MicroImaging Newsletter Sept 09:Layout 1 - Carl Zeiss

Microscopy from Carl Zeiss
MicroImaging
Newsletter
9/09
Join Us for
an Evening Under
the Stars at
Neuroscience 2009
Superresolution
Superresolution microscopes put cutting
edge discoveries at your fingertips
Over 100 years ago, Ernst Abbe determined
the resolution limit of a light
microscope. This minimum value of about
220 nm holds true for such modern day
light microscopes as simple camera
systems through high end laser scanning
confocal microscopes. This means that
two structures that are closer together
than 220 nm appear as one unified
structure in your current microscope.
We are developing instruments based on
two Superresolution techniques:
Superresolution Structured Illumination
Microscopy (SR-SIM) and Photoactivation
Localization Microscopy (PAL-M). These
systems promise to yield images beyond
what is capable with your current system.
And with Carl Zeiss’ easy-to-use
software, hands-on training, and extensive
support network, you can be sure to
hit the ground running with publication
quality images.
To celebrate the introduction of our new
Superresolution systems, we will be hosting an
evening in Chicago during the annual meeting
of the Society for Neuroscience. The event will
feature talks by two leading scientists in the field
of Superresolution microscopy: Dr. Eric Betzig
(Howard Hughes Medical Institute, Janelia Farm)
and Dr. Rainer Heintzmann (Kings College
London). Prior to the talks, relax and socialize in
the gallery with complimentary drinks and appetizers
to the sound of live jazz music. The event
begins at 6 pm on Tuesday, October 20th.
Reservations are required and can be
made at www.zeiss.com/neuroscience.
Contents
Superresolution
Evening Under the Stars
Xtreme Field of View SEM
Requirements for
Live Cell Imaging
Product News
Recently, there has been a small explosion
of papers as researchers have broken
that resolution limit with various modifications
of fluorescence light microscopy.
The potential these so-called
Superresolution microscopes bring to
research caused the technique to be identified
as Method of the Year 2009 by
Nature Methods (January 2009, Vol 6,
No. 1).
Bring Superresolution to your current
experiments: SR-SIM can be
used on conventional samples
When an image containing regularly
spaced structures is overlayed with a structured
pattern, simple interference pattern,
also called Moiré fringes, can be seen. In a
microscope system, if the frequency of the
grid is at or near the resolution limit of the
microscope, these Moiré fringes will con-
…more on page 2
Resources

Xtreme Field of
View Scanning
Electron
Microscopy
The ZEISS SEM Visualization Engine (SEM-
VE) enables the acquisition of a variety of
scanning electron microscopy images (SE,
BSE, STEM, etc.) over large fields of view
(>1 gigapixel) at nanometer scale resolution.
In the neuroscience field, researchers
are using SEM-VE to map large neural networks
including synaptic junctions -
requiring nanometer-resolution – over
cubic millimeter volumes.
10 µm
A single acquisition extreme
field-of-view low-voltage STEM image
(2 nm pixel size) of a rat hippocampus
acquired in a field-emission SEM.
(Sample courtesy of J. Mendenhall,
Univ. of Texas at Austin)
The SEM-VE adapts to any standard
ZEISS SmartSEM platform, and can
extend the number of pixels acquired
in an image by 150 times, without
sacrificing ultimate resolution. This
dramatically improves automation and
throughput by reducing stage motion
requirements that can be critical to laborious
image stitching routines.
MicroImaging Newsletter
Superresolution
…continued from page 1
tain high frequency information that is
normally irresolvable. By analyzing these
Moiré fringes in Fourier space, this higher
frequency information can be decoded and
unveiled in the final image. Using this
technique of Superresolution Structured
Illumination Microscopy (SR-SIM), the resolution
can be doubled in both XY and Z.
As SR-SIM works with any fluorophore (eg.
Alexa dyes, DAPI, Cy dyes) or fluorescent
protein (eg. GFP, YFP, mCherry, mTomato)
and does not require special sample
preparation, SR-SIM can easily be incorporated
into your current experiments to produce
4-color, Superresolution images in
3D. In addition, with acquisition speeds at
1-2 frames per second, Superresolution
imaging of live cells undergoing slower
movements is possible.
Experience resolution approaching
that of electron microscopy with PAL-M
As stated, two points of light appear as
one if they are closer than 220 nm in a
traditional fluorescent microscope. The
idea behind PAL-M is to image these two
points separately, determine their exact
XY locations, and then overlay them in a
post-processing step. This technique is
possible with photoactivable or photoconvertible
fluorescent highlighters
whereby low powers of activation light
affect a very small population of the
sample. By repeating a process of activating
a few molecules, imaging these
molecules, then bleaching or switching
them off, a pointillist image is constructed
over time with resolution limits down
to 20 nm. Z-resolution down to 100 nm
is achieved with TIRF illumination using
our new PAL-M 100x/1.57 NA objective
lens. With a Carl Zeiss Superresolution
specialist available to train you and your
lab on the logistics of fluorophores, sample
preparation, imaging, and analysis,
you can expect images approaching EMresolution
while still using relatively easy
sample preps and fluorophore labels.
Accurately correlate confocal, SR-
SIM, and PAL-M images from the
same structures Only Carl Zeiss can
offer you both SIM and PAL-M on the
same microscope stand as a confocal
microscope. With the same software, you
can easily switch between confocal or
either of the Superresolution techniques.
Compare and contrast the same structures
with all three techniques to cross-examine
your data and build more convincing scientific
models. Additionally, LSM 710s can
be integrated with a Superresolution system,
allowing existing owners to extend
their imaging capabilities.
www.zeiss.com/superresolution
2
Learn more about SEM-VE and brain
mapping at: www.zeiss.com/nts

Since the 1970s scientists have tried to
capture life on screen. Such attempts
range from observing cultured cell lines to
time-lapse photography of developing
early embryos to deep brain imaging in
waking-state small mammals. Although
imaging of living specimens introduces
many challenges, only living samples can
accurately portray biology’s natural
state. Although the number and sophistication
of the tools utilized for live cell
microscopy has increased enormously,
there are many parameters which must be
carefully considered when planning such
an experiment.
Five basic requirements must be satisfied
in order to conduct a successful live cell
experiment:
1 Although the goal in live cell imaging
is to observe the natural state of the
cells or organisms, most experiments
require artificial introduction of a
label for proteins, sub-cellular components,
or particular cell types. The
development of an array of fluorescent
proteins and dyes has provided
biomedical research with enormous
flexibility of markers that extend now
to photo-activatable and -switchable
versions that can be used, for example,
in cell lineage experiments.
2 Having introduced specific marker(s)
into the cells or organism, the second
critical step is to excite or activate
them with minimal illumination.
This reduces the potential for photoxicity
and resulting damage or
death to the sample. Examples of
this include high speed LED illumination
systems for widefield imaging
and AOTF controlled or directly modulated
solid-state lasers employed in
confocal microscopes, which both
work well to image cells with the
gentlest light possible.
MicroImaging Newsletter
Live Cell Imaging:
Five Basic Requirements
3 Use of a detection device with the
highest possible efficiency ensures that
the lowest illumination intensity can be
used. CCD cameras have improved
remarkably over the last decade with
quantum efficiencies greater than 85%
whereas EMCCDs allow for very high
acquisition rates combined with high
efficiency. Unitary detectors, such as
the PMTs used in point scanning confocal
microscopes, have not improved to
the same extent as CCDs, but the use
of materials such as gallium arsenide
phosphide (GaAsP) and significant
reductions in dark current have
enabled these systems to be much
more applicable to live cell imaging.
4 A live cell experiment only works if
the cells are kept alive - often for
days. To achieve this, an incubation
system may be required to control
temperature, pH, CO 2 , O 2 , and
humidity levels. The most refined systems
include computer control and
recording of these parameters.
5 Last, but not least, the sample has to
be held in focus over the term of the
experiment. Two techniques are now
commonly used. Patterns formed with
infra-red light are projected onto the
underside of the coverglass and a
small camera linked to the microscope’s
focus mechanism is used to
hold the pattern in focus, thereby holding
the cells in focus. Alternatively, a
laser (typically in a laser scanning
microscope) can be used to image the
undersurface of the coverglass in
reflected light mode, thereby providing
an absolute reference from which the
focus can be returned to the sample.
If you have any questions on getting
started with live cell imaging or
with your current experiments, send
an email to Support@zeiss.com.
Products
VivaTome: Fast
optical sectioning
made easy
and affordable
With the new VivaTome, Carl Zeiss
introduces an innovative concept for fast
acquisition of optical
sections. Using the
technique of aperture
correlation, VivaTome
combines the light
efficiency of structured
illumination
with the speed of a
spinning disk. A grid
pattern with high
transmission efficiency
is used to modulate
the excitation light, therefore,
VivaTome can be operated with a standard
white light source. All information necessary
to calculate an optical section is captured
simultaneously in one single image,
efficiently excluding motion artifacts.
Furthermore, a standard widefield image
can be calculated from the same data –
so all the information from your sample
is accessible in a single shot. Two different
grid patterns allow the use of a wide
variety of different
objectives with optimal
sectioning performance.
VivaTome
allows image capture
at high frame
rates suitable for
imaging of living
cells and for observation
of dynamic
events in developmental
processes.
The simple and compact
instrument can be mounted on the
camera port of a variety of upright and
inverted ZEISS microscopes. Field
upgrades with current systems are also
possible. It is fully integrated in the powerful
AxioVision Software package, opening
up new possibilities in imaging of
dynamic events. 3

MicroImaging Resources
MicroImaging Newsletter
Zeiss on
Your Campus:
Join us for
a new lecture
series on live
cell microscopy
With over 1000 participants in 47 locations,
the recently completed North American
Zeiss on Your Campus (ZOYC) Tour 2009 on
3D Imaging Techniques was a great success.
Our goal in these workshops was to
enable scientists to fully optimize the imaging
parameters using their current systems,
as well as understand the various available
3D techniques in order to make informed
decisions when selecting a microscope system
for a given experiment. This was
achieved through a short series of informative
lectures and hands-on sessions.
Based on the positive feedback from this
endeavor, we will launch another educational
tour in 2010. This new tour will focus on imaging
of living cells and animals, a topic which
has been strongly requested by the scientific
community. Our lectures and hands-on sessions
with living samples will include topics
such as balancing resolution and speed during
image acquisition, cell viability and photo-toxicity,
and presenting 4D data sets. Some of the
locations we will be visiting in 2010 include:
• Columbia University
• McGill University
• Stanford University
• St. Jude’s Children’s Research Hospital
• University of California at Irvine
• University of Michigan
• University of Pennsylvania
• And more…
Please visit www.zeiss.com/zoyc to
view the full list of locations with dates and
to reserve your spot.
Zeiss Online Campus
Wondering how to correctly analyze
your images for colocalization Curious
how FRET Biosensors work Do you
have questions about the latest
developments in fluorescent proteins
Carl Zeiss answers such questions at
www.zeiss.com/campus. Developed
in collaboration with Mike Davidson of
Service & Support
Our in-house service and support teams
work mainly behind the scenes; but if
something goes wrong, our people make
the difference. We are pleased to introduce
team members in this, as well as
upcoming newsletters.
Working in the application support
group, Tim Pratt answers customer questions
about image analysis and Laser
Microdissection systems. Having done his
PhD in Molecular Genetics, he has a solid
background in laboratory benchwork and
experimental design. Tim has been with
Carl Zeiss for over four years and participates
in many internal training sessions
and external scientific meetings to keep
his knowledge of microscopy and related
techniques current.
Tim and his peers in the support group
use remote access tools which enable
them to connect to any system and troubleshoot
most questions immediately.
They answer questions ranging from
Florida State University, our site is an
online reference guide for microscopy,
digital imaging, and related applications.
The combination of interactive tutorials,
review articles, and comprehensive lists
of published references make this site the
best place to learn more about leading
edge techniques in microscopy.
“Why is my camera image in the wrong
color” to “How do I correct shading
in my image” to “Can I image a particular
fluorophore with my current system”
Tim and his colleagues can be
reached Mon – Fri from 9 am – 8 pm
Eastern time on the Carl Zeiss
support line at 1-800-509-3905.
They can also be reached by email at
support@zeiss.com.
Carl Zeiss MicroImaging, Inc.
One Zeiss Drive
Thornwood, NY 10594
1-800-233-2343
micro@zeiss.com
www.zeiss.com/micro
Customer Service: 1-800-233-2343
Systems Service: 1-800-633-6610
Application Support: 1-800-509-3905
Editors: Duncan McMillan, Dr. Maya Everett,
and Dr. Jochen Tham
Production:
Carl Zeiss MicroImaging, Inc., Thornwood, NY
For more information, to request literature
on our products, or to remove your
name from our mailing list, please email
micro@zeiss.com