Focus On Super-Resolution

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Nikon is focused on providing resolution improvement technologies suited towards a variety of applications.

For hundreds of years optical microscopy was relegated to the diffraction-limited regime, unable to resolve details smaller than approximately 200 nm (in XY) and 500 nm (in Z). That limit has been shattered, spawning a number of techniques and culminating in the 2014 Nobel Prize in Chemistry being awarded to pioneers in super-resolution microscopy. We can now image with twice the resolution as previous with structured illumination microscopy (SIM), and about 10x greater resolution with Stochastic Optical Reconstruction Microscopy (STORM). Enhanced resolution is even being extracted from confocal instruments by using sub-Airy Unit pinhole values and deconvolution. Nikon is proud to bring a number of different super-resolution technologies to market, and is focused on overcoming the many unique challenges for its successful adoption in difficult experimental systems.

Structured Illumination Microscopy (SIM)

A user-friendly super-resolution technology easily applied towards a variety of sample types.

Super-resolution structured illumination microscopy (SIM) is a revolutionary imaging technique for doubling the resolution of a widefield microscope in 3D. Structured illumination is performed with beam interference at the sample plane, producing a diffraction-limited and sinusoidal pattern of light-dark stripes. The single spatial frequency present in the pattern mixes with the various spatial frequencies comprising the sample structure. In short, spatial frequencies usually outside of the microscope bandpass may be artificially down-modulated via frequency mixing, allowing for their indirect detection and subsequent restoration in post-processing. SIM may provide up to a 2x improvement over the optical resolution of a typical widefield microscope in x, y, and z.

  • Nikon’s new N-SIM S system uses a spatial light modulator (SLM) for pattern generation, unlocking acquisition rates of up to 15 frames per second. The increase in acquisition rate is especially useful for live cell imaging, but also shortens the time required for acquiring z-stacks and other dimensions. The N-SIM S is capable of simultaneous or sequential multicolor imaging, 3D-SIM, 2D-SIM, and 2D TIRF SIM with a variety of objectives and immersion types.
  • The N-SIM E system is a value implementation of the original N-SIM system, providing 3D-SIM imaging with 3 laser wavelengths using a single diffraction grating.
  • Learn about reflected light imaging of nanoparticles with N-SIM in our application note.

Stochastic Optical Reconstruction Microscopy (STORM)

A powerful super-resolution technique for pushing optical resolution improvement to its limits.

STochastic Optical Reconstruction Microscopy (STORM) and similar techniques exploit the concept of single molecule localization to improve optical resolution by about a factor of 10 compared to widefield imaging. A variety of photochemical techniques exist for “switching off” most of the fluorophores labeling a given sample. By “switching on” a small number of fluorophores at a time, single emission events can be easily identified, and their center position statistically fitted to a sub-diffraction limited voxel. Users may expect about 20 nm resolution in XY and 70 nm in Z. Note that STORM is implemented in conjunction with specialized sample preparations and buffer systems for inducing switching.

  • Nikon’s N-STORM system features a proprietary z-stacking routine based on our Perfect Focus System (PFS) that allows users to image 5+ μm deep in the sample and with variable TIRF/oblique illumination.
  • Nikon’s new 100x Silicone Immersion Lens (NA = 1.35) is designed for live cell STORM and other single molecule imaging experiments to realize superior spherical aberration correction in aqueous media. Also ideal for imaging thicker samples and live specimen using N-SIM S.
  • The N-STORM is compatible with other localization microscopy techniques. Learn about performing DNA-PAINT experiments with N-STORM in our application note.

Deconvolution for Enhanced Resolution (ER) of Confocal Data

The ER module for Nikon A1 model confocal microscopes allows for near-super-resolution imaging.

While it has long been known that deconvolution analysis can be used to improve the signal-to-noise and optical resolution of confocal microscopy data, only recently has it become practical to do so routinely. Years ago deconvolution of even modestly sized datasets required overnight processing, precluding widespread use by non-specialists. Modern computers, improved algorithms, and GPU acceleration have greatly decreased the time required to perform deconvolution, making it a simple addition to almost any imaging workflow. Nikon’s enhanced resolution (ER) software module provides deconvolution algorithms designed for point-scanning confocal data, and using specialized point-spread function (PSF) models for Nikon’s CFI series objectives.

Download our Science eBook on confocal and super-resolution microscopy and learn more!

Auto-Correction Collar Objectives

New objectives feature automated correction collar alignment, essential for correcting spherical aberrations in sensitive super-resolution experiments.

Spherical aberration is one of the most common optical aberrations limiting the performance of high-resolution microscopies. Many objective lenses include a correction collar to correct for spherical aberration caused by variations in cover glass thickness and the refractive index of the sample medium. However, correction collar adjustment can be tedious to perform (especially for multiple samples) and the optimal position difficult to confidently identify. Nikon’s Auto-Correction Collar (ACC) series of objectives for super-resolution microscopy ensure the highest degree of spherical aberration correction for your sample in combination with an automated adjustment routine in the NIS-Elements software. ACC objectives may be used to maximize the resolution performance of any system, with versions available for our 100x HP Apo TIRF oil, 100x SR Apo TIRF oil, and 60x SR Plan Apo water immersion objectives.

Exceptional Stability for Super-Resolution

The Eclipse Ti2-E motorized inverted microscope, featuring Perfect Focus, is an optimized platform for super-resolution imaging.

Realizing the full benefit of super-resolution is not possible without ensuring the utmost system stability. A small degree of focal drift, normally unnoticeable with traditional techniques, could be enough to compromise super-resolution data collection. The Ti2-E inverted research microscope platform features an all-new cam-based Z focusing mechanism which eliminates XY drift to provide an ultra stable platform for demanding applications. The Ti2-E also incorporates Nikon’s latest generation of Perfect Focus System (PFS4) which is the industry leading focus-locking technology. Utilizing a linear encoder and a high-speed feedback mechanism, PFS4 corrects focus drifts caused by temperature changes and mechanical vibrations. The new modular design of PFS4 separates the detector portion from the nosepiece to reduce burden on the z-drive and heat output has been virtually eliminated to provide an incredibly stable imaging environment.