Cell Biology

Many interesting cellular structures are smaller than the optical diffraction limit of ~200-300 nm. These include the substructures of most organelles and all macromolecular machines, channels, and receptors.

Asking and answering questions about the molecular organization, interactions, and stoichiometry of these and other structures requires imaging of labeled structures at resolutions below the diffraction limit of light.
 

Conventional Microscopy Techniques in Cell Biology

Conventional light microscopy methods, like widefield and confocal microscopy, can image and identify specifically labeled cellular structures. However, they are limited in their ability to image below the optical diffraction limit. Alternatively, electron microscopy (EM) can achieve resolutions down to 0.1-0.2 nm laterally, but specific labeling or quantitative measurement of molecules within the cell is very challenging.
 

Super-Resolution Fluorescence Microscopy for Cell Biology

Super-resolution microscopy supports high-resolution imaging—down to 20 nm laterally and 50 nm along the optical axis —of specifically labeled cellular structures. It thereby bridges the gap between conventional light and electron microscopy (EM) techniques.

Mitochondria imaged with conventional fluorescence microscopy (left) vs. SMLM super-resolution microscopy (right) *Right: Mitochondria stained for TOM20 with Alexa 647, imaged with SMLM (dSTORM).

The high resolution (20 nm laterally) paired with the ability to image specifically labeled structures makes SMLM a powerhouse solution in cell biology research. Discoveries made with SMLM include the visualization of local movements of chromatin domains in HeLa cells, the eightfold symmetry within the nuclear pore complex of Xenopus oocytes and the radial ninefold symmetry of the centriolar protein CEP164, and the organization of connected tubular structures within the endoplasmic reticulum[1, 2].
 

Examples of Nanoscale Cellular Structures Suitable for SMLM Research

Super-resolution microscopy supports high-resolution imaging—down to 20 nm laterally and 50 nm along the optical axis —of specifically labeled cellular structures. It thereby bridges the gap between conventional light and electron microscopy (EM) techniques.

The resolution needed to visualize structures of interest must be taken into consideration when deciding if SMLM is the right solution. Some examples of the sizes of diffraction-limited structures suitable for imaging with SMLM are provided. Examples* include (but are not limited to):

• Diffusion channel of nuclear pore complex (40-50nm in length)
• Nucleoid in mitochondria (~100 nm in diameter)
• Microtubules (24 nm in thickness)
• Ribosome (~20-30 nm in diameter)
• Bacterial heat shock protein DegP (20 nm in diameter)
• Cylindrical pyrenoid tubules in the chloroplast (107 ± 26 nm in diameter)
• Endocytic pits in mouse cells (86±2.4nm in diameter)
• Tubular segments of cristae in rat liver mitochondria (30-40 nm in diameter)
• Plasma membrane caveolae (50-100 nm in diameter)
• Hook of the bacterial flagellum (~55 nm in length)

_{*Examples of nanoscale substructures and their dimensions are from 'The database of Useful Biological Numbers'. Link: https://bionumbers.hms.harvard.edu/search.aspx}

Of the super-resolution microscopy approaches, Single-Molecule Localization Microscopy (SMLM) is arguably the best-suited for cell biology research.

Specifically, SMLM is the best method for studying specific nanoscale structures within cells when the research question requires:

• Visualization of molecules located down to 20 nm apart;
• Labeling of multiple specific molecular structures; or
• Quantitative data collection at this scale.
   

SMLM is already being used to answer previously unexplored questions in cell biology. SMLM is ideal for imaging multiple specific structures in 3D and at high resolution, as well as quantifying data. See the sample data and descriptions, below, to learn more about research applications that can be uniquely achieved with SMLM:

Motion Models and Particle Tracking

With SMLM, the movement of molecules within a cell can be imaged in real-time with high spatial resolution. A successful example of single-particle tracking in a bacterial cell with SMLM showed that the actin homologue, MreB, moves in a circumferential pattern around the bacterial cell, driven by cell wall synthesis[4].

_{Right: Two experiments monitoring mitochondrial dynamics, labeled with (1) an orange HaloTag® dye (549) and (2) a photactivatable far-red dye (PA-JF-646®).}

Fixed Sample Imaging

Microtubules acquired on the VXL - dSTORM images of fixed microtubules labeled with Alexa 647 using primary/seconday antibody labeling.

Live Cell Imaging

Acquired on the VXL - Imaging of live BSC1 cells labeled with Alexa 647 transferrin.

Bruker's Vutara VXL offers best-in-class ease of use and imaging depth. Key features supporting the needs of cell biology researchers include:

• Automated Workflows: SRX software makes it easy to acquire the necessary data sets, and workflows allow the user to focus on the biological question instead of tinkering with complicated microscope settings.
• Superior 3D Imaging and Sample Flexibility: Proprietary bi-plane detection provides three-dimensional information and enables imaging within tissue slices.
• Unlimited Multiplexing: The integrated Microfluidics Unit allows sequential labeling of an unlimited number of fluorescent probes.
• Expert Applications Support: Vutara VXL users can receive personalized guidance for sample prep optimization specific to their research objectives.