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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 moiré 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
N-SIM image (Slice 3D-SIM mode)
Conventional widefield image
• 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
N-SIM image (Slice 3D-SIM mode)
Conventional widefield image
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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)
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Various observation modes
TIRF-SIM/2D-SIM mode
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
TIRF-SIM image
Conventional TIRF image
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
N-SIM image (Slice 3D-SIM mode)
conventional widefield image
• 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
Stack 3D-SIM mode (Volume view)
Stack 3D-SIM mode (Maximum projection)
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Simultaneous two-wavelength super-resolution imaging
By attaching two EMCCD cameras to the microscope with the optional Two Camera Imaging Adapter*, simultaneous two-wavelength super-resolution imaging with excitation of 488 nm and 561 nm is possible.
Two Camera Imaging Adapter (for N-SIM)
• *The actual product may differ slightly in design.
• *Andor Technology Ltd.
Growth cone of NG108 cell expressing GFP-LifeAct (F-actin, green) and mCherry-tubulin (microtubules, red)
Photos courtesy of: Dr. Kaoru Katoh, The National Institute of Advanced Industrial Science and Technology (AIST)
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5-color multi-laser super-resolution capability
LU5 N-SIM 5 Laser Module is a modular system with up to five lasers enabling super resolution in five colors. Multi-color capability is essential to the study of dynamic interactions of multiple proteins of interest at the molecular level.
Co-localization images of a target protein of VGEF signaling (Cy3) and its ubiquitin E3 ligase (FITC)
Unprecedentedly detailed structure of the nuclear body can be observed
Mode: Slice 3D-SIM, Z-stack
Objective: CFI Apochromat TIRF 100x oil (NA 1.49)
Photographed with the cooperation of: Drs. Hidetaka Ohnuki and Shigeki Higashiyama, Ehime University Graduate School of Medicine
3D reconstruction image
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The Principle of the Structured Illumination Microscopy
Analytical processing of recorded moiré patterns produced by overlay of a known high spatial frequency pattern, mathematically restores the sub-resolution structure of a specimen.
Illumination with a known, high spatial frequency pattern allows for the extraction of super-resolution information from the resulting moiré fringes.
Utilization of high spatial frequency laser interference to illuminate sub-resolution structure within a specimen produces moiré fringes, which are captured. These moiré 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 moiré pattern images
An image of moiré 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 moiré 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).
Fig. A: Resolution is limited by the NA of the objective
Fig. B: The product of Structured Illumination and normally un-resolvable specimen structure produce recordable moiré fringes containing the specimen information at double the conventional resolution limit.
Fig. C: Images with resolutions equivalent to those captured with objective lenses with approximately double the NA are achieved.
Objectives for super-resolution microscopes
The SR (super resolution) objectives have been designed to provide superb optical performance with Nikon’s super-resolution microscopes.
The adjustment and inspection of lenses using wavefront aberration measurement have been applied to yield optical performances with the lowest possible asymmetric aberration.
CFI SR Plan Apochromat IR 60x WI
CFI SR Apochromat TIRF 100x oil
Introducing Nikon Technology that enables Super Resolution Microscope N-SIM
Super-resolution live-cell imaging that goes beyond the limitations of the optical microscope.
• Super Resolution Microscope N-SIM
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