Live-Cell Imaging with Super-Resolution Microscopy

Super-Resolution Microscopy transforms Live-Cell Imaging, offering unprecedented glimpses into cellular mechanisms with nanoscale precision. This cutting-edge technique is revolutionizing the understanding of biological processes, paving the way for breakthroughs in medical research.

Image Credit: Konstantin Kolosov/

Image Credit: Konstantin Kolosov/

Introduction to Super-Resolution Microscopy

The journey of microscopy began in the late 16th century with the invention of the compound microscope.¹ Over the centuries, scientists have been able to peer deeper into the world of the minuscule, revealing the complex structures of cells and microorganisms. However, the resolution of traditional light microscopy was limited by the diffraction limit, as described by Ernst Abbe.²

The advent of super-resolution techniques at the turn of the 21st century marked a revolutionary departure from this limitation. Techniques such as STED (Stimulated Emission Depletion)³, PALM (Photoactivated Localization Microscopy)⁴, and STORM (Stochastic Optical Reconstruction Microscopy)⁵ have enabled scientists to observe structures at the nanoscale, far beyond the diffraction limit of light.

Super-resolution microscopy encompasses several techniques, but they all share the basic principle of circumventing the diffraction limit to achieve higher resolution. STED uses a de-excitation laser to minimize the area of fluorescence, allowing for imaging at a higher resolution.³ PALM and STORM rely on the precise localization of individual fluorescent molecules that are switched on and off, building a composite image that reveals structures at a nanometer scale (10-30nm).⁴,

The Leap to Live-Cell Imaging

Traditional microscopy techniques offer high resolution but cannot be used to image living cells. They involve fixing cells, which prevents capturing cell dynamics. In contrast, live-cell imaging studies cells in their growth medium, providing real-time spatio-temporal insights into cellular behavior.

This technique transforms snapshots into movies, visualizing and quantifying cellular processes over time. Observing cellular processes in real-time is crucial because it can reveal transient events that might be missed in end-point assays. It allows for the study of cellular structures in their native environment and provides insights into the dynamics and kinetics of cellular processes, such as protein interactions, trafficking, and signaling pathways, making them less prone to experimental artifacts.

Live-cell imaging has become an essential tool for comprehending dynamic molecular events in single cells and cellular networks.

Technological Advancements in Live-Cell Imaging

Recent technological advancements have significantly improved live-cell super-resolution imaging. Innovations such as improved fluorescent probes, faster and more sensitive cameras, and sophisticated computational algorithms have enabled researchers to image living cells with minimal disturbance to their natural state.

Recent advancements in super-resolution microscopy, particularly the MINFLUX technique, have revolutionized our ability to image at the nanoscale. MINFLUX combines minimal photon fluxes with standard fluorescence microscopy to achieve 1-3 nm 3D resolution, making molecule-scale imaging broadly accessible.⁶,

Innovations such as synchronized beam steering and active-feedback stabilization allow for nanometer-precise localization of fluorophores in real-time. This method significantly outpaces traditional camera-based localization techniques like PALM/STORM, offering faster tracking capabilities.⁶,⁷ With these breakthroughs, MINFLUX has opened new avenues for investigating life sciences at the nanometric level.⁶,

Other advancements in super-resolution microscopy involve the use of nanoprobes and photostable fluorescent nanomaterials.⁸ These new probes have reduced inconveniences like photobleaching and phototoxicity associated with PALM, STORM, and STED. These innovations enable lower light power usage, enhanced photostability, and improved long-term imaging of live samples at high resolution.⁸

Case Studies: Breakthroughs in Cellular Biology

A good example of how Super-Resolution Microscopy is aiding in disease research is the study by Barabás et al. (2021).⁹ The study focuses on neurotrophin receptors, specifically TrkA and p75NTR, which are vital for neuronal survival and are known to be altered in Alzheimer's disease (AD).⁹

By employing live-cell single-molecule imaging techniques, the researchers examined the behavior of these receptors on the surface of live neurons derived from human-induced pluripotent stem cells (hiPSCs) of familial AD (fAD) patients with presenilin 1 (PSEN1) mutations, as well as from non-demented control subjects.⁹

The findings demonstrate that the movement of TrkA and p75NTR on the cell surface and the activation of their related signaling pathways, specifically PI3K/AKT, is disrupted in neurons from fAD patients compared to controls.⁹ This altered receptor trafficking suggests a potential mechanism contributing to the neuronal dysfunction observed in AD.⁹

The study underscores the importance of understanding receptor dynamics in disease conditions, which could lead to the development of new therapeutic approaches for AD.⁹ The ability to observe these cellular processes in real-time has profound implications for disease research and drug development. It will help in the direct observation of the effects of pharmacological agents on cells, providing a powerful tool for drug screening and development.

Challenges and Considerations

Live-cell imaging is a complex technique that faces technical challenges such as maintaining cell health under artificial conditions, controlling the imaging environment, and minimizing phototoxicity and photobleaching.¹⁰ Precise labeling of cellular components without affecting cell function, managing focus drift, and balancing imaging speed with resolution are also critical.¹⁰

Additionally, the large data volumes require efficient management, and advanced microscopy techniques demand high sensitivity and expertise.¹⁰ Overcoming these hurdles is essential for accurate real-time observation of cellular processes.¹⁰

Ethical considerations in live-cell imaging include ensuring informed consent for the use of human-derived cells, protecting donor privacy, managing data responsibly, respecting commercialization boundaries, and providing equitable access to personalized medicine.¹¹ Additionally, research must adhere to guidelines, especially when involving vulnerable populations or sensitive biological materials.¹¹


Super-resolution microscopy has revolutionized live-cell imaging, offering nanoscale insights into cellular processes and driving advances in medical research. By enabling real-time visualization of dynamic cellular events, this technology has enhanced our understanding of disease mechanisms and facilitated the development of novel therapies.

Future prospects include overcoming technical challenges to improve cell viability and imaging quality and managing large data sets. As techniques like MINFLUX advance, it is expected that even more precise imaging will be developed, fostering breakthroughs in cellular biology and structural biology.


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  2. Beyond the diffraction limit. (2009). Nature Photonics, 3(7), 361–361.
  3. Oracz J, et al. (2017). Photobleaching in STED nanoscopy and its dependence on the photon flux applied for reversible silencing of the fluorophore. Scientific Reports, 7(1).
  4. Hess S. T, et al. (2006). Ultra-High Resolution Imaging by Fluorescence Photoactivation Localization Microscopy. Biophysical Journal, 91(11), 4258–4272.
  5. Rust M. J, et al. (2006). Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nature Methods, 3(10), 793–796.
  6. New super-resolution microscope combines Nobel-winning technologies (2020); Physics World. Physics World. [Online]
  7. Schmidt R, et al.(2021). MINFLUX nanometer-scale 3D imaging and microsecond-range tracking on a common fluorescence microscope. Nature Communications, 12(1).
  8. Liu Y, et al.(2021). Shedding New Lights Into STED Microscopy: Emerging Nanoprobes for Imaging. Frontiers in Chemistry, 9.
  9. Barabás K, et al. (2021). Live-Cell Imaging of Single Neurotrophin Receptor Molecules on Human Neurons in Alzheimer's Disease. International Journal of Molecular Sciences, 22(24), 13260.
  10. Maintaining Live Cells on the Microscope Stage. (n.d.). Nikon's MicroscopyU. [Online]
  11. de Jongh D, et al. (2022). Organoids: a systematic review of ethical issues. Stem Cell Research & Therapy, 13(1).

Further Reading


Last Updated: Feb 13, 2024

Deliana Infante

Written by

Deliana Infante

I am Deliana, a biologist from the Simón Bolívar University (Venezuela). I have been working in research laboratories since 2016. In 2019, I joined The Immunopathology Laboratory of the Venezuelan Institute for Scientific Research (IVIC) as a research-associated professional, that is, a research assistant.


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