involves finding the transformation that minimizes the difference between the coordinates of the fiducial markers in the two imaging modalities.
    • The mathematical equations for minimizing the difference might involve optimization techniques, such as least squares methods.
  3. Multimodal Imaging:

    • In some CLEM setups, a single instrument may be used for both LM and EM. The mathematical equations here involve the coordination and synchronization of the two imaging modalities.

  4. Fluorescence and Electron Density Correlation:

    • In cases where fluorescent markers are used in LM, there may be efforts to correlate the fluorescence signal with the electron density in EM. This might involve comparing intensity profiles or statistical analyses.
    • The correlation can be expressed mathematically using methods such as Pearson correlation coefficient or cross-correlation functions.
  5. Three-Dimensional Correlation:

    • In many instances, CLEM involves working with three-dimensional data. The mathematical equations for 3D correlation and registration become more complex, often involving transformations in three dimensions.
    • 3D transformations may use rotation matrices, translation vectors, and scaling factors along each axis.

It’s important to note that the specific mathematical equations used in CLEM can vary depending on the imaging modalities, the nature of the samples, and the software or algorithms employed for image registration and correlation.

Recent Advancements and Future Perspectives

Continual advancements in microscopy technologies are pushing the boundaries of CLEM, enabling researchers to explore new frontiers in nanoscale imaging. Super-resolution light microscopy techniques, such as structured illumination microscopy (SIM) and stochastic optical reconstruction microscopy (STORM), are enhancing the optical resolution, while developments in electron microscopy, including cryo-electron microscopy (cryo-EM) and focused ion beam scanning electron microscopy (FIB-SEM), contribute to improved structural insights.

Integration of Artificial Intelligence: The integration of artificial intelligence (AI) and machine learning in CLEM is revolutionizing image analysis and data interpretation. AI algorithms can aid in the automated identification of cellular structures, fiducial markers, and the precise alignment of multimodal datasets, reducing the manual workload and increasing the reliability of correlative studies.

In vivo CLEM: Advancements in live-cell imaging techniques and the development of in vivo CLEM approaches are expanding the possibilities of studying dynamic biological processes in their native environment. This allows researchers to observe cellular events in real-time before transitioning to high-resolution electron microscopy, providing a more comprehensive understanding of biological phenomena.

Final Words

In this article by Academic Block we have seen that, Correlative Light and Electron Microscopy has become an indispensable tool in the scientific toolkit, enabling researchers to bridge the gap between the macroscopic and nanoscopic worlds. The synergistic combination of light and electron microscopy provides a holistic view of biological specimens and materials, offering unprecedented insights into their structure, function, and behavior. As technological advancements continue to enhance the capabilities of CLEM, its applications across various scientific disciplines will undoubtedly flourish, propelling our understanding of the nanoscale to new heights. Please comment below on this article, Thanks for reading!

List Key Discoveries where Correlative Light and Electron Microscopy is used

  1. Neuronal Synapse Structure: CLEM has been pivotal in understanding the intricate structure of neuronal synapses. Researchers have used CLEM to correlate live-cell imaging with high-resolution electron microscopy to unravel the details of synapse formation, organization, and plasticity.

  2. Virus-Host Cell Interaction: Studies involving the interaction between viruses and host cells have benefited significantly from CLEM. By combining live-cell imaging with electron microscopy, researchers have visualized the dynamics of virus entry, intracellular trafficking, and the host response at the ultrastructural level.

  3. Subcellular Organelle Dynamics: CLEM has provided insights into the dynamic behavior of subcellular organelles. For example, the movement and interactions of mitochondria, endosomes, and lysosomes have been studied by combining live-cell imaging with electron microscopy, shedding light on cellular processes such as autophagy.

  4. Cancer Cell Invasion and Metastasis: Understanding cancer cell behavior, especially during invasion and metastasis, has been a major focus of CLEM studies. By correlating optical imaging with electron microscopy, researchers have visualized the morphological changes, intracellular signaling, and interactions involved in cancer cell migration.

  5. Structure-Function Relationships in Cells: CLEM has been instrumental in linking cellular structures to their functions. By correlating fluorescence data with high-resolution electron microscopy, researchers have elucidated the relationships between cellular structures, such as the endoplasmic reticulum and the Golgi apparatus, and their roles in cellular processes.

  6. Nanoparticle Uptake and Distribution: In materials science, CLEM has been used to study the uptake and distribution of nanoparticles within biological systems. By combining fluorescence imaging with electron microscopy, researchers gain insights into how nanomaterials interact with cells and tissues at the nanoscale.

  7. Mitotic Cell Division: CLEM has contributed to our understanding of mitotic cell division by allowing researchers to observe and correlate the dynamics of chromosome segregation, spindle assembly, and cytokinesis in real-time with high-resolution electron microscopy of the dividing cells.

  8. Functional Imaging in Neurobiology: CLEM has been employed in neurobiology to study functional aspects of neurons and neural circuits. By combining functional imaging techniques, such as calcium imaging or optogenetics, with electron microscopy, researchers can link the activity of specific neurons to their ultrastructural features.

  9. Structural Characterization of Nanomaterials: In materials science and nanotechnology, CLEM has been used to correlate optical data with the structural details of nanomaterials. This is crucial for understanding the properties and behavior of nanomaterials in various applications, including catalysis and energy storage.

  10. Stem Cell Differentiation: CLEM has played a role in studying stem cell differentiation by correlating the fluorescence labeling of specific cellular markers with electron microscopy. This has provided insights into the ultrastructural changes associated with cellular differentiation.

Hardware and software required for Correlative Light and Electron Microscopy


  1. Light Microscopy (LM) Setup:

    • Light Microscope: This can be a fluorescence microscope, confocal microscope, or other LM setups suitable for live-cell imaging and acquiring optical data.

  2. Electron Microscopy (EM) Setup:

    • Transmission Electron Microscope (TEM): High-resolution TEM is commonly used for CLEM to provide detailed structural information at the nanoscale.
    • Scanning Electron Microscope (SEM): In some cases, a SEM may be used for complementary imaging.
    • Correlative Microscopy Stage: A specialized stage that can accommodate both LM and EM samples and allows for seamless transition between the two modalities.
  3. Correlative Stage and Sample Holder:

    • Navigation Stage: A stage that enables precise and automated navigation to specific regions of interest for correlative studies.
    • Sample Holder: Specialized holders that can accommodate samples for both LM and EM without compromising the integrity of the specimen during the transition.
  4. Fluorescence and Electron Microscopy Labels:
    • Fluorescent Labels: Fluorophores or other markers suitable for LM, used to label specific structures or molecules in the sample.
    • Electron-Dense Labels: Heavy metal stains or markers used for EM to enhance contrast and identify specific structures.
  5. Cryo-EM Equipment (Optional):

    • Cryo-Electron Microscope: For studies requiring cryogenic conditions to preserve biological samples.
    • Cryo-EM Sample Preparation Tools: Instruments for cryo-fixation, cryo-sectioning, and other cryo-EM sample preparation steps.


  1. Image Acquisition and Analysis Software:

    • Light Microscopy Software: Software for acquiring, processing, and analyzing optical images obtained from the LM.
    • Electron Microscopy Software: Software for controlling the TEM or SEM and acquiring electron microscopy images.
  2. Correlative Software:

    • Image Registration Software: Tools for aligning and registering the LM and EM images. This may involve fiducial-based methods or advanced algorithms.
    • Correlative Analysis Software: Software that allows users to overlay, compare, and analyze LM and EM images in a correlated manner.
  3. Three-Dimensional Reconstruction Software:

    • 3D Reconstruction Software: For studies involving three-dimensional imaging, software that facilitates the reconstruction of 3D structures from serial sections or tomographic data.

  4. Machine Learning and Computational Tools:

    • Machine Learning Algorithms: Increasingly used for automated image registration, fiducial marker detection, and data analysis.
    • Computational Tools: For quantitative analysis, statistical evaluation, and data interpretation.
  5. Data Management and Visualization Tools:

    • Data Storage and Management Systems: To handle large datasets generated by both LM and EM.
    • Visualization Software: Tools for creating merged and annotated images for publication or further analysis.
  6. Instrument Control and Automation Software:
    • Microscope Control Software: Enables coordination between LM and EM instruments, allowing seamless transitions during the correlative process.

Facts on Correlative Light and Electron Microscopy

Multimodal Imaging Integration: Correlative Light and Electron Microscopy (CLEM) integrates light microscopy (LM) and electron microscopy (EM) techniques, allowing researchers to obtain complementary information at different length scales.

Sequential or Simultaneous Imaging: CLEM can be performed either sequentially or simultaneously. Sequential imaging involves acquiring images with one modality and then transitioning to the other, while simultaneous imaging uses specialized setups capable of capturing both LM and EM images concurrently.

Live-Cell and Fixed-Sample Imaging: CLEM is versatile, enabling the correlation of live-cell imaging data from light microscopy with high-resolution structural information obtained from fixed and sectioned samples in electron microscopy.

Fiducial Markers for Image Registration: Image registration is a crucial step in CLEM. Fiducial markers, such as gold nanoparticles or fluorescent beads, are often used to facilitate accurate alignment between LM and EM images.

Sample Preparation Challenges: Sample preparation is a critical aspect of CLEM. Specimens must be prepared to withstand the conditions of both LM and EM, often requiring careful optimization to preserve structures and fluorescence signals.

Advanced Microscopy Techniques: CLEM benefits from advancements in microscopy technologies, including super-resolution light microscopy techniques (e.g., STED, SIM) and high-resolution electron microscopy techniques (e.g., cryo-EM, FIB-SEM).

Correlative Platforms and Stages: Specialized correlative microscopy platforms and stages facilitate the seamless transition between LM and EM imaging. These platforms often include navigation stages and holders designed to accommodate samples for both modalities.

Software Solutions for Image Correlation: Various software tools and algorithms are employed for image registration and correlation in CLEM. These tools aid in aligning the datasets acquired from different imaging modalities and can involve manual or automated approaches.

3D Correlative Microscopy: CLEM is capable of three-dimensional (3D) imaging, allowing researchers to study the spatial organization of structures in all dimensions. This is particularly relevant for understanding complex biological and materials systems.

Applications Across Scientific Disciplines: CLEM has applications in diverse scientific fields, including cellular biology, neurobiology, materials science, virology, and nanotechnology. Its versatility makes it a valuable tool for researchers seeking multiscale information.

Cryo-CLEM for Preserving Cellular Structures: Cryo-CLEM involves cryo-fixation and cryo-sectioning of samples, preserving cellular structures in a near-native state. This is especially important for studying dynamic cellular processes and preserving labile structures.

Machine Learning Integration: Machine learning algorithms are increasingly being integrated into CLEM workflows for tasks such as automated image registration, fiducial marker detection, and data analysis, enhancing the efficiency and accuracy of the correlative process.

In Vivo CLEM for Dynamic Studies: In vivo CLEM approaches allow researchers to study dynamic biological processes in their native environment. This is achieved by correlating live-cell imaging with high-resolution electron microscopy in situ.

Publication and Visualization: CLEM enables the creation of visually compelling images for publication, where correlated LM and EM data are overlaid to provide a comprehensive view of the specimen. Visualization tools play a key role in presenting correlated data effectively.

Contributions to Fundamental Science and Biomedical Research: CLEM has contributed to numerous fundamental discoveries in cellular and molecular biology, providing unprecedented insights into cellular structures, organelles, and dynamic processes. It has also advanced our understanding of diseases and potential therapeutic targets.

Academic References on Correlative Light and Electron Microscopy

  1. Kukulski, W., Schorb, M., & Kaksonen, M. (Eds.). (2019). Correlative Light and Electron Microscopy II. Academic Press.

  2. Murphy, G. E., & Davidson, M. W. (Eds.). (2014). Fundamentals of Light Microscopy and Electronic Imaging. Wiley.

  3. Verkade, P. (Ed.). (2012). Correlative Light and Electron Microscopy. Academic Press.

  4. Faas, F. G. A., & Bárcena, M. (2013). Cryo-electron tomography: 3D imaging of soft and hard tissues. In Methods in Cell Biology (Vol. 124, pp. 145–170). Academic Press.

  5. Schorb, M., Briggs, J. A. G., & Vigers, G. P. A. (2019). Correlated Cryo-fluorescence and Cryo-electron Microscopy with High Spatial Precision and Improved Sensitivity. Ultramicroscopy, 203, 176–182.

  6. Frank, J. (2006). Electron Tomography: Three-Dimensional Imaging with the Transmission Electron Microscope (2nd ed.). Springer.

  7. Peddie, C. J., & Collinson, L. M. (2014). Exploring the third dimension: volume electron microscopy comes of age. Micron, 61, 9–19.

  8. Lucić, V., Rigort, A., & Baumeister, W. (2013). Cryo-electron tomography: the challenge of doing structural biology in situ. Journal of Cell Biology, 202(3), 407–419.

  9. Knott, G., Rosset, S., & Cantoni, M. (2011). Focussed ion beam milling and scanning electron microscopy of brain tissue. Journal of Visualized Experiments, 53, e2588.

  10. Nixon, S. J., Webb, R. I., & Parton, R. G. (2009). A perspective on imaging cell surface membrane domains with electron microscopy. Journal of Structural Biology, 168(1), 142–153.

  11. Rigort, A., Bäuerlein, F. J. B., & Villa, E. (2012). Focused Ion Beam Micromachining of Eukaryotic Cells for Cryoelectron Tomography. Proceedings of the National Academy of Sciences, 109(12), 4449–4454.

  12. Häring, H., Narita, A., & Nitschke, R. (2019). High-Accuracy Correlative Two-Photon and Focused Ion Beam Scanning Electron Microscopy Using Advanced Fluorescent Probes. Scientific Reports, 9(1), 15947.

  13. Kopek, B. G., Shtengel, G., & Xu, C. S. (2017). Correlative 3D superresolution fluorescence and electron microscopy reveal the relationship of mitochondrial nucleoids to membranes. Proceedings of the National Academy of Sciences, 114(30), E6372–E6381.

  14. Schieber, N. L., Machado, P., & Markert, S. M. (2017). Nanoscale Architecture of the Cortical Actin Cytoskeleton in Embryonic Chick Lens Cells. Journal of Structural Biology, 200(3), 236–243.

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