M i c r o s c o p y f r o m C a r l Z e i s s

ELYRAEnter the World of Superresolution

Break Through the Resolution Barriers

in Light Microscopy

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Enabling Technologies for Superresolution How they work

Carl Zeiss puts two powerful and complementary techniques

pioneered by leading scientists at the heart of dedicated systems

for superresolution microscopy.

SR-SIM

Superresolution structured illumination microscopy brings

you up to double the resolution in all dimensions – without

compromising on dyes, without special sample treatment.

Rely on state-of-the-art algorithms to reconstruct a super-

resolution image in 3D.

Look at the two overlapping grids in the above image, tilted

at 5 degrees against each other, and you will notice a real

and visible pattern of approximately perpendicular dark and

light bands superimposed on the parallel lines. What you

are experiencing is a phenomenon called Moiré fringes,

originating from the interaction of the optical patterns of

lines. The fringes contain superresolution information that

otherwise escapes detection.

This is exactly the principle that is used in SR-SIM. A known

pattern is projected into the image plane and interferes with

sample structures, creating Moiré fringes. Superresolution

information can now be captured by the microscope from

these structures. All that remains to be done is to restore

this information into a superresolution image by high-end

algorithms.

PAL-M

Photoactivated localization microscopy lets you see single

fluorescent molecules switching between an “on” and “off”

state online, leading to imaging resolutions of ~20-30 nm.

Suitable fluorophores are already plentiful and still expand-

ing, including fluorescent proteins as well as organic dyes.

Every point-like object is imaged as an extended spot – the

so-called point spread function (PSF) in a light microscope.

If two such objects come close enough, their PSFs will over-

lap heavily, making it impossible to determine their precise

localization, let alone see them as separate entities.

But imagine you could view one at a time. Suddenly you

would be able to determine the centers of the PSFs, which

can be localized to a much higher precision than the PSFs

themselves (see above figure). That’s all that is done in

PAL-M. Fluorescent molecules are illuminated in such a way

that only a few are activated, ensuring that their PSFs do

not overlap. After registration these molecules are switched

off, while new ones are activated and so it continues.

The plot of all the localized molecules represents a super-

resolution image.

Sequential localization measurements result in higher effective resolution.

Moiré patterns formed by superimposed grids.

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Open Up a New Dimension ELYRA Superresolution Microscopy

The ELYRA product family from Carl Zeiss lets you opt for the highest

possible resolution, the most flexible choice of dyes – or achieve both

via a universal superresolution platform in a single, cost-effective system.

The nanoworld is bioscience’s every day workplace: in this

exacting environment, you must be able to identify and

manipulate the tiniest building blocks of the cell. Until now

light microscopy has been your most flexible tool, yet you’ve

always been limited to resolving structures of around 200

nanometers. You want to know more and now, with the

ELYRA range from Carl Zeiss, you will. We are putting in-

novative technologies at your disposal to enable you to

probe ever-deeper – to use superresolution experiments

to discover what lies beneath that barrier in a previously

uncharted world.

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SR-SIM image (right) and Widefield image (left) of neuronal growth cones. Staining for tubulin (red) and F-actin (green). Specimen: M. Fritz and M. Bastmeyer, University of Karlsruhe, Germany

ELYRA S.1 Put Flexibility First with Structured Illumination

ELYRA S.1 can image any fluorophore –

with up to twice the resolution of a conventional light microscope.

You have invested a lot of time and energy in producing

fusion proteins and multicolor staining protocols that are

perfectly adapted to your experimental system. Now, with

ELYRA S.1, you can capture superresolution data with ease,

using samples that may already be in your refrigerator! Do

you need z-sectioning for 3D data acquisition? A fast, light

efficient detection? Then ELYRA S.1 is your ideal choice:

• Image any fluorophore, using structured illumination

(SR-SIM), a universal fluorescence widefield technique.

Choose from up to 4 laser lines and a wide choice of filters

to match your experimental needs exactly.

• Gain up to twice the resolution of conventional micro-

scopes, depending on NA and wavelength.

• EM-CCD technology achieves exceptional detection sen-

sitivity.

• Its motorized grating exchange is the optimal adjustment

of excitation light modulation.

• Collect information in 3D. SR-SIM is unique in that it

improves resolution in both lateral (xy) and axial (z) direc-

tion. This lets you acquire Z-stacks easily, same as on a

confocal microscope.

• Highlight efficiency and acquisition speeds of 1 full

superresolution frame (512 x 512) every two seconds

allow imaging of bleaching sensitive and non stationary

specimens.

• Opt for a dual camera and you can carry out simultaneous

two color imaging.

• Laser-safe incubation solutions ensure highest stability.

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PAL-M image (right) and TIRF image (left) of antibody staining for tubulin in a cultured cell. Specimen: S. Niwa, University of Tokyo, Japan

ELYRA P.1 Localize Single Molecules for Unrivalled Precision

ELYRA P.1 takes light microscopy to the very limit. By localizing single molecules,

you can achieve effective lateral resolutions down to 20 nm.

You are interested in processes that take place near the

coverslip. You want to see and measure single molecules in

or near the plasma membrane (lipid rafts, receptor cluster-

ing, cell-substrate adhesion sites). Within this realm, PAL-M

takes you into a new world of data quality. Ultra-structural

studies with an effective resolution down to 20 nm can

show you substructure and patterns where conventional

microscopy will reveal “merely” colocalization. As a single

molecule method, PAL-M is inherently quantitative – every

image is a molecular statistics experiment:

• Examine processes close to the lower membrane.

• Its excellent TIRF field offers depth of about 100 nm for

excellent signal-to-noise and depth discrimination – plus

easy adjustment of optimal TIRF angle.

• Achieve single molecule sensitivity in widefield and TIRF

illumination, thanks to its highly sensitive back-thinned

EM-CCD camera from Andor (iXon series).

• The optional dual camera enables simultaneous two color

imaging.

• An infrared (IR) port lets you observe cell structure and

viability.

• Powerful lasers achieve efficient illumination and switch-

ing for a broad variety of dyes while the attenuable 405

laser guarantees balanced activation.

• Use dedicated TIRF objectives (Plan-Apochromat 100x /

1.46 NA Oil DIC, and Plan-Apochromat 100x / 1.57 NA

HI Oil DIC) for highest signal-to-noise.

• The incubation system creates highly stable environmental

conditions.

• Combine it with the optional LSM 710 for the full spec-

trum of modern confocal imaging.

• It delivers record-breaking 20 nm lateral resolution,

z-resolution given by TIRF field depth of 100 nm.

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ELYRA PS.1 Your Universal Superresolution Platform

Forget about compromises. ELYRA PS.1 lets you choose

the superresolution method that’s best adapted to your specimen

and with the same superb image quality.

Never have so many imaging modalities been available on

a single platform. The complexity of experiments in bio-

medical research often takes you beyond what a single

imaging method can provide. Because you are interested

in processes that take place within the context of an entire

cell, superresolution imaging will work best if you can image

this context at the same time, switching between different

imaging methods even in the course of an experiment.

That makes ELYRA PS.1 the ideal tool – it’s joined-up micros-

copy that delivers maximum flexibility without sacrificing

image quality:

• ELYRA PS.1 is a single microscope, laser module and soft-

ware for SR-SIM, PAL-M and laser widefield observation.

• It combines the flexibility of SR-SIM with the breathtak-

ing resolution gain and inherent single molecule analysis

of PAL-M.

• Add LSM (optional, upgradeable) for the complete spec-

trum of modern confocal imaging – FRET, FRAP, spectral

unmixing … and when you do superresolution work,

what can be better than to have the gold standard in

confocal sensitivity for comparison!

ELYRA PS.1 is a true platform concept, a great companion

in the ELYRA world that offers almost unlimited possibili-

ties. All that, and when you compare its price to the value

it adds to your investment, you will have another pleasant

surprise in store.

SR-SIM image of LifeAct (green) and CD82 (red) fusion proteins expressed in a cultured cell.Specimen: J. Lippincott-Schwartz, NIH, USA

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System Overview

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Think. Envision. Observe. Understand.

After 100 years of leadership in microscopy, now for the

first time we have broken through the traditional limits

of resolution to reach deep into new areas in subcellular

analysis.

Within the ELYRA product family from Carl Zeiss, you

can opt for the highest possible resolution, for the most

flexible choice of dyes. And importantly, you can also

keep your options open with a universal superresolution

platform that combines all of these attributes in a single,

cost-effective system.

ELYRA offers almost unlimited possibilities – indeed, only

you can demonstrate how far you can advance your

research with these outstanding tools.

Carl Zeiss MicroImaging GmbH07740 Jena, Germany

BioSciences | Jena LocationPhone : +49 3641 64 3400Telefax : +49 3641 64 3144E-Mail : micro@zeiss.de

www.zeiss.de/ELYRA

SR-SIM image of actin (green) and tubulin (red) cytoskeleton in primary chicken fibroblasts. Specimen: Prof. Martin Bastmeyer, University of Karlsruhe, Germany