Super- Resolution Microscopy (SRM) Imaging Services

Introduction

Super-Resolution Microscopy (SRM) represents a paradigm shift in the realm of optical microscopy. Traditional optical microscopes are constrained by the diffraction limit, which restricts their resolving power. However, SRM techniques transcend this limitation, enabling the visualization of structures at the nanometer scale. This revolutionary advancement has been acknowledged with a Nobel prize in 2014 and has since seen a rapid evolution with diverse applications in scientific research.

Applications of Super-Resolution Microscopy (SRM) Imaging in Scientific Research

SRM has found profound applications in biomedical research, particularly in elucidating intricate cellular and molecular processes. For instance, the dynamic processes of plant growth and development have been explored using SRM, providing insights into plant subcellular compartments, cells, tissues, and organs. Moreover, SRM's ability to capture rapid subcellular processes in real-time has been a boon for researchers aiming to understand the nuances of cellular dynamics.

Stochastic Optical Reconstruction Microscopy (STORM) Imaging

STORM is a prominent SRM technique that utilizes stochastic activation of fluorophores to achieve super-resolution. By pinpointing the exact location of individual molecules, STORM creates high-resolution images that are beyond the reach of conventional microscopy. Recent studies have employed STORM to investigate the spatial distribution of nuclear molecules, offering insights into the organization of proteins and histone marks at the nanometric resolution.

Fig.1 The principle of STORM¹.

Stimulated Emission Depletion (STED) Imaging

STED microscopy is another groundbreaking SRM technique that employs a de-excitation beam to suppress fluorescence in regions outside the focal spot. This results in a narrowed effective point spread function, enabling super-resolution imaging. Real-time STED has been applied to study the motion of synaptic vesicles, providing a deeper understanding of synaptic transmission processes.

Fig.2 Three principles underlying STEM microscopy².

Structured Illumination Microscopy (SIM) Imaging

SIM is a technique that employs patterned illumination to enhance spatial resolution. By capturing multiple images with varying illumination patterns, SIM reconstructs a high-resolution image. Recent applications of SIM have been directed towards imaging bacterial cells, revealing intricate details of the cytokinetic FtsZ protein and its organization.

Fig.3 The schematic diagram of the proposed SIM with partially coherent illumination³.

Fluorescence Photoactivation Localization Microscopy (FPALM) Imaging

FPALM is a super-resolution technique that relies on the photoactivation of fluorescent molecules. By localizing individual molecules, FPALM reconstructs images with nanometer precision. This technique has been instrumental in studying cellular structures and physiological processes, offering insights that were previously unattainable with traditional microscopy.

Fig.4 Biplane FPALM setup and axial resolution⁴. (a) Lasers are coupled into a conventional microscope for widefield activation and excitation of photoactivatable fluorescent molecules. (b) Histogram of localized caged fluorescein-labeled antibodies on a coverslip as a function of z-axis position.

Photoactivation Localization Microscopy (PALM) Imaging

PALM, similar to FPALM, is a technique that also relies on the photoactivation of fluorescent molecules. However, the nuances in the methodology and applications can differ. PALM has been widely used in various research areas to provide detailed insights into cellular and molecular structures at the nanoscale.

Fig.5 PALM analysis of protein clustering at the cell surface⁵.

FAQs

Q1: What is the principle behind Super-Resolution Microscopy?

A: SRM techniques surpass the diffraction limit of traditional optical microscopy by employing various methods like stochastic activation of fluorophores or patterned illumination.

Q2: How does STORM differ from STED?

A: While STORM relies on the stochastic activation and localization of fluorophores, STED uses a de-excitation beam to suppress fluorescence outside the focal spot.

Q3: What applications does SRM have in scientific research?

A: SRM has diverse applications, from studying plant growth and development to visualizing rapid subcellular processes and investigating cellular structures at the nanometer scale.

Q4: Are there any limitations to SRM techniques?

A: While SRM offers enhanced resolution, challenges like phototoxicity, sample preparation, and the need for specific fluorescent probes can pose limitations.

Reference

1. Zhi, Yanan, Benquan Wang, and Xincheng Yao. "Super-resolution scanning laser microscopy based on virtually structured detection." Critical Reviews™ in Biomedical Engineering 43.4 (2015).
2. Dodgson, James, et al. "Super-resolution microscopy: SIM, STED and localization microscopy." Advanced microscopy in mycology (2015): 47-60.
3. Wen, Kai, et al. "Structured illumination microscopy with partially coherent illumination for phase and fluorescent imaging." Optics Express 29.21 (2021): 33679-33693.
4. Juette, Manuel F., et al. "Three-dimensional sub–100 nm resolution fluorescence microscopy of thick samples." Nature methods 5.6 (2008): 527-529.
5. Owen, Dylan M., and Katharina Gaus. "Imaging lipid domains in cell membranes: the advent of super-resolution fluorescence microscopy." Frontiers in plant science 4 (2013): 503.