Description
The Stochastic Optical Reconstruction Microscope allows fluorescence imaging to be performed with 20 nm resolution in x/y and ~40 nm in z (using a MicAO 3DSR Adaptive Optics device). Samples can be maintained at room temperature in a hydrated environment (Solent incubator), so the apparatus finds many applications in biological physics (e.g. live cells) and soft-condensed matter physics (e.g. peptide aggregates). There are four solid state lasers available that can be used to illuminate a sample at 405, 488, 561 and 647 nm. Three dimensional STORM imaging is possible using ellipsoidal point spread function engineering with the Adaptive Optics device. A Photometrics Evolve 512 EMCCD and a Hamamatsu ORCA Flash v2 sCMOS are available for image detection. The apparatus can also be used for dynamic single molecule fluorescence experiments and a number of microfluidic platforms are available. A Total Internal Reflection Fluorescence (TIRF) modality is available using an Olympus UAPON 100 1.49NA OTIRF lens. The apparatus is built on an Olympus IX71 inverted microscope frame, with a PRIOR ProScan III sample stage and a MadCity Labs C-focus system to compensate for thermal drift in the z-plane. The laser light is delivered through a vibrated optical fibre to scramble any coherent speckle artefacts.
Uses / Applications
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.
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.
Specification
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.
