Super Resolution MicroscopesA brand new era insuper resolution

N-SIM S
Structured illumination microscopy
15 fps image acquisition
Lateral resolution of ~115 nm
Axial resolution of ~269 nm

2The N-SIM S Super Resolution Microscope utilizes a unique high-speed structured illumination system to achieveacquisition speeds of up to 15 fps*, enabling fast biological processes to be captured at twice the spatial
resolution of conventional light microscopes (~115nm** in XY).
The N-STORM Super Resolution Microscope achieves a 10-fold improvement in resolution compared toconventional light microscopes (~20 nm in XY), enabling observation at the true molecular level.
The N-SIM S and N-STORM can be easily combined within the same imaging system for greater flexibility innanoscale imaging experiments. They can be further combined with confocal microscope system such as the
AX to create a single versatile platform for multi-scale imaging. Powered at the core by the industry-leading
Ti2 inverted microscope with its ultra-flexible design, nano-precision Z-drive and Perfect Focus System, thepossibilities for experimental design are limitless.
* TIRF-SIM/2D-SIM mode, 512 x 512 pixels, 2 msec exposure time** F WHM of 100 nm bead images collected in 3D-SIM mode, using 488 nm excitation laser. In TIRF-SIM mode, 86 nm is achieved using 40 nm beads
excited by a 488 nm laser.

N-STORM
Stochastic optical reconstruction microscopy
Lateral resolution of ~20 nm
Axial resolution of ~50 nm

3Lamellipodia of NG108 cell labeled with Alexa Fluor® 488 for actin (green) and TRITC-Phalloidin for microtubules (red).
Photo courtesy of: Drs. Shizuha Ishiyama and Kaoru Katoh, The National Institute of Advanced Industrial Scienceand Technology (AIST)

LLC-PK1 cell labeled with DAPI for nucleus (blue), Alexa Fluor® 488 for microtubules (green) and TRITC-Phalloidin for actin (red).
Photo courtesy of: Dr. Kaoru Katoh, The National Institute of Advanced Industrial Science and Technology (AIST)

See life in super resolution

4

Synaptonemal complexes of C. elegans pachytene germ cells labeled with anti-SYP-1 antibodies.
Photo courtesy of: Tyler Machovina and Dr. Judith Yanowitz, Magee-Womens Research Institute.

Malaria parasite surface (MTIP) labeled with Alexa Fluor ® 488 (green), Erythrocyte membrane (Band 3) labeled with
Alexa Fluor ® 568 (red), DNA labeled with DAPI (blue)
Scientific Reports DOI:10.1038/s41598-018-22026-0
Photo courtesy of: Drs. Masayuki Morita, Eizo Takashima, Tadahiro Iimura, Takafumi Tsuboi, Proteo-Science Center,
Ehime University

The N-SIM S combines innovative structured
illumination microscopy technology with
unparalleled optics to double the resolutionof conventional light microscopes. Withacquisition speeds of up to 15 fps, the N-SIM S
enables high-speed super resolution imagingof dynamic events in live cells.

❖ Captures rapid changes in live cells at 15 fps*
❖ Twice the resolution of conventional lightmicroscopes (approx. 115 nm**), utilizing
structured illumination microscopy❖ Automatic switching between different
illumination modes❖ Two-channel TIRF-SIM acquisition❖ Acquires larger fields of view (66 µm x
66 μm***)❖ Compatible with easy-to-use dry objectives❖ Simultaneous two-channel imaging (optional)* TIRF-SIM/2D-SIM mode, 512 x 512 pixels, 2 msec exposure time** F WHM of 100 nm bead images collected in 3D-SIM mode, using 488 nm excitation
laser. In TIRF-SIM mode, 86 nm is achieved using 40 nm beads excited by a 488
nm laser.
*** Field of view using a 100X objective.

5

Endosomes of COS7 cell labeled with YFP. Rapid movement of endosomes is captured at high resolution.
Image acquisition speed: 6 fps
Imaging mode: 3D-SIM
Image courtesy of: Yasushi Okada, M.D., Ph.D., Department of Physics, Graduate School of Science, The University of Tokyo
Scan the QR code to view a video illustrating super-resolution and widefield images.

Capture rapid changes in live cells
High-speed super-resolution imaging at 15 fps
Nikon’s new high-speed structured illumination system utilizes a novel pattern modulation technology to generate fast and precise switching of
illumination patterns. The N-SIM S Super Resolution Microscope achieves incredible acquisition speeds (up to 15 fps*), enabling super-resolution
time-lapse imaging of live cells and intracellular dynamics. Discover a new level of live-cell imaging with the N-SIM S.
* TIRF-SIM/2D-SIM mode, 512 x 512 pixels, 2 msec exposure time

6

Growth cone of NG108 cell labeled with GFP-Lifeact for F-actin. Formation of actin mesh is captured at high-speed.
Image acquisition speed: 10 fps
Imaging mode: TIRF-SIM
Image courtesy of: Drs. Minami Tanaka and Kaoru Katoh, The National Institute of Advanced Industrial Science and Technology (AIST)
Scan the QR code to view a video illustrating super-resolution and widefield images.

Histone H2B-GFP expressing in a HeLa cell. Visualization of fine movements of chromatin domains in different locations.
Image acquisition speed: 3.9 fps
Imaging mode: 3D-SIM
Image courtesy of: Yuko Sato, Ph.D. and Hiroshi Kimura, Ph.D., Cell Biology Center, Institute of Innovative Research, Tokyo Institute of Technology
Scan the QR code to view a video illustrating super-resolution and widefield images.

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Two-color TIRF-SIM imaging of growth cone of NG108 cell labeled with Alexa Fluor ® 488 for F-actin (green) and Alexa Fluor ® 555 for microtubules (orange)
Reconstructed image size: 2048 x 2048 pixels (66 μm x 66 μm with a 100X objective)
Sample courtesy of: Drs. Shizuha Ishiyama and Kaoru Katoh, The National Institute of Advanced Industrial Science and Technology (AIST)

Easily switch between imaging modes
for optimal results
Automatic switching between illumination modes
Newly-developed, high-speed structured illumination technology not only enables fast acquisition rates but also automatic switching between
illumination modes and automated optimization of structured illumination patterns for different wavelengths and magnifications. This expanded
automation enables fast 2-color TIRF-SIM imaging as well as multiplexing of different SIM modalities. The N-SIM S provides easy-to-use, streamlined
workflows, whether it be for single-mode or multi-modal imaging experiments.

Acquire larger fields of view
The N-SIM S can acquire super-resolution images with a large field of view of 66 µm square. This larger imaging area enables very high throughput for
applications/samples that benefit from larger fields of view, such as a neurons, reducing the amount of time and effort required to obtain data.

8

Simultaneous two-channel imaging
Simultaneous two-color imaging is possible by utilizing an optional Two Camera Imaging Adaptor* and two sCMOS cameras.
*Hamamatsu Photonics K.K.

Immediately before drug application

10 minutes after

20 minutes after

30 minutes after

Growth cone of NG108 cells. Fascin (green): GFP-Fascin, Actin (red): LifeAct-KO Objective Lens : CFI SR HP Apochromat TIRF 100xC Oil
Using image splitting optical system, dual color time-lapse images were captured after application of drug solution. The time course was recorded that fascin was dissociated from theactin bundles and filamentous actin changed their pattern. Co-localization of fascin and actin and dissociation of fascin were well demonstrated. Optic aberrations and image distortion
were adjusted to be less than 30 nm.
Images courtesy of: Dr. Minami Tanaka, Dr. Kaoru Katoh, Biomedical Research Institute Molecular Neurobiology Group, National Institute of Advanced Industrial Science andTechnology

2D-SIM mode/TIRF-SIM modeThis mode captures super-resolution 2D images at high speed with incredible contrast. The TIRF-SIM mode enables Total Internal Reflection
Fluorescence observation at double the resolution of conventional TIRF microscopes, facilitating a greater understanding of molecular interactions at
the cell surface.

3D-SIM mode
The 3D-SIM mode generates structured illumination patterns in three dimensions to deliver a two-fold improvement in lateral and axial resolution.
Two reconstruction methods (“slice” and “stack”) are available to optimize results for application requirements (e.g. sample thickness, speed, etc.).
3D-SIM image

Conventional widefield image

Bacillus subtilis bacterium stained with membrane dye Nile Red (red), and expressing the cell division protein DivIVA fused to GFP (green). The superresolution microscope enables accurate localization of the protein during division.
Reconstruction method: Slice
Photos courtesy of: Drs. Henrik Strahl and Leendert Hamoen, Centre for Bacterial Cell Biology, Newcastle University

Width: 26.19 μm, Height: 27.11 μm, Depth: 3.36 μm

Width: 16.00 μm, Height: 13.36 μm, Depth: 6.00 μm

Mouse keratinocyte indirectly immunolabeled for keratin intermediatefilaments and visualized with Alexa Fluor ® 488 conjugated secondary
antibodies.
Reconstruction method: Stack
Photo courtesy of: Dr. Reinhard Windoffer, RWTH Aachen University

Human U2OS cell in mitosis metaphase
The cell is labeled green (kinetochore protein CENP-B), red (alpha-tubulin) andblue (DNA).
Reconstruction method: Stack
Photo courtesy of: Dr. Alexey Khodjakov, Wadsworth Center, Albany NY

9

“N-SIM provides theresolution necessary to
identify and evaluate thestructural organization of
the nuclear lamina*1, *2.
Its ease of use and stableperformance has made N-SIM
an integral research tool in mylaboratory.”*1 Mol Biol Cell. 2015 Nov 5; 26(22):4075-86.
*2 Nature . 2017 Mar 9; 543(7644):261-264.

Dr. Robert D. Goldman
Ellison Foundation Senior Scholar, Stephen Walter Ranson Professor,
Chair, Dept. of Cell & Mol. Biol., Feinberg School of Medicine,Northwestern University

Lamin B1 (red) and Lamin C (green) form separate but interacting meshworks within the lamina of the embryonic fibroblast nucleus of a mouse. Prepared for double indirect
immunofluorescence and imaged by 3D-SIM. The meshwork structure was computationally derived from LB1 (cyan) and LC (magenta) 3D-SIM fluorescence data using steerable filters.*1
Photos courtesy of: Drs. Takeshi Shimi and Mark Kittisopikul

The principle of Structured Illumination Microscopy
Analytical processing of recorded moiré patterns,
produced by overlaying a known high spatial
frequency pattern, mathematically restores the
sub-resolution structure of a specimen.
Utilization of high spatial frequency laser interference to illuminate subresolution structures 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, theunknown specimen information can be recovered to achieve resolution
beyond the limit of conventional light microscopes.

Illumination with a known, high spatial
frequency pattern allows for the
extraction of super-resolution informationfrom the resulting moiré fringes.

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 andorientations 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 theconventional resolution limit.
Create super-resolution images by processing multiple images

Capture multiple images with structured illumination that is shifted in phase.
Repeat this process for three different angles. This series of images are then processed using advanced algorithms to
obtain super-resolution images.

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, andspatial frequencies of structure beyond the optical system apertureare 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).

Fig. A: Resolution is limited by
the NA of the objective.

10

When this “super-resolution” information is then mathematicallycombined 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. B: The product of Structured Illumination andnormally 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 withapproximately double the NA are achieved.

N-SIM S system diagram

D-LEDI Fluorescence LED
illumination system

Ti2-E with double layer
configuration with
Perfect Focus Unit

TI2-LA-HTIRF H-TIRF modulewith TI2-LA-FL Epi-Fl module

LU-NV/LUD-H/
Ziva Light Engine
Series Laser Unit

TI2-LA-NS2 N-STORM module 2
with TI2-LA-FL Epi-Fl module**

Piezo Z stage

TI2-FTQ2
Motorized filter cube turretSide port***Vibration isolated table

NIS-Elements AR /NIS-Elements C*

TI2-N-SIM-S
N-SIM S
illuminator

N-SIM Shield box

C-DA C-mount adapter

PC

NIS-A 6D and
N-SIM analysis

ORCA Fusion BT sCMOS camera(Hamamatsu Photonics K.K.)

Motorized N-STORM kit *** Required when used with confocal system** Required when configured with N-STORM*** Supplied with microscope main body

N-SIM S Specifications
Lateral resolution (FWHM of beads in xy)

115 nm*1 in 3D-SIM mode, 86 nm*2 in TIRF-SIM mode

Axial resolution (FWHM of beads in z)

269 nm*1 in 3D-SIM mode

Image acquisition time

Up to 15 fps (TIRF-SIM/2D-SIM, 2 msec exposure time)

Reconstructed image size

1024 x 1024 pixels, 2048 x 2048 pixels

Imaging mode

TIRF-SIM
2D-SIM
3D-SIM (Reconstruction method: slice, stack)Multi-color imaging

Up to 6 colors

Simultaneous multi-color imaging

Two colors

Compatible Laser

LU-NV series laser unit
Standard: 405 nm, 488 nm, 561 nm, 640 nm
Option: 445 nm
LUD-H series laser unit (TIRF-SIM)
Standard: 405 nm, 488 nm, 561 nm, 640 nm
Option: 445 nm, 515 nm

Ziva Light Engine series laser unit (2D/3D-SIM)
405 nm, 446 nm, 476 nm, 518 nm, 545 nm, 637 nm

Compatible microscope

Motorized inverted microscope ECLIPSE Ti2-E
Perfect Focus System
Motorized XY stage with encoders
Piezo Z stage

Objective

CFI SR HP Plan Apochromat Lambda S 100XC Sil (NA1.35)
CFI SR HP Apochromat TIRF 100XC Oil (NA 1.49)
CFI SR HP Apochromat TIRF 100XAC Oil (NA 1.49)
CFI SR Plan Apochromat IR 60XC WI (NA 1.27)
CFI SR Plan Apochromat IR 60XAC WI (NA 1.27)
CFI Plan Apochromat Lambda 60XC (NA 0.95)*3
CFI Plan Apochromat Lambda 40XC (NA 0.95)*3

Camera

ORCA Fusion BT sCMOS camera (Hamamatsu Photonics K.K.)

Software

NIS-Elements AR/NIS-Elements C (for Confocal Microscope AX/AX R)
Both require additional software modules NIS-A 6D and N-SIM Analysis

Operating conditions

20 ºC to 28 ºC ( ± 1.5 ºC)*1 These values are measured using 100 nm diameter beads excited by a 488 nm laser. Actual resolution is dependent on laser wavelength and optical configuration.
*2 This value is measured using 40 nm diameter beads excited by a 488 nm laser. Actual resolution is dependent on laser wavelength and optical configuration.
*3 Supports 2D-SIM and 3D-SIM (slice reconstruction).

11

a: Hippocampal neurons and glia in culture
3D-STORM imaging of actin labeled with Alexa Fluor ® 647 Phalloidin using depth-code pseudo color. Image “a” shows
four types of actin organization, from bottom left to top right: the cell body of a neuron, a glial cell with stress fibers,
a neuronal dendrite with spines, and an axon.
Photos courtesy of: Dr. Christophe Leterrier, NeuroCyto team, NICN CNRS-AMU UMR7259, Marseille, France

b: Growth cone of a neuron in culture

Experience the nanoscale universe

12

c: Glia in a neuronal culture

d: COS cells

STochastic Optical Reconstruction Microscopy(STORM) reconstructs a super-resolution
image by combining precise localization
information for individual fluorophores incomplex fluorescent microscope specimens.
The N-STORM takes advantage of Nikon’s
powerful Ti2-E inverted microscope andapplies high-precision multi-color localization
and reconstruction in three dimensions (xyz) toenable super-resolution imaging at tenfold theresolution of conventional light microscopes(up to 20 nm in xy). This powerful technology
enables visualization of molecular interactions
and organizations at the nanoscopic
scale, opening up new worlds of scientificunderstanding.

❖ 10 times the resolution of conventional lightmicroscopes in x, y and z directions❖ Dynamic super resolution imaging at themolecule level❖ Multi-color imaging capability❖ High definition, high density images❖ Large image acquisition area

13

The N-STORM is compatible with DNAPAINT technology, which exploits
transient binding and unbinding offluorescently labeled DNA strands to theircomplementary target strands, creating
signal blinking to enable super-resolution
imaging.

3D DNA-PAINT image of the nuclear lamina protein
Lamin A/C in a fixed CV-1 cell
Image depth: 5.1 μm
Step size: 100 nm(a) 3D image with color-coded z position.
(b) 3D volume rendering.
(c-h) R epresentative individual z planes, depth
denoted in bottom left

Tenfold increased resolution in x, y and z
Up to 50 nm axial resolution
In addition to lateral super-resolution, the N-STORM utilizes proprietary methods to achieve a tenfold enhancement in axial resolution overconventional light microscopes and provide nanoscale information in 3D.
The 3D-Stack function allows multiple 3D-STORM images from different Z positions to be captured and stitched into one image to create thicker
STORM images.
5 µm

Tubulin of BSC-1 cell labeled with Alexa Fluor ® 647

A human fibroblast labeled with EdU-Alexa Fluor ® 647 to visualize
DNA with 3D-STORM.
Photo courtesy of: Jason Otterstrom, Ph.D., Melike Lakadamyali,
Ph.D., The Institute of Photonic Sciences (ICFO), Castelldefels, Spain

14

Primary cell culture of Drosophila brain
3D-STORM image of EdU-labeled DNA in Drosophila melanogaster neuroblast
Photo courtesy of: Anna Oddone, Ph.D., Melike Lakadamyali, Ph.D. group,
The Institute of Photonic Sciences (ICFO), Castelldefels, SpainUp to 20 nm lateral resolution
The N-STORM utilizes high-precision localization information from thousands of individual fluorophores present in a field of view tocreate breathtaking “super-resolution” images, exhibiting spatial resolution 10 times greater than conventional light microscopes.
N-STORM images

5 µm

1 µm

200 nm1 µm

200 nmConventional widefield images

5 µm

Human cervical cancer cells (HeLa S3) labeled with Alexa Fluor ® 647 (NUP153) and ATTO 488 (TPR)
Photos courtesy of: Dr. Michael W. Davidson, National High Magnetic Field Laboratory, Florida State University

N-STORM images

5 µm

1 µm

200 nm1 µm

200 nmConventional widefield images

5 µm

African green monkey kidney cells (BSC-1) labeled with Alexa Fluor ® 647 (Tubulin) and ATTO 488 (Calreticulin)
Photos courtesy of: Dr. Michael W. Davidson, National High Magnetic Field Laboratory, Florida State University

15

Dynamic super resolution imaging
Newly developed optics and illumination systems, optimized for sCMOS technology, have increased image acquisition speeds by up to 10 times. Withacquisition times reduced from minutes to seconds*, dynamic events in live specimens can now be captured with molecular level resolution.
* Using high-speed mode (20 μm x 20 μm imaging area)

0sec

4sec

8sec

20sec

◀

◀

◀

◀

16sec

12sec

24sec

28sec

◀

◀

◀

◀

◀

Time-lapse STORM images of African green monkey kidney cell (BSC-1) labeled with Mito-Tracker Red (Mitochondria).
Imaging speed: 500 fps
28 sec time-lapse imaging with 2 sec interval
Scale bar: 0.2 μm

Multi-color imaging
capability
Multi-color super-resolution imaging can be
carried out using both activator-reporter pairs
for sequential activation imaging and activatorfree labels for continuous activation imaging.
This flexibility allows users to easily gain criticalinsights into the localization and interaction
properties of multiple proteins at the molecularlevel.

3-color STORM image of a CV-1 cell stained with antibodies
against alpha-tubulin (Alexa Fluor ® 647; magenta),
caveolin (Alexa Fluor ® 555; red), and with Alexa Fluor ®
488-phalloidin (green) for f-actin.

16

High definition, highdensity images
Newly developed excitation optics andimproved image acquisition rates provide
increased molecule localization density,
resulting in clearer images of macromolecular
structures.

5 µm
Tubulin of BSC-1 cell labeled with Alexa Fluor ® 647,
acquisition time: 20 seconds

512 pixels
sCMOS

Large image
acquisition area

256 pixels

EM-CCD

256 pixels

512 pixels

New intermediate zoom lenses in theimaging system have been developed andoptimized for a wide field of view. The
wide-view mode achieves 80 μm x 80 μm,
a 4-fold increase in imaging area compared
to previous models.

10 µm

4 times wider imaging area, 80 μm x 80 μm (wide-viewmode). Conventional imaging area of 40 μm x 40 μm also
shown for comparison.
Sample: Mitochondria TOM20 conjugated with Alexa
Fluor ® 647

17

The principle of STochastic Optical Reconstruction Microscopy
STochastic Optical Reconstruction Reconstruction of N-STORM images using localization information
Microscopy (STORM) reconstructs of individual fluorophores
Conventional fluorescent microscopy
a super-resolution image
by combining high-accuracy
localization information of
Excite all fluorophores
Individual localization information cannot be detectedindividual fluorophores in three
N-STORM processing
dimensions and multiple colors
N-STORM uses stochastic activation of relatively
small numbers of fluorophores using very lowintensity light. This random stochastic “activation”
of fluorophores allows temporal separation ofindividual molecules, enabling high precision
Gaussian fitting of each fluorophore image in XY.
By utilizing special 3D-STORM optics, N-STORM canalso localize individual molecules along the Z-axis
with high precision. Computationally combining
molecular coordinates in three dimensions resultsin super-resolution 3D images of the nanoscopic
world.

Activates with very
low-intensity light

Detects the center location

RepeatExcites with stronglight
Activates with very
low-intensity light

Detects the center location

Excites with strong
light

Super
resolution
image

Plot detected localization
information

High-precision Z-axis position detection
Using a cylindrical lens that asymmetrically condenses light beams in either X
or Y direction, Z-axis molecule locations can be determined with an accuracyof about 50 nm. Location in Z is determined by detecting the orientation of theastigmatism-induced stretch in the X or Y direction and the size of the out-offocus point images. 3D fluorescent images can be reconstructed by combining
the determined Z-axis location information with XY-axis location information.

Single spot image

400

200

0

-200

-400 Z-axis location(nm)

Variety of photoswitchable probes and labeling approaches for high localization accuracy
Various types of activator-reporter pairs and activator-free labels are available. The activator-reporter dye pair approach provides consistent
localization accuracy between channels by leveraging the same reporter dye for different channels. Each dye pair consists of an activator dye andreporter dye, with the activator dye regulating the activation state of the reporter dye. Activator-free labels consist of imaging dye only, enabling
simple labeling and sample preparation techniques such as conventional indirect immunofluorescence using conventional dye-conjugated antibodies.
Activator-reporter pair

Alexa Fluor ® 405

Alexa Fluor ® 647

Secondary antibody

Primary
antibody
Target molecule

Activator-free label
Alexa Fluor ® 647

Dye for activator Dye for reporter
Alexa Fluor ® 405

Alexa Fluor ® 647

Cy2

Alexa Fluor ® 647

Cy3

Alexa Fluor ® 647

An activator-reporter pair consists of a shorter-wavelengthdye for activation and a longer-wavelength dye for detection.
Creation of three-color, super-resolution images is possible
with multiple dye-pairs.

Sequential activation illumination with activator-reporter pair
STEP 1 M ost activator-reporter dye pairs are converted to a non-emissive state bycombining them with high intensity light and specialized imaging buffer additives.
Alexa Fluor ®
405

Dye for imaging
Secondary antibody

Alexa Fluor ® 647, CF™ 647, Cy5, ATTO 647N
CF™ 568, Cy3B, Alexa Fluor ® 568, Alexa Fluor ® 555
CF™ 488, ATTO 488, Alexa Fluor ® 488

Primary antibody

Target molecule

An activator-free label requires only a reporter dye forimage acquisition. Creation of three-color, super-resolution
images is possible using dyes with well-separated emission
spectra.

Continuous activation illumination with activator-free label
STEP 1 A ctivator-free reporter dye is converted to a non-emissive state by combining it withhigh intensity light and specialized imaging buffer additives.

Alexa Fluor ® 647
Target molecule

Alexa Fluor ® 647

STEP 2	Absorption of light by an activator results in transfer of energy to a nearby
reporter dye, accelerating the transition of the reporter dye from a non-emissive
to a ground state. The use of spectrally distinct activator dyes allows for theuse of the same reporter dye for multiple imaging channels.
Alexa
Fluor ®
405

Target molecule

STEP 2	The reporter dye will stochastically relax to the ground state and then be excited
to the fluorescent state by ongoing high intensity illumination.

Alexa Fluor ® 647
Target molecule

STEP 3	High intensity illumination results in fluorescence emission from the activatedreporter dye.

Alexa Fluor ® 647

18

Alexa Fluor ® 647

Target molecule

Target molecule
RepeatAlexa Fluor ®
405

Target molecule

Alexa Fluor ® 647

RepeatN-STORM system diagram
D-LEDI Fluorescence LED
illumination system

Ti2-E with single layer
configuration with
Perfect Focus Unit

TI2-LA-NS2 N-STORM module 2,
TI2-LA-TIRF-E motorized TIRF module/
TI2-NS2-MU N-STORM
Magnification Change Unit

Piezo Z stage
LU-NV/LUD-H
Series Laser Unit

Vibration isolated table

ManualN-STORM Kit

iXON Ultra DU-897U
EMCCD camera(Andor Technology Ltd.)

ORCA Fusion BT sCMOS camera(Hamamatsu Photonics K.K.)

Motorized
N-STORM kit

PC

NIS-Elements AR /NIS-Elements C*

NIS-A 6D and
N-STORM analysis* Required when used with confocal system

N-STORM Specifications
XY resolution

Approximately 20 nm

Z-axis resolution

Approximately 50 nm

Imaging mode

2D-STORM (normal mode, continuous mode)
3D-STORM (normal mode, continuous mode), 3D-Stack function

Max. field of view

80 μm x 80 μm

Acquisition speed

Up to 500 Hz

Multi-color imaging

Up to 3 colors

Compatible laser

LU-NV series laser unit
405 nm, 488 nm, 561 nm, 647 nm
LUD-H series laser unit
405 nm, 488 nm, 561 nm, 640 nm

Compatible microscope

Motorized inverted microscope ECLIPSE Ti2-E
Perfect Focus System
Motorized XY stage with encoders
Piezo Z stage

Objective

CFI SR HP Plan Apochromat Lambda S 100XC Sil (NA1.35)
CFI SR HP Apochromat TIRF 100XC Oil (NA 1.49)
CFI SR HP Apochromat TIRF 100XAC Oil (NA 1.49)
CFI HP Plan Apochromat VC 100X Oil (NA 1.40)

Camera

ORCA Fusion BT sCMOS camera (Hamamatsu Photonics K.K.)
iXON Ultra DU-897U EMCCD camera (Andor Technology Ltd.)

Software

NIS-Elements AR/NIS-Elements C (for Confocal Microscope AX/AX R)
Both require additional software modules NIS-A 6D and N-STORM Analysis

Operating conditions

20 ºC to 25 ºC ( ± 0.5 ºC)

19

Ultra-stable platforms for superresolution imaging
Slight changes in temperature and minor vibrations in the imaging environment can greatly impact focus stability, which in turn can be detrimental
to super-resolution imaging. The ECLIPSE Ti2-E motorized inverted research microscope has been designed with dramatically improved focus stability
and an automatic real-time focus correction system to eliminate focus drift, enabling faithful visualization of nanoscopic cell details.

20Real-time focus correction with PFS
The Perfect Focus System (PFS) maintains focus by automatically tracking and maintaining the desired Z position. PFS corrects focus drift, caused byminute temperature changes and vibrations, in real time. The detector portion of the PFS is separated from the nosepiece to minimize mechanicalload and heat transfer, further reducing the potential for Z-drifts.
Focal plane of interest
Coverslip interface

PFS measuring unit

Objective
LED
Dichroic

Sensor

PFS nosepiece

Near-IR
light
Observation light path

Offset lensCameraHigh-stability Z-focusing mechanism

Auto correction collar

The durable body of the Ti2-E provides a highly stable platform forsuper-resolution microscopes. The Ti2-E minimizes vibrations bydownsizing the Z-focusing mechanism and positioning it adjacent to
the nosepiece, providing the superior Z-focusing precision and stability
required for super-resolution imaging.

Super-resolution imaging is highly sensitive to spherical aberrations.
An automatic correction collar enables easy and precise correction
collar adjustment to compensate for spherical aberrations, ensuringconsistently high quality super-resolution images.

Focusing mechanism

21

Highest performance optics for superresolution imaging
Nikon has developed dedicated objectives for super-resolution imaging to realize bright and precise nanoscale images.
The SR objectives are aligned and inspected using wavefront aberration measurement technologies to ensure the lowest possible asymmetricaberration and superb optical performance required for super-resolution imaging.

22

Silicone immersion objectives
Silicone immersion objectives use high viscosity silicone
oil with a refractive index close to that of live cells as animmersion liquid. Because of this improved refractive
index compatibility, these objectives can provide
improved photon collection capability and resolution
when performing super-resolution imaging deeper into
the specimen. They exhibit superior chromatic aberration
correction and high transmittance over a broad range of
wavelengths.

CFI SR HP Plan Apochromat
Lambda S 100XC Sil

Silicon immersion objective

Oil immersion objective

N-SIM image
Sample: Mouse brain section labeled with tdTomato expressing neurons

0.2 µmScan the QR code toview a video

2 µm

N-STORM images (approx. 6.5 μm depth)
Left: CFI SR HP Plan Apochromat Lambda S 100XC Sil, Right: CFI SR HP Apochromat TIRF 100XC Oil

Oil immersion objectives

Water immersion objectives

These objectives provide the high numerical apertures required for
N-STORM imaging. The HP objectives are compatible with the ultrahighpower lasers required for inducing rapid photoswitching of fluorophores.
They provide improved axial chromatic aberration correction to achieve thehighest level of precision in localization and image alignment for 3D multicolor STORM imaging.

These objectives correct chromatic aberration over a wide range of
wavelengths up to near-IR, enabling high-resolution deep imaging of live
cells. The AC-type objectives that support the Auto Correction Collar of theTi2-E microscope allow precise and easy adjustment of the correction collar.

CFI SR HP Apochromat
TIRF 100XC Oil

CFI HP Plan Apochromat
VC 100X Oil

CFI SR Plan Apochromat
IR 60XC WI

CFI SR HP Apochromat
TIRF 100XAC Oil

CFI SR Plan Apochromat
IR 60XAC WI

Dry objectives
The N-SIM S is compatible with dry objectives, making both super-resolution imaging and confocal imaging
available without switching lenses. Low-magnification, wide field-of-view dry lenses enable high resolution
observation even at the periphery of sample tissues.
*Dry objectives support 2D-SIM and 3D-SIM (slice reconstruction) only
CFI Plan Apochromat
Lambda 60XC

Model

Immersion

NA

Silicone oil

1.35

0.31-0.29 (0.30*): 23˚C, 0.30-0.28 (0.29*): 37˚C

Manual

CFI SR HP Apochromat TIRF 100XC Oil

Oil

1.49

0.16-0.10 (0.12*): 23˚C, 0.15-0.09 (0.12*): 37˚C

Manual

CFI SR HP Apochromat TIRF 100XAC Oil

Oil

1.49

0.16-0.10 (0.12*): 23˚C, 0.15-0.09 (0.12*): 37˚C

Auto

CFI HP Plan Apochromat VC 100X Oil

Oil

1.40

0.13

CFI SR HP Plan Apochromat Lambda S 100XC Sil

W.D. (mm)

CFI Plan Apochromat
Lambda 40XC

Correction collar

CFI SR Plan Apochromat IR 60XC WI

Water

1.27

0.18-0.16 (0.17*)

CFI SR Plan Apochromat IR 60XAC WI

Water

1.27

0.18-0.16 (0.17*)

ManualAuto

CFI Plan Apochromat Lambda 60XC

Dry

0.95

0.21-0.11 (0.15*)

Manual

CFI Plan Apochromat Lambda 40XC

Dry

0.95

0.25-0.16 (0.21*)

Manual*With cover glass thickness of 0.17 mm

23Unified acquisition and analysis
software platform
NIS-Elements, Nikon’s unified software platform, provides an intuitive workflow for super-resolution imaging. Combined withgraphical programming tools such as JOBS and illumination sequence, as well as powerful analysis and visualization tools, NISElements creates a comprehensive operating environment that can be fully customized for a variety of application requirements.

24

Image Acquisition

Display & Processing

N-SIM S

N-SIM S

Image acquisition setting

Image reconstruction

The N-SIM S can easily switch
between 2D-SIM, 3D-SIM and
TIRF-SIM modes. The JOBS
flexible imaging sequence
option enables seamless image
acquisition between the N-SIM
S, N-STORM and confocal
microscopes.

Auto settings allow the software to automatically select the mostappropriate reconstruction parameters for acquired images in order toreconstruct N-SIM S images. Users can further optimize reconstruction
by manually adjusting these parameters. The reconstruction view allows
users to preview the results of the selected reconstructed parameters on
the current/selected frame, enabling efficient reconstruction parameter
determination.

N-STORM

N-STORM

Image acquisition setting

Batch processing analysis

The N-STORM can easily switch
between 2D-STORM and
3D-STORM image acquisition
modes.

Multiple N-STORM images can be simultaneously analyzed.

Real-time display of localizations per frame

3D display

During N-STORM image
acquisition, the number of
localized fluorescent molecules
is displayed in real time using
images and graphs. Clicking
the Auto LP (Auto Laser Power)
button automatically adjusts
laser power, depending on thenumber of localized fluorescentspots.

A major feature of the N-STORM is 3D super-resolution image acquisition
and analysis. Acquired images can be displayed at any angle after
analysis.

25Explore the nano world with
N-SIM S / N-STORM
Seamless switching between imaging modalities for multi-scale
experiments
Combining the N-SIM S and N-STORM with a single Ti2-E inverted microscope expands the functionality of each individual technology. The N-SIM S
enables acquisition of thicker volume images, thereby providing a more comprehensive molecular landscape for interpreting the single-molecule level
data acquired by the N-STORM.
Confocal image
N-SIM image
The N-SIM S and/or N-STORM can be further combined witha confocal microscope such as the AX. A desired location
in a sample can be specified in a low-magnification/large
FOV confocal image and acquired in super-resolution bysimply switching the imaging method. Combining a confocal
microscope with a super-resolution system can provide amethod for gaining larger contextual views of the superresolution information.

E. coli (XL1-Blue) expressing SGFP2
Photos courtesy of: Drs. Takahisa
Suzuki and Ikuo Wada, Fukushima
Medical University School of
Medicine
5 µm“Simultaneous equipment of the A1+ Confocal Microscope and the N-SIM Super Resolution Microscope with a single
imaging system enables multi-mode analysis. This multi-mode combination is advantageous in the study that requires
wide and detailed views in a consistent manner. To analyze giant cells such as cultured mature osteoclasts with diametersof a few hundred microns, the user can acquire an image with a wide view using the A1+, select the area to be observed
from that wide image, then switch to the N-SIM for ultrafine observation. Electron microscopy had been commonly used
to observe podosomes that assemble to form ring-like structures called actin rings that are functionally associated withcellular adhesion and locomotion of osteoclasts. The N-SIM optically resolves individual podosomes, and enables thequantitative evaluation of osteoclast functions by analysis of the temporal and spatial dynamics of podosomes.”
Dr. Tadahiro Iimura
Division of Bio-Imaging, Proteo-Science Center (PROS),
Division of Analytical Bio-Medicine, Advanced Research Support Center (ADRES), Ehime University”We found that CCR5, the target molecule for treatment of HIV infections, exists in osteoclasts, and that the functional loss of CCR5 impairs the bone-resorption activity of osteoclasts, resulting
in a lower incidence of osteoporosis.* This finding suggests that CCR5-antagonist treatment may prevent bone-destructive diseases such as osteoporosis as well as HIV transmission.“*Nature Communications DOI: 10.1038/s41467-017-02368-5

Podosome (actin) labeled with Alexa Fluor® 568 (red), tubulin labeled with Alexa Fluor® 488 (green) and nucleus labeled with DAPI (blue)
Left: Large FOV confocal image, Middle: Projection of Z-stack confocal images, Right: Super-resolution image
Photos courtesy of: Drs. Ji-Won Lee and Tadahiro Iimura, Division of Bio-Imaging, Proteo-Science Center (PROS), Ehime University

26

Mouse brain section (hippocampus CA1 region)
immunostained against CB1 cannabinoid receptors using
Alexa Fluor ® 647. With STORM imaging, the membranes ofhollow axon terminals are more sharply observed.
Photos courtesy of: Barna Dudok Ph.D., Laszlo Barna
Ph.D., and Istvan Katona Ph.D., Laboratory of Molecular
Neurobiology, Institute of Experimental Medicine of the
Hungarian Academy of Sciences

Confocal image

3D-STORM image

2 µm

HEK cells expressing an eGFP-CB1 fusion construct wereimaged with both confocal and 3D-STORM modules on thesame imaging platform. CB1 was counter-stained using
secondary antibodies labeled with a Cy3-Alexa Fluor ® 647
tandem dye pair for STORM imaging. GFP fluorescence wasimaged using the confocal module.
Membrane structures are visible at a higher resolution in
the STORM image than in the confocal image. In addition,
intracellular membrane structures that are not visible inthe confocal image due to limitations in dynamic range andresolution are visible in the STORM image.
Photos courtesy of: Barna Dudok Ph.D., Laszlo Barna
Ph.D., and Istvan Katona Ph.D., Laboratory of Molecular
Neurobiology, Institute of Experimental Medicine of the
Hungarian Academy of Sciences

Confocal image

2 µm

N-STORM image“STORM imaging is the most important tool for our research as it is the only technique that allows us to measure
the precise position and distribution of important signaling molecules within the chemical synapse on the nanoscale.
But this data alone is like looking at stars in a dark sky. We need to use confocal microscopy to show in which
context these molecular changes are taking place. After the physiological experiments we use confocal imaging
for the morphological characterization of the cell type and the synapse. Then, we switch to STORM mode to gather
information on the signaling molecules responsible for the physiological signaling. In this way we can correlate themolecular data, the anatomical data and the physiological data from the very same synapse. And this is very exciting!”
Dr. Istvan KatonaLaboratory of Molecular Neurobiology, Institute of Experimental Medicine of the Hungarian Academy of Sciences

Correlated confocal-STORM images demonstrating cannabinoid receptor localizations (green) on axon terminals of a hippocampal GABAergic interneuron (cyan). The images weretaken on a combined N-STORM/C2 confocal system
Photos courtesy of: Dr. Barna Dudok, Laboratory of Molecular Neurobiology, Institute of Experimental Medicine of the Hungarian Academy of Sciences

27

N-SIM S layout

N-STORM layout
Vibration isolated table
N-STORM
Laser unit

PC rack
Vibration isolated table
N-SIM S

1000

Laser unit

1000

PC rack

1500

1500
2750

400

2750
673

673

904.5

800

1471.5
1354

904.5

798

1540

1354

400

Unit: mm

Specifications and equipment are subject to change without any notice or obligation on
the part of the manufacturer. May 2021 ©2010-21 NIKON CORPORATIONWARNINGWARNING-LASER RADIATIONAVOID EXPOSURE TO BEAM
CLASS 3B LASER PRODUCTTO ENSURE CORRECT USAGE, READ THE CORRESPONDING
MANUALS CAREFULLY BEFORE USING YOUR EQUIPMENT.

Monitor images are simulated.
Alexa Fluor® and Cy are registered trademarks of Thermo Fisher Scientific, Inc.
Company names and product names appearing in this brochure are their registered trademarks or trademarks.
N.B. Export of the products* in this brochure is controlled under the Japanese Foreign Exchange and Foreign
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*Products: Hardware and its technical information (including software)

Total Power 500mW MAX.
CW 400〜700nm
IEC/EN60825-1 : 2007, 2014

Complies with FDA performance standards
for laser products except for
deviations pursuant to Laser Notice
No. 50, dated June 24, 2007

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Total Power 1500mW MAX.
CW 370〜790nm
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Complies with FDA performance standards
for laser products except for
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