Introduction:
Stochastic Optical Reconstruction Microscopy (STORM) on a Nikon Ti-2 LFOV platform for 2D/3D single-molecule localization, achieving ~10× the precision of conventional light microscopy (accuracy down to ~20 nm in XY, ~50 nm in Z).
Main features and applications:
In STochastic Optical Reconstruction Microscopy (STORM) photoswitchable molecules are randomly activated from a dark state to an emissive state, where they are detected, followed by a switch back to a dark state. The process is repeated until most fluorophores have been imaged. From each activation event, the localization of the individual molecules are determined in the subsequent reconstruction. This enables the following:
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Super-resolution mapping of molecular interactions and nanoscale organization.
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Multicolor imaging possible by using different sets of photoswitchable fluorophores.
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3D localization possible (in z) as well using a special cylindrical lens.
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High-speed acquisition modes for smaller fields (~10 s per frame over ~20 µm × 20 µm).
Specifications
Microscope body:
Nikon Ti-2 with N-STORM optics, software-controlled TIRF illumination and Perfect Focus System (PFS).
Compatible sample holders:
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Standard ~25 x 75 mm glass slides + chambered glass slides
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Culture dishes w. diameter: ~30 – 40 mm
Objectives:
Objectives include high-power (HP), super-resolution (SR), high-NA oil/silicon immersion objectives as well as standard objectives.
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10× / NA 0.30 Plan Fluor
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20× / NA 0.40 LWD (3.1 mm), Ph1
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100× / NA 1.35 SR, HP, Plan Apo, Sil
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100× / NA 1.49 SR, HP, Apo TIRF, Oil
Light Sources / filters:
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LU-NV laser unit: 405 nm, 488 nm, 561 nm, 647 nm.
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Standard and TIRF-grade filters for STORM imaging.
Camera:
Hamamatsu ORCA-Flash 4.0 sCMOS w. field of view up to ~80 µm × 80 µm (STORM is typically performed using cropped view, i.e. 512x512 or 256x256).
Stage & Environment:
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Motorized stage and objective for automated multi-point imaging
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Nikon Perfect Focus System (PFS) for hardware-based autofocus
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Prior piezo Z-stage for fast/precise imaging in Z
Software:
NIS-Elements 6.10 with ND acquisition and JOBs.
Location:
V10-602a-0