Super Resolution Microscopy

Traditional fluorescence microscopy can only image features down to a resolution of about 200 nanometers. Advances in modern imaging approaches, however, have broken the diffraction limit of light, giving rise to super resolution microscopy. Imaging methods using super resolution techniques allow researchers to observe structures at the nanoscale, providing exceptional resolution and insights into the molecular landscape. These techniques in combination with specific ligands (e.g, Janelia Fluor® HaloTag® Ligands) have revolutionized cell biology by allowing researchers to observe and analyze molecular processes with unprecedented detail.

Common terms related to super resolution microscopy are described below:

Blinking: Light emitting species including organic dye molecules, fluorescent proteins, and semiconductor molecules switch between an “on” (fluorescent) and “off” (non-fluorescent) state. Blinking may be inherent to certain fluorophores or can be prompted by specific imaging conditions.

Triplet State: Refers to a specific energy state of a fluorophore. It is a higher energy state than the ground state but is characterized by a different spin state compared to the singlet state. The difference in spin state makes the return to ground state (and light emission) less likely or slower and can contribute to photobleaching.

Diffraction Limit: The limit of the resolving power of a conventional light microscope, which is determined by the wavelength of light and the numerical aperture of the microscope lens. In other words, this is the minimum size of a spot to which a lens can focus light.

Quantum Yield: The ratio of the number of protons emitted to the number of photons absorbed. Quantum yield describes the efficiency of a fluorophore to convert the excitation wavelength into fluorescence and is independent of instrument settings. In microscopy, quantum yield impacts a fluorophore’s brightness and photostability as well as the overall image quality.

Cell Biology

Super resolution microscopy techniques, such as STED and STORM, can reveal structures within cells at a much finer detail than traditional fluorescence microscopy. This includes the organization of organelles, cytoskeletal structures, and membrane complexes. This improved spatial distribution of molecules within complexes provides insights into the stoichiometry and organization of molecular assemblies.

Neuroscience

STORM and STED can be used to map the distribution and organization of synaptic proteins, dendritic spines, and neural networks at an unprecedented resolution, aiding in the understanding of synaptic function and neural circuitry.

Pathogen-Host Interactions

STORM allows for the detailed study of the interactions between pathogens (such as viruses and bacteria) and their host cells, providing insights into the mechanisms of infection and immune response at the molecular level.

Direct Imaging (STED Specific)

Unlike techniques that rely on post-acquisition computational reconstruction (such as STORM), STED provides real-time super-resolution images. This direct imaging capability simplifies the process and reduces potential artifacts introduced during image reconstruction.

What is STORM Microscopy?

Stochastic Optical Reconstruction Microscopy (STORM) is a super-resolution microscopy technique that surpasses the diffraction limit of visible light, enabling the visualization of biological structures at the nanometer scale. STORM utilizes the principles of single-molecule localization to achieve high spatial resolution.

How Does STORM Microscopy Work?

The core principle of STORM microscopy relies on the precise localization of individual fluorophores. By temporally separating the activation and imaging of single fluorophores within a densely labeled sample, STORM constructs high-resolution images through iterative cycles of activation, imaging, and localization. The main components and mechanisms of STORM are described below:

Fluorophores: Specially designed photoswitchable or photoactivatable fluorophores are crucial for STORM. These fluorophores can be toggled between a fluorescent and a non-fluorescent state using specific wavelengths of light.

Activation and Imaging: The fluorophores are stochastically activated in sparse subsets, ensuring that only a few emit fluorescence at any given time. This prevents overlap of the emission spots, allowing for precise localization.

Single-Molecule Localization: Each activated fluorophore’s position is determined with high precision by fitting the point spread function (PSF) of its emitted light to a Gaussian profile. The coordinates of individual fluorophores are then recorded.

Image Reconstruction: After many cycles of activation and imaging, a super-resolution image is reconstructed from the accumulated positions of thousands to millions of single fluorophores.

STORM Microscopy Advantages

High Spatial Resolution

Achieves lateral resolution of 10–30 nm and axial resolution of 50–60 nm, which is among the highest of all super resolution methods.

Single-Molecule Detection

STORM relies on the precise localization of individual fluorescent molecules and enables the detection and analysis of individual proteins all the way down to single molecule resolution.

Quantitative Data

The technique provides quantitative information about the number and spatial distribution of molecules within a sample, aiding in understanding molecular stoichiometry and the organization of molecular complexes.

STORM Microscopy Disadvantages

Complex Sample Preparation

Due to the requirement of photoswitchable fluorophores, a limited number of dyes and fluorescent proteins are compatible with this technique.

Slow Image Acquisition

STORM is highly time-intensive, as it relies on capturing many sequential images to reconstruct a final super-resolved image. This also makes this technique less suitable for dynamic processes or live-cell imaging.