The interactions within and between the organelles inside our cells could hold the key to understanding complex diseases such as cancer and neurodegeneration.
Recent advances in imaging techniques are now uncovering the hidden dynamics of these cellular organelles, revealing a new frontier in biology and medicine.
Image Credit: Komsan Loonprom/Shutterstock.com
The Importance of Organelle Behavior
Cells are dynamic systems that carry out a wide range of biochemical processes through specialized subcellular structures known as organelles. These structures are fundamental to maintaining cellular homeostasis, and their dysfunction is implicated in numerous diseases, including cancer, neurodegeneration, and metabolic disorders.
Understanding organelle behavior — how they interact, reorganize, and function under various conditions — is crucial for both basic biology and translational medicine.1
While genomic and proteomic approaches have deepened our knowledge of cellular composition and molecular functions, they fall short of capturing the spatial and temporal intricacies of intracellular dynamics.
Microscopy continues to be an indispensable tool for visualizing subcellular structures, and recent advances in super-resolution and live-cell imaging are providing unprecedented insights into organelle function and behavior.2
Live-Cell Imaging with Super-Resolution Microscopy
Breakthroughs in Imaging Technology
Traditional light microscopy has long been constrained by the diffraction limit, which restricts resolution to approximately 200–300 nm. This limitation has historically prevented researchers from visualizing nanoscopic cellular structures such as nucleosomes (~10 nm) and the synaptic cleft (~20 nm).2
However, super-resolution microscopy techniques have overcome these barriers, enabling the visualization of organelles and their interactions with remarkable precision.
Several super-resolution approaches have emerged, each offering unique advantages. Structured illumination microscopy (SIM) enhances resolution by approximately twofold and is widely used for live-cell imaging.
Stimulated emission depletion (STED) microscopy employs a targeted depletion laser to achieve resolutions below 50 nm.2
Single-molecule localization microscopy or SMLM techniques, such as photoactivated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM), achieve even higher resolution by precisely localizing individual fluorophores or fluorescent tags.2
Expansion microscopy (ExM) represents another innovation in the field, and it can physically enlarge the sample using a swellable hydrogel to enable nanoscale imaging using conventional microscopes.
Moreover, when combined with STORM or SIM, ExM has achieved resolutions as fine as 4 nm. These techniques have significantly enhanced our ability to study organelle architecture and dynamics.2,3
Beyond spatial resolution, advancements in live-cell imaging have also enabled the observation of organelle behavior in real-time. Intravital microscopy (IVM) has facilitated the direct visualization of cellular processes within living organisms, providing valuable insights into biological phenomena in their native physiological context.4
Mitosis in an animal cell Under the Microscope
Insights into Major Cellular Organelles
These groundbreaking advances in imaging techniques, including super-resolution and live-cell imaging, have revealed new aspects of organelle function and interaction, especially about the mitochondria, endoplasmic reticulum (ER), and lysosomes.
As the primary sites of adenosine triphosphate (ATP) production, mitochondria play a central role in cellular metabolism. Advanced imaging techniques using new fluorescent probes have enabled real-time tracking of mitochondrial ATP dynamics, revealing how metabolic shifts, such as glucose depletion, affect ATP distribution.1
These studies have highlighted metabolic heterogeneity across cell types, including the distinct ATP responses observed in cancer cells, providing deeper insight into cellular energy regulation and their link to diseases.5
The ER is integral to protein synthesis, lipid metabolism, and calcium signaling. Super-resolution microscopy has helped visualize ER network remodeling and its interactions with other organelles.2
High-throughput single-particle trajectory analysis, which is based on the paths of ions and other single particles as they move inside cells and between organelles, has provided quantitative insights into ER-lysosome interactions and revealed the intricate details of membrane reorganization and intracellular transport mechanisms.3
Additionally, as key regulators of cellular degradation and recycling, lysosomes have also been extensively studied using advanced imaging probes. Advances in super-resolution imaging have allowed the observation of lysosome-mitochondria interactions and uncovered their role in metabolic adaptation and cellular stress responses.6
Moreover, novel lysosome-targeted biosensors have been developed to monitor lysosomal function in real-time, aiding in drug discovery and disease modeling.1
Microscopy and Artificial Intelligence
Recent research and advances in areas overlapping imaging technology and artificial intelligence (AI) are further transforming the landscape of cell biology research. AI-driven approaches enhance the speed, accuracy, and accessibility of imaging data analysis and reduce reliance on manual processing.7
In electron microscopy, machine learning is being leveraged for image segmentation, denoising, and resolution enhancement. Furthermore, deep learning algorithms are being applied to reconstruct high-resolution images from lower-resolution data, optimizing imaging workflows and improving data interpretability.
Additionally, convolutional neural networks (CNNs) have demonstrated exceptional performance in identifying and quantifying cellular structures, facilitating automated characterization of organelle morphology and defects.7
The primary bottleneck in high-resolution imaging now lies in data analysis rather than image acquisition. However, AI-based automation is streamlining image processing, accelerating data interpretation, and enabling researchers to extract meaningful biological insights more efficiently.7
How Light-Sheet Microscopy Has Evolved With Cutting-Edge Technology
Future Directions in Organelle Imaging
Recent advancements in imaging technology have revolutionized our ability to study organelle dynamics with unprecedented detail.
As these technologies continue to evolve, they will not only enhance our fundamental understanding of cell biology but also pave the way for new diagnostic and therapeutic strategies in disease research.
Several key areas are shaping the future of organelle imaging. Researchers are continuing to refine super-resolution techniques to achieve even greater spatial resolution.
Additionally, innovations in probe design and imaging modalities aim to improve contrast, specificity, and temporal resolution for more precise visualization of organelle dynamics.7,8
Furthermore, researchers have suggested that combining super-resolution microscopy with complementary techniques, such as electron microscopy and cryo-electron tomography, will provide a more comprehensive view of cellular structures.2,7
AI-driven tools are poised to play an increasingly central role in automating image processing and data interpretation. Machine learning models will continue to evolve, enhancing the accuracy of segmentation, tracking, and quantitative analysis of organelle behavior.7
There is also a growing emphasis on engineering novel fluorescent and biosensor probes to enable real-time tracking of organelles under physiological conditions.1,4
As imaging technologies improve, their application in disease modeling and precision medicine is also proposed to expand. High-resolution imaging of patient-derived cells and tissues will provide valuable insights into disease mechanisms, biomarker discovery, and therapeutic development.4
References
- Chin, M. Y., Espinosa, J. A., Pohan, G., Markossian, S., & Arkin, M. R. (2021). Reimagining dots and dashes: Visualizing structure and function of organelles for high-content imaging analysis. Cell chemical biology, 28(3), 320–337. https://doi.org/10.1016/j.chembiol.2021.01.016
- Bond, C., Santiago-Ruiz, A. N., Tang, Q., & Lakadamyali, M. (2022). Technological advances in super-resolution microscopy to study cellular processes. Molecular cell, 82(2), 315–332. https://doi.org/10.1016/j.molcel.2021.12.022
- Parutto, P., Heck, J., Lu, M., Kaminski, C., Avezov, E., Heine, M., & Holcman, D. (2022). High-throughput super-resolution single-particle trajectory analysis reconstructs organelle dynamics and membrane reorganization. Cell reports methods, 2(8), 100277. https://doi.org/10.1016/j.crmeth.2022.100277
- Choo, Y. W., Jeong, J., & Jung, K. (2020). Recent advances in intravital microscopy for investigation of dynamic cellular behavior in vivo. BMB reports, 53(7), 357–366. https://doi.org/10.5483/BMBRep.2020.53.7.069
- Depaoli, M. R., Karsten, F., Madreiter-Sokolowski, C. T., Klec, C., Gottschalk, B., Bischof, H., Eroglu, E., Waldeck-Weiermair, M., Simmen, T., Graier, W. F., & Malli, R. (2018). Real-Time Imaging of Mitochondrial ATP Dynamics Reveals the Metabolic Setting of Single Cells. Cell reports, 25(2), 501–512.e3. https://doi.org/10.1016/j.celrep.2018.09.027
- Wang, H., Fang, G., Chen, H., Hu, M., Cui, Y., Wang, B., Su, Y., Liu, Y., Dong, B., & Shao, X. (2022). Lysosome-Targeted Biosensor for the Super-Resolution Imaging of Lysosome-Mitochondrion Interaction. Frontiers in pharmacology, 13, 865173. https://doi.org/10.3389/fphar.2022.865173
- Botifoll, M., Pinto-Huguet, I., & Arbiol, J. (2022). Machine learning in electron microscopy for advanced nanocharacterization: current developments, available tools and future outlook. Nanoscale horizons, 7(12), 1427–1477. https://doi.org/10.1039/d2nh00377e
- Wu, H., Zhao, Y., Zhou, X., Wu, T., Qian, J., Wu, S., Liu, Y., & Zuo, C. (2024). Super-resolution microscopy reveals new insights into organelle interactions. Advanced imaging, 1(3), 032001. https://doi.org/10.3788/ai.2024.20004
Further Reading