Equipments Details
Description
Overview:
Stochastic Optical Reconstruction Microscopy (STORM) is a cutting-edge super-resolution imaging technique that enables fluorescence microscopy to exceed the diffraction limit conventional light microscopy (∼200 nm). Achieving lateral resolutions of ~20 nm and axial resolutions of ~40 nm, STORM reconstructs high-resolution images by localizing individual fluorescent molecules with high spatial precision allowing researchers to study molecular architecture and interactions at an unprecedented level of detail. This is accomplished by using light of different wavelengths to switch fluorophores between "on" (fluorescent) and "off" (non-fluorescent) states in a controlled and cyclical fashion.
In each imaging cycle, only a sparse subset of fluorescent molecules in the field of view is activated so that their individual emissions do not spatially overlap. Each activated molecule appears as a diffraction-limited spot (PSF), which is then fitted computationally (often with a Gaussian function) to determine the precise x–y coordinates of that single fluorophore, typically with nanometer precision. By repeating this process across thousands of cycles—each activating a different, random subset of fluorophores—a comprehensive dataset of molecule localizations is compiled. This dataset is then reconstructed into a super-resolution image with spatial detail far beyond the classical optical diffraction limit.
More technical detail/ detailed description:
STORM imaging in this setup is implemented on an Olympus IX71 inverted microscope frame equipped with a UAPON 100× 1.49 NA OTIRF oil immersion objective lens configured in epiillumination geometry. This high numerical aperture lens provides a diffraction-limited resolution of approximately 230 nm at 560 nm excitation, though STORM itself improves upon this by localizing individual fluorophores far more precisely. Samples are illuminated via a combination of up to four solid-state lasers operating at 405, 488, 561, and 647 nm. The laser beams are merged using dichroic mirrors and delivered through an optical fiber to the microscope’s back port.
To reduce speckle and coherent artefacts caused by laser interference, the optical fiber is mechanically vibrated during operation. The resulting scrambled illumination ensures a uniform excitation field in the imaging plane, crucial for consistent and artifact-free imaging. Light reaches the sample through the high-NA objective, and emitted fluorescence is captured via either a Hamamatsu ORCA Flash 4.0 v2 sCMOS or a Photometrics Evolve 512 EMCCD camera, depending on the experiment's requirements.
The cameras are interfaced with high-speed data acquisition systems that include solid-state drives (SSDs) to accommodate fast data writing. This enables high-frame-rate imaging at up to 100 frames per second, which is essential to minimize motion blur and photobleaching artifacts during long imaging sequences. With a 100× objective, the effective pixel size in the sample plane is ~130 nm, and exposure times are typically set to 10 ms. This allows for rapid capture of dynamic molecular events, which is particularly useful in time-resolved experiments, such as those probing the structural dynamics of peptide aggregates or live-cell imaging.
To maintain focus stability and correct for thermal drift in the axial (z) direction, the setup includes a MadCity Labs C-focus system. Additionally, 3D-STORM imaging is made possible through the integration of a MicAO 3DSR adaptive optics module, which uses engineered elliptical PSFs to localize emitters in three dimensions. This configuration supports detailed 3D reconstructions of complex biological and soft matter structures.
Environmental control during imaging is achieved using a Solent incubator, which allows samples to be maintained in a hydrated, room-temperature environment, making the system well-suited for live-cell and soft matter studies. Furthermore, the apparatus supports Total Internal Reflection Fluorescence (TIRF) imaging, offering high-contrast imaging of surface-adjacent regions such as cell membranes.
Capabilities/ key specifications:
The STORM microscopy system offers a powerful suite of capabilities for high-resolution imaging and molecular-scale investigation. It enables fluorescence imaging with lateral resolution down to ~20 nm and axial resolution of ~40 nm, surpassing the diffraction limit by nearly an order of magnitude. The system supports both 2D and 3D imaging through the use of adaptive optics and engineered point spread functions, allowing for precise spatial mapping of structures in complex biological and soft matter samples. Multicolor imaging is achievable using four solid-state lasers at 405, 488, 561, and 647 nm, enabling multiplexed analysis of different molecular species within the same sample.
Uses/ applications:
The system is equipped for dynamic, time-resolved single-molecule experiments, thanks to high-speed cameras capable of capturing up to 100 frames per second and is supported by real-time thermal drift correction for stability in the z-axis. Additional modalities such as Total Internal Reflection Fluorescence (TIRF) expand its utility for membrane-proximal studies. The apparatus can also be used for dynamic single molecule fluorescence experiments and a number of microfluidic platforms are available.
With compatibility for live-cell imaging in a controlled, hydrated environment and integration with microfluidic platforms, this system provides a highly versatile and sensitive platform for nanoscale investigations in biological physics, molecular biology, and materials science.
Instrument(s) make and model:
Olympus IX71 inverted microscope frame with PRIOR ProScan III sample stage and a MadCity Labs C-focus system to compensate for thermal drift in the z-plane.
UAPON 100× 1.49 NA OTIRF oil immersion objective lens.
405 nm, 488 nm, 561 nm, and 647 nm solid state lasers.
Hamamatsu ORCA Flash 4.0 v2 sCMOS and Photometrics Evolve 512 EMCCD camera.
MicAO 3DSR adaptive optics module with MicAOv1.3 software.
Solent Scientific 3-ORIO7 Incubation chamber.
Stochastic Optical Reconstruction Microscopy (STORM) is a cutting-edge super-resolution imaging technique that enables fluorescence microscopy to exceed the diffraction limit conventional light microscopy (∼200 nm). Achieving lateral resolutions of ~20 nm and axial resolutions of ~40 nm, STORM reconstructs high-resolution images by localizing individual fluorescent molecules with high spatial precision allowing researchers to study molecular architecture and interactions at an unprecedented level of detail. This is accomplished by using light of different wavelengths to switch fluorophores between "on" (fluorescent) and "off" (non-fluorescent) states in a controlled and cyclical fashion.
In each imaging cycle, only a sparse subset of fluorescent molecules in the field of view is activated so that their individual emissions do not spatially overlap. Each activated molecule appears as a diffraction-limited spot (PSF), which is then fitted computationally (often with a Gaussian function) to determine the precise x–y coordinates of that single fluorophore, typically with nanometer precision. By repeating this process across thousands of cycles—each activating a different, random subset of fluorophores—a comprehensive dataset of molecule localizations is compiled. This dataset is then reconstructed into a super-resolution image with spatial detail far beyond the classical optical diffraction limit.
More technical detail/ detailed description:
STORM imaging in this setup is implemented on an Olympus IX71 inverted microscope frame equipped with a UAPON 100× 1.49 NA OTIRF oil immersion objective lens configured in epiillumination geometry. This high numerical aperture lens provides a diffraction-limited resolution of approximately 230 nm at 560 nm excitation, though STORM itself improves upon this by localizing individual fluorophores far more precisely. Samples are illuminated via a combination of up to four solid-state lasers operating at 405, 488, 561, and 647 nm. The laser beams are merged using dichroic mirrors and delivered through an optical fiber to the microscope’s back port.
To reduce speckle and coherent artefacts caused by laser interference, the optical fiber is mechanically vibrated during operation. The resulting scrambled illumination ensures a uniform excitation field in the imaging plane, crucial for consistent and artifact-free imaging. Light reaches the sample through the high-NA objective, and emitted fluorescence is captured via either a Hamamatsu ORCA Flash 4.0 v2 sCMOS or a Photometrics Evolve 512 EMCCD camera, depending on the experiment's requirements.
The cameras are interfaced with high-speed data acquisition systems that include solid-state drives (SSDs) to accommodate fast data writing. This enables high-frame-rate imaging at up to 100 frames per second, which is essential to minimize motion blur and photobleaching artifacts during long imaging sequences. With a 100× objective, the effective pixel size in the sample plane is ~130 nm, and exposure times are typically set to 10 ms. This allows for rapid capture of dynamic molecular events, which is particularly useful in time-resolved experiments, such as those probing the structural dynamics of peptide aggregates or live-cell imaging.
To maintain focus stability and correct for thermal drift in the axial (z) direction, the setup includes a MadCity Labs C-focus system. Additionally, 3D-STORM imaging is made possible through the integration of a MicAO 3DSR adaptive optics module, which uses engineered elliptical PSFs to localize emitters in three dimensions. This configuration supports detailed 3D reconstructions of complex biological and soft matter structures.
Environmental control during imaging is achieved using a Solent incubator, which allows samples to be maintained in a hydrated, room-temperature environment, making the system well-suited for live-cell and soft matter studies. Furthermore, the apparatus supports Total Internal Reflection Fluorescence (TIRF) imaging, offering high-contrast imaging of surface-adjacent regions such as cell membranes.
Capabilities/ key specifications:
The STORM microscopy system offers a powerful suite of capabilities for high-resolution imaging and molecular-scale investigation. It enables fluorescence imaging with lateral resolution down to ~20 nm and axial resolution of ~40 nm, surpassing the diffraction limit by nearly an order of magnitude. The system supports both 2D and 3D imaging through the use of adaptive optics and engineered point spread functions, allowing for precise spatial mapping of structures in complex biological and soft matter samples. Multicolor imaging is achievable using four solid-state lasers at 405, 488, 561, and 647 nm, enabling multiplexed analysis of different molecular species within the same sample.
Uses/ applications:
The system is equipped for dynamic, time-resolved single-molecule experiments, thanks to high-speed cameras capable of capturing up to 100 frames per second and is supported by real-time thermal drift correction for stability in the z-axis. Additional modalities such as Total Internal Reflection Fluorescence (TIRF) expand its utility for membrane-proximal studies. The apparatus can also be used for dynamic single molecule fluorescence experiments and a number of microfluidic platforms are available.
With compatibility for live-cell imaging in a controlled, hydrated environment and integration with microfluidic platforms, this system provides a highly versatile and sensitive platform for nanoscale investigations in biological physics, molecular biology, and materials science.
Instrument(s) make and model:
Olympus IX71 inverted microscope frame with PRIOR ProScan III sample stage and a MadCity Labs C-focus system to compensate for thermal drift in the z-plane.
UAPON 100× 1.49 NA OTIRF oil immersion objective lens.
405 nm, 488 nm, 561 nm, and 647 nm solid state lasers.
Hamamatsu ORCA Flash 4.0 v2 sCMOS and Photometrics Evolve 512 EMCCD camera.
MicAO 3DSR adaptive optics module with MicAOv1.3 software.
Solent Scientific 3-ORIO7 Incubation chamber.
Facility keywords
- microscope
Research Beacons, Institutes and Platforms
- Photon Science Institute
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