Test Samples for Optimizing STORM Super-Resolution Microscopy

Prepare a simple test sample by coating glass with fluorescent dextran to create a uniform reference surface. This baseline sample is used to assess basic microscope performance and optical properties.

Prepare actin filament networks on glass coverslips as a structured test sample. This sample provides known filament geometry to validate resolution and assess image reconstruction accuracy.

Prepare microsphere reference beads that serve as fiducial markers for tracking microscope drift during acquisition. These beads enable spatial calibration and drift correction in subsequent processing steps.

Label EGF receptors in fixed cells as a biological test sample. This demonstrates STORM performance on actual cellular structures and validates the protocol on relevant biological targets.

Perform microscopy data acquisition on prepared samples using Alexa 647 photoactivatable dye. Collect raw single-molecule localization sequences under optimized illumination and detection conditions.

Process raw STORM acquisition data through rainSTORM software to localize individual molecules and generate super-resolution images. Apply reconstruction algorithms to achieve final 30-50 nm resolution.

Use fiducial microsphere bead positions tracked throughout acquisition to correct for microscope stage drift. Implement box-tracking and drift correction algorithms within rainSTORM to refine image accuracy.

Analyze reconstructed images from all three test samples to assess microscope resolution, accuracy, and consistency. Compare measured versus expected features to validate system optimization and performance.