Molekulare Zellbiologie

SR-SIM: Super-Resolution Structured Illumination Microscopy (SR-SIM) is a widefield imaging technology in which a grid is projected into the laser beam to generate a stripe pattern through interference of diffraction orders and superimposed on the specimen while capturing images. The grid pattern is shifted and rotated between the capture of each image, following processing using a specialized algorithm. High-frequency information can be extracted from the raw data to produce a reconstructed image with an axial resolution up to 150 nm. SR-SIM can be combined with live-cell imaging.
Fig: Super-resolution image of human neuroblastoma cells showing the endoplasmic reticulum in red (mCherry-ER) and mitochondria in green (GFP-TOM20).

FRAP: Fluorescent recovery after photobleaching (FRAP) is an imaging method that is based on laser-scanning microscopy (LSM). It is able to determine the kinetics of diffusion in a living biological specimen, usually by photobleaching a small area with a focused, intense laser pulse followed by monitoring fluorescent recovery driven by active transport or Brownian motion. The repopulation into the bleached area can reveal information about protein interaction partners, organelle continuity and protein trafficking especially into larger protein assemblies or protein aggregates.
Fig: FRAP experiment in human neuroblastoma cells expressing mitochondrial GFP-TOM20. Photobleaching was performed by a high energy 405 nm laser pulse in the red ROI (region of interest), green and blue ROIs served as control regions.

Imaging of photoconvertible proteins: The imaging of proteins fused to Dendra2 provides a unique set of advantageous properties, including optical pulse labeling after irreversible photoconversion from green to red. Upon intense 405 nm or 488 nm light irradiation, Dendra2 undergoes irreversible photoconversion, reflected by a decrease in green and appearance of red fluorescence, resulting in a red-to-green fluorescence ratio of about 4000. This technique is efficiently used for tracking movements of cells, organelles, or proteins and provides an ideal tool for real-time tracking of protein dynamics especially considering protein degradation and fate.
Fig: Photoconversion experiment in human neuroblastoma cells expressing an aggregation-prone protein fused to photoconvertible DDR2. The spectra of the DDR2-fusion protein prior to conversion (Ex490/Em507) are similar to that of GFP and shift to red after conversion (Ex553/Em573). In this experiment, the recruitment of newly synthesized protein to an existing cytoplasmic protein aggregate is visualized by live-cell imaging.

Intraorganellar calcium imaging: This method utilizes the genetically engineered calcium-measuring organelle-entrapped protein indicator proteins (CEPIAs). The spatio-temporal resolution of CEPIA makes it possible to resolve the Ca²⁺ import into individual mitochondria while simultaneously measuring ER and cytosolic Ca²⁺ in a live-cell imaging context. Variants of these Ca²⁺ sensors show a broad range of affinities and display a large dynamic range, allowing superior temporal resolution.
Fig: Calcium live-cell imaging experiment in human neuroblastoma cells expressing R-CEPIA1er (red) and CEPIA2mt (green). The cells were constantly perfused with fresh medium in a flow chamber. Upon application of medium containing 10 µM histamine, Ca²⁺ release from the ER and its uptake by mitochondria can be visualized.

PALM: Photoactivated localization microscopy (PALM) is a super-resolution technique that allows resolution beyond the diffraction limit. It relies on the controlled activation and sampling of sparse subsets of photoconvertable fluorescent molecules and integrates thousands of images to increase the spatial resolution of the optical microscope by at least an order of magnitude (featuring 10 to 20 nm resolution). This facilitates the investigation of biological processes close to the molecular scale.
Fig: 3-Channel photoactivated localization microscopy (PALM) image reconstructed from 5000 single images per channel. Small punctate structures formed by two different nuclear proteins (green and blue) are visualized with a spatial resolution of approx. 40 nm. The nuclear membrane protein Lamin A is shown in red. Scale bar: upper panel 2.5 µm, lower panel 0.5 µm.