Temporal resolution of 0.6 sec/frame enables super-resolution time-lapse imaging of dynamic live cell events

In structured illumination microscopy (SIM), the unknown cellular ultra-structure is elucidated by analyzing the moire pattern produced when illuminating the specimen with a known high-frequency patterned illumination. Nikon¡¯s Structured Illumination Microscope (N-SIM) realizes super resolution of up to 115 nm in multiple colors. In addition, it can continuously capture super-resolution images at a temporal resolution of 0.6 sec/frame, enabling the study of dynamic interactions in living cells.
Live-cell imaging at double the resolution of conventional optical microscopes

N-SIM utilizes Nikon¡¯s innovative new approach to ¡°structured illumination microscopy¡± technology. By pairing this powerful technology with Nikon¡¯s renowned CFI Apochromat TIRF 100x oil objective (NA 1.49), N-SIM nearly doubles (to approximately 115 nm*) the spatial resolution of conventional optical microscopes, and enables detailed visualization of the minute intracellular structures and their interactive functions.

*Excited with 488 nm laser, in 3D-SIM mode

Microtubules in B16 melanoma cell labeled with YFP
Objective: CFI Apochromat TIRF 100x oil (NA 1.49)
Image capturing speed: approximately 1.8 sec/frame (movie)
Photographed with the cooperation of: Dr. Yasushi Okada, Laboratory for Cell Polarity Regulation, Quantitative Biology Center, RIKEN

Endoplasmic reticulum (ER) in living HeLa cell labeled with GFP Objective: CFI Apochromat TIRF 100x oil (NA 1.49)
Image capturing speed: approximately 1.5 sec/frame (movie)
Photographed with the cooperation of: Dr. Ikuo Wada, Institute of Biomedical Sciences, Fukushima Medical University School of Medicine
Temporal resolution of 0.6 sec/frame?amazingly fast super-resolution microscope

N-SIM provides ultra fast imaging capability for Structured Illumination techniques, with a time resolution of up to 0.6 sec/frame, which is effective for live-cell imaging (with TIRF-SIM/2D-SIM mode; imaging of up to approximately 1 sec/frame is possible with Slice 3D-SIM mode).
Dynamics of mitochondria stained with Mito-Tracker red
Cristae in mitochondria are visualized and the dynamics of mitochondria can be observed. Mode: Slice 3D-SIM mode
Objective: CFI Apochromat TIRF 100x oil (NA 1.49)
Image capturing interval: approximately 1 sec. (movie)
Various observation modes


This mode captures super-resolution 2D images at high speed with incredible contrast. TIRF-SIM mode takes advantage of Total Internal Reflection Fluorescence observation at double the resolution as compared to conventional TIRF microscopes, facilitating a greater understanding of molecular interactions at the cell surface.
Plasma membrane of B16 melanoma cell labeled with YFP
Objective: CFI Apochromat TIRF 100x oil (NA 1.49)
Photographed with the cooperation of: Dr. Yasushi Okada, Laboratory for Cell Polarity Regulation, Quantitative Biology Center, RIKEN

3D-SIM mode

Two modes are available. Slice 3D-SIM mode allows axial super-resolution imaging with optical sectioning at 300 nm resolution in live-cell specimens; Stack 3D-SIM mode can image thicker specimens with higher contrast than Slice 3D-SIM mode.
Bacillus subtilis bacterium stained with membrane dye Nile Red (red), and expressing the cell division protein DivIVA fused to GFP (green).
The superior resolution of N-SIM system allows for accurate localization of the protein during division.
Photos courtesy of: Drs. Henrik Strahl and Leendert Hamoen, Centre for Bacterial Cell Biology, Newcastle University

Mouse keratinocyte labeled with an antibody against keratin intermediate filaments and stained with an Alexa 488 conjugated second antibody.
Photos courtesy of: Dr. Reinhard Windoffer, RWTH Aachen University
5 laser multi-color super-resolution capability

LU5 N-SIM 5 Laser Module is a modular system with up to five lasers enabling true multi-color super resolution. Multi-color capability is essential to the study of dynamic interactions of multiple proteins of interest at the molecular level.
 CFI P Achromat objective series           CFI TU Plan Fluor EPI P objective
 (for diascopic illumination)                    series (for episcopic illumination)
The Principle of the Structured Illumination Microscopy

Analytical processing of recorded moire patterns produced by overlay of a known high spatial frequency pattern, mathematically restores the sub-resolution structure of a specimen.

Utilization of high spatial frequency laser interference to illuminate sub-resolution structure within a specimen produces moire fringes, which are captured. These moire fringes include modulated information of the sub-resolution structure of the specimen.
Through image processing, the unknown specimen information can be recovered to achieve resolution beyond the limit of conventional optical microscopes.

Create super-resolution images by processing multiple moire pattern images

An image of moire patterns captured in this process includes information of the minute structures within a specimen. Multiple phases and orientations of structured illumination are captured, and the displaced "super resolution" information is extracted from moire fringe information. This information is combined mathematically in "Fourier" or aperture space and then transformed back into image space, creating an image at double the conventional resolution limit.

Utilizing high-frequency striped illumination to double the resolution

The capture of high resolution, high spatial frequency information is limited by the Numerical Aperture (NA) of the objectives, and spatial frequencies of structure beyond the optical system aperture are excluded (Fig. A). Illuminating the specimen with high frequency structured illumination, which is multiplied by the unknown structure in the specimen beyond the classical resolution limit, brings the displaced "super resolution" information within the optical system aperture (Fig. B).
When this ¡°super-resolution¡± information is then mathematically combined with the standard information captured by the objective lens, it results in resolutions equivalent to those captured with objective lenses with approximately double the NA (Fig. C).

Objectives for super-resolution microscopes

The SR (Super Resolution) objectives have been designed for new applications that break the diffraction barrier.
The most recent optical designs and the best selection of optical glasses have been applied to yield optical performances with the lowest possible remaining spherical and cylindrical aberrations.

  Diascopic/Episcopic illumination type