Milestones:Super-Resolved Fluorescence Microscopy, 1992

Title

Super-Resolved Fluorescence Microscopy, 1992

Citation

The first super-resolution image of a biological sample was obtained in 1992 by exciting and collecting light diffracted in the near field of the sample. This breakthrough achievement, called super-resolved fluorescence microscopy, exploited the properties of evanescent waves and made single-molecule microscopy possible. Its successful use in imaging single fluorophores inspired applications in cell biology, microbiology, and neurobiology.

Street address(es) and GPS coordinates of the Milestone Plaque Sites

600 Mountain Avenue, New Providence, NJ 07974, U.S.A., 40.684031,-74.401783

Details of the physical location of the plaque

On wall inside entrance lobby to the left of main entrance.

How the plaque site is protected/secured

The plaque will be placed prior to entering the building and thus visitors do not need to pass through security.

Historical significance of the work

Introduction

Optical microscopy, the use of light to visualize objects, has been a cornerstone of scientific discovery for centuries. However, for over a century, its resolution was fundamentally limited by the diffraction limit, a physical constraint imposed by the wave nature of light. This limit dictated that two objects closer than half the wavelength of light could not be distinguished, effectively blurring fine details and hindering the study of microscopic structures. This limit prevented researchers from resolving structures smaller than approximately 200 nanometers, effectively obscuring the fine details of cellular processes and molecular interactions.

The Diffraction Limit and its Implications

Ernst Abbe, in the late 19th century, formulated the diffraction limit, which states that two objects closer than half the wavelength of light cannot be distinguished as separate entities. This limit effectively restricted optical microscopy to resolving structures larger than approximately 200 nanometers. While electron microscopy offered higher resolution, it was not suitable for studying living cells due to its invasive nature. The diffraction limit hindered the ability to visualize crucial biological processes, such as the intricate organization of cellular components, the dynamics of molecular interactions, and the mechanisms of disease development.

Breaking the Diffraction Barrier: The Birth of Near-Field Scanning Optical Microscopy (NSOM)

In the late 1980s and early 1990s, a revolutionary technique emerged that challenged the diffraction limit: Near-field Scanning Optical Microscopy (NSOM). Unlike conventional microscopes that rely on far-field light, NSOM operates in the near field, exploiting the properties of evanescent waves. These waves, which decay exponentially with distance from the source, can interact with objects at a much smaller scale than the wavelength of light.

Pioneering Work at Bell Labs: Eric Betzig and the Tapered Fiber Probe

Eric Betzig, a researcher at Bell Labs in Murray Hill, New Jersey, played a pivotal role in the development of NSOM. He and his colleagues developed a novel approach using a tapered fiber probe, which allowed for significantly higher resolution and signal intensity compared to previous NSOM designs. This breakthrough, published in Science in 1991, demonstrated the potential of NSOM for high-resolution imaging of various materials, including biological samples.

A Pivotal Moment in 1992: The First Super-Resolution Image of a Biological Sample

A pivotal moment in the history of microscopy occurred in 1992. Researchers led by Betzig, building upon the advancements made with NSOM, successfully obtained a super-resolution image of a biological sample. This breakthrough, achieved using a tapered fiber probe and advanced imaging techniques, demonstrated the ability to overcome the diffraction limit and visualize microscopic structures with unprecedented detail. This achievement marked a significant shift in the field, inspiring future applications in cell biology, microbiology, and neurobiology.

Single-Molecule Localization Microscopy (SMLM) and the Quest for Even Higher Resolution

While NSOM offered a significant improvement in resolution, the quest for even higher resolution continued. The development of single-molecule localization microscopy (SMLM) techniques, such as Photoactivated Localization Microscopy (PALM) and Stochastic Optical Reconstruction Microscopy (STORM), further revolutionized the field. These techniques, inspired by the early work on NSOM, allowed for the localization of individual fluorescent molecules with unprecedented precision, pushing the resolution limit to the nanoscale.

Path to a Nobel Prize

In 2014, Eric Betzig, along with Stefan W. Hell and W.E. Moerner, were awarded the Nobel Prize in Chemistry for their groundbreaking work in developing super-resolved fluorescence microscopy. Betzig's development of PALM (Photo-Activated Localization Microscopy), which uses photo-switchable fluorescent molecules to overcome the diffraction limit, was a key factor in this recognition. PALM has enabled scientists to visualize the intricate organization of cellular components, track the dynamics of molecular interactions, and uncover the mechanisms of disease development with unprecedented detail.

Impact and Applications of Super-Resolved Fluorescence Microscopy

The development of super-resolved fluorescence microscopy, inspired by the early work on NSOM and culminating in the Nobel Prize-winning work of Eric Betzig, Stefan W. Hell, and W.E. Moerner, has revolutionized our understanding of biological systems. This breakthrough has enabled scientists to see beyond the diffraction limit, revealing the intricate details of life at the molecular level. Super-resolved fluorescence microscopy is a powerful tool that continues to transform our understanding of life and inspire new discoveries and technological advancements. Super-resolved fluorescence microscopy has had a profound impact on various fields of science, including cell biology, microbiology, neurobiology, and medicine. It has enabled researchers to:

Visualize the intricate organization of cellular components: Super-resolution microscopy has revealed the precise arrangement of proteins within cells, providing insights into the complex machinery of life.
Track the dynamics of molecular interactions: By visualizing the movement of individual molecules, researchers can now study the intricate dance of proteins and other biomolecules within cells, revealing the mechanisms of cellular processes.
Uncover the mechanisms of disease development: Super-resolution microscopy has provided new insights into the molecular basis of diseases, such as cancer, neurodegenerative disorders, and infectious diseases.
Develop new diagnostic and therapeutic tools: The ability to visualize cellular processes at the nanoscale has opened new avenues for developing more precise diagnostic tools and targeted therapies.

Obstacles that needed to be overcome

Lenses and optical microscopes have been employed for centuries to examine small objects including biological samples. However, the resolution of these optical devices had been limited by the diffraction limit or 1/2 the wavelength of the light being used to examine the object.

The fundamental obstacle (and achievement) was how to be able to image beyond the diffraction limit of imaging which was accomplished in two ways: first, it used near field exploiting evanescent waves for imaging; and second, it used a novel tapered fiber to scan across the sample to develop the image.

Features that set this work apart from similar achievements

Near Field Optical Microscopy was a significant advance in the state of the art in imaging at the time it was discovered (1992) which when exploited with novel fluorescence excitation techniques established the Super-Resolved Fluorescence Microscopy breakthrough. First, it dramatically increased the resolution as compared with optical microscopes using lenses in the far field for imaging. For the first time, details of objects were revealed as never before. And second, it did not use electron microscopes for imaging which avoided damaging the samples under test, especially living biological samples, allowing the discovery to have wide use in bio-imaging ever since.

Significant references

Harris T. D. et al., "Super-Resolution Imaging Spectroscopy" Applied Spectroscopy, v. 48, no 1, 1994 pp 14A-21A
Betzig, Eric; Chichester, Robert J. (November 26, 1993). "Single Molecules Observed by Near-Field Scanning Optical Microscopy". Science. 262 (5138): 1422–1425.
Betzig, Eric; Trautman, Jay K. (July 10, 1992). "Near-Field Optics: Microscopy, Spectroscopy, and Surface Modification Beyond the Diffraction Limit". Science. 257 (5067): 189–195. <br>

Papers on beyond diffraction limit and 1992 observation of single molecules:

Betzig, E., & Trautman, J. K. (1>Supporting Texts992). Near-field optics: Microscopy, spectroscopy, and surface modification beyond the diffraction limit. Science, 257(5075), 189–195. (This paper describes Betzig's early work on near-field microscopy.)
Betzig, E., & Chichester, R. J. (1993). Single molecules observed by near-field scanning optical microscopy. Science, 262(5138), 1422–1425. (This paper demonstrates the ability to image single molecules using near-field microscopy.)

Eric Betzig's Work on PALM

Betzig, E., Patterson, G. H., Sougrat, R., Lindwasser, O. W., Olenych, S., Bonifacino, J. S., ... & Hess, H. F. (2006). Imaging intracellular fluorescent proteins at nanometer resolution. Science, 313(5793), 1642–1645. (This landmark paper describes the development of PALM.)

Other Key Super-Resolution Microscopy Techniques:

Hell, S. W. (2003). Toward fluorescence nanoscopy. Nature Biotechnology, 21(9), 1347–1355. (This paper describes the development of STED microscopy.)
Rust, M. J., Bates, M., & Zhuang, X. (2006). Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nature Methods, 3(10), 793–795. (This paper describes the development of STORM.)

General Background on Super-Resolution Microscopy:

Schermelleh, L., Heintzmann, R., & Leonhardt, H. (2010). A guide to super-resolution fluorescence microscopy. Journal of Cell Science, 123(21), 3791–3797. (This review provides a comprehensive overview of super-resolution microscopy techniques.)
Huang, B., Bates, M., & Zhuang, X. (2009). Super-resolution fluorescence microscopy. Annual Review of Biochemistry, 78, 993–1016. (This review discusses the principles and applications of super-resolution microscopy.)

Nobel Prize Information:

The Nobel Prize in Chemistry 2014. (2014). NobelPrize.org. Retrieved from https://www.nobelprize.org/prizes/chemistry/2014/summary/ (This page provides information about the Nobel Prize)
Betzig, E; Trautman, J.K.; Harris, T.D.; Weiner,J.S. "Breaking the Diffraction Barrier: Optical Microscopy on a Nanometric Scale" Science 251 (5000)1468
Nobel Lecture "Single Molecules, Cells, and Super-Resolution Optics", December 8, 2014

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