ApoTome - Carl Zeiss International

ApoTome
Microscopy from Carl Zeiss
Suddenly Everything Looks Different
The Standard for Brilliant Images
in 3D Fluorescence Imaging
Änderungen vorbehalten.
Gedruckt auf umweltfreundlich
chlorfrei gebleichtem Papier.
60-2-0002/d – gedruckt 04.08

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1) Triple fluorescence of a drosophila embryo, lower left:
conventional fluorescence image, upper right: ApoTome mode
Frank Josten and Michael Hoch, University of Bonn, Germany
2) ApoTome mode and conventional image, hippocampus of
the rat, triple fluorescence, maximum projection of a 3D image
stack, Plan-APOCHROMAT 63x/1.4
Eberhard Fuchs and Susanne Bauch, GPC Göttingen, Germany
3) ApoTome mode and conventional image, multiple myeloma,
triple fluorescence, C-APOCHROMAT 63x/1.2
Peter Hutzler and Karin Bink, GSF Neuherberg, Germany
4) 3D image stack, upper left: conventional image, lower right:
ApoTome mode, Drosophila melanogaster embryo,
double fluorescence, Plan-NEOFLUAR 40x/0.75
Christian Klämbt, University of Münster, Germany
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Contents
The Quality Factor:
ApoTome
Where the Exceptional is Standard in 3D Imaging 4-5
The Performance Factor:
A Module That is More Than the Sum of Its Advantages 6-7
The Image Quality Factor:
The Visible Difference in Fluorescence Imaging 8-9
The System Factor:
Precise Interplay Reveals Everything in Great Detail 10-11
The Intelligence Factor:
AxioVision and ApoTome
New Perspectives for Life Sciences 12-14
The Function Factor:
Optical Sections by Means of Structured Illumination 15-17
System Solutions from Carl Zeiss
ApoTome with Axio Observer and Axio Imager 18-19
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The Quality Factor: ApoTome.
Where the Exceptional is Standard in 3D Imaging.
Innovation in 3D Imaging has a good name: ApoTome. With ApoTome Carl Zeiss
triggered a small revolution in fluorescence microscopy. Since then everything has
been different.
From an innovation to the standard
in life science research
In the meantime ApoTome has established itself as the
standard method in high-end research in Life Sciences.
Since the introduction of ApoTome, the problems associated
with stray light from other focal planes have become
a concern of the past. Carl Zeiss now offers outstanding
technology for the generation of optical sections in 2D
and 3D Fluorescence Imaging. Exceptional performance
became the standard.
• Exceptional in terms of contrast, image quality and
resolution – with an optical section thickness of one
Rayleigh unit
• Exceptional ease of operation – extremely simple and
flexible adaptation
• Exceptional in its seamless integration – easy to implement
in existing Carl Zeiss system solutions
Optical sections for every application
With ApoTome for the upright system solution
Axio Imager and for the inverted research platform
Axio Observer the new generation in light microscopy
sets a new standard in 3D Imaging: optical sections in
even complex applications; fascinating images, brilliant
image quality, and undreamed-of possibilities for scientific
analysis.
Awarded the R&D 100 Award and the Innovation Price
Photonics Circle of Excellence in 2003

AxioImager system with ApoTome, AxioCam MRm and AxioVision
The name
ApoTome, the innovation in 3D Imaging
The module
Slider technology for the Carl Zeiss Microscopes
Axiovert 200, Axio Observer, Axioplan 2 imaging*
and Axio Imager
The performance
New image quality and elevated contrast in the conventional
fluorescence microscope
Deblurred optical sections in thicker samples
The price
A cost-effective leap forward into an exceptional performance
category of digital microscopy
The principle
Sophisticated technology for artifact-free structured
illumination
The positions
Practice-oriented 2-in-1 slider for conventional fluorescence
(position 1) and the ApoTome mode (position 2)
for sophisticated digital microscopy
The operation
Quickly integrated
Simple control principle due to high-performance
technology
Problem-free working in the familiar system environment
The fluorescence
Open for the entire spectrum of possible fluorescence dyes
The system
Perfectly coordinated with the high-performance components
in the Carl Zeiss complete system: ApoTome –
microscope – camera – software – PC
The variants
Cost-effective as complete system or as an upgrade for
your existing system
*All Axioplan 2 imaging microscopes delivered after February 2000. Serial
numbers: from 35 11 000001; from 35 10 000001; from 35 02 000001
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The Performance Factor: A Module That is
More Than the Sum of Its Advantages.
ApoTome is more than just the sum of its advantages, i.e. brilliant image quality,
performance and speed, and an open spectrum of fluorescence dyes. Together
with its ease of operation and practice-oriented design, all these benefits add up
to a definitive increase in the performance of your Digital Imaging workstation.
1A: the image quality
One Rayleigh unit of optical section depth – this value
represents highest optical resolution with an excellent
signal-to-noise ratio. In the z-direction, the visible resolution
is increased by a factor of 2 compared to conventional
fluorescence microscopy. This allows generation of
optical sections and 3D image reconstructions from the
specimen. This is particularly crucial for thick specimens
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where it is also necessary to achieve optimum resolution
and contrast in the x-, y-plane. The optimized algorithm
for precise, artifact-free images is yet another benefit. In
a nutshell, this means you will be working with maximum
contrast, excellent image quality, deblurred image planes
and, as a consequence, with more visible information for
your analyses, documentations and publications.
Optimized: the speed
Three raw images are acquired for each optical section.
The results are calculated online in less than 80 msec –
depending on the image size used. The resulting image
is immediately displayed on the monitor and is immediately
available for further processing. Multichannel fluorescence
and Z-stack images are acquired automatically.
Flexible: optimum results with every objective
Different applications require different objectives. In
combination with ApoTome the best possible resolution
is always achieved. If needed, the grid in ApoTome can
be quickly and easily changed. A grid that is ideal for
the objective and the application can be selected from
three grids with different grid frequencies. The advantage
is that in each case an optimum result with an optical
section depth of approximately one Raleigh unit is
achieved.
1) Conventional, 2) ApoTome mode
Mouse omentum (mesentery), double fluorescence,
Plan-APOCHROMAT 20x/0.75
Mike Tighe, Kim Kusser and Troy Randall, Trudeau Institute,
New York, USA

Innovative LED fluorescent light source Colibri, fast,
flexible, without mechanical switching
Position 1: Iris diaphragm, position 2: ApoTome mode
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No limits: the fluorescence dyes and light sources
ApoTome can be combined with all light sources used
in fluorescence microscopy. HBO illumination is ideal as
an economical entry-level solution. The metal halogenide
lamp HXP 120 does not require adjustment, and offers a
long service life and a very homogenous illumination. The
brand new Colibri LED light source from Carl Zeiss generates
exceptionally high-contrast fluorescence images
with a high dynamic range. Extremely long-lasting LEDs
emit only the desired wavelength range. Consequently,
undesired light does not even have to be suppressed at
all. ApoTome provides nearly unlimited flexibility in the
selection of fluorescence dyes. System technology does
not determine whether you work with DAPI, FITC, rhodamine,
Cy5 or with vital dyes such as GFP or mRFP. You
decide.
Plug and go: ease of operation
Simply slide ApoTome into the field diaphragm plane
of the reflected-light beam path of your microscope.
ApoTome offers you two positions: position 1 – the iris
diaphragm for the Koehler illumination in the reflectedlight
beam path; position 2 – the ApoTome grid for optical
sections with brilliant image quality. The operation
is just as simple as the insertion. Set ApoTome to the
appropriate position, iris diaphragm or ApoTome mode –
depending on what your application requires. The slider
remains in your microscope and is thus always ready to
use and protected.
Advanced technology: the image acquisition modes
Advanced technology for brilliant images and greatest
sample protection: ApoTome calculates your images
directly online, which allows you immediate access to
the optical sections. However, ApoTome also allows the
preparation of raw data. Thus, you can also process images
generated with this module offline with different
settings. The advantage is that images of very sensitive
specimens must only be acquired once. With the aid of
a specially adapted deconvolution, the resolution of the
raw data can be further increased for perfect results with
every sample.
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The Image Quality Factor: The Visible Difference
in Fluorescence Imaging.
Experience the difference: ApoTome brings Fluorescence Imaging to a new level
of performance – with excellent image quality, fascinating images, and new
perspectives for science and research.
Decoding gene functions
In Drosophila research, wild type and mutant embryos are
frequently compared with each other in various stages of
development. In the case of the mutants, genes playing a
specific role in developing or maintaining bodily functions
have been manipulated. The comparative study allows
conclusions to be drawn about gene functions in certain
1) Conventional, 2) ApoTome mode
Drosophila melanogaster embryo, double fluorescence, EC Plan-NEOFLUAR 40x/1.3
George Scaria, University of Illinois at the Chicago Medical School, USA
developmental processes. The images below show the
development of Drosophila melanogaster embryos at a
relatively early stage subsequent to fertilization. All the
cells are in the same cycle of cell division. In the ApoTome
mode, the spindle microtubules (red), which separate the
chromosomes (blue), can be clearly identified.

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1) Conventional, 2) ApoTome mode
Sprouting axons of a dorsal root
ganglia explant, triple fluorescence,
Plan-APOCHROMAT 20x/0.75
Investigation of neuronal circuits
Studies in developmental neurobiology include the examination
of how nerve cells developing in a certain part
of the body connect with cells in a completely different
part of the body. The connection is made by means of
long cell processes, known as axons, which, by exploring
their environment, e.g., along a chemical gradient, find
their way through the tissue to their target cells. This process
occurs primarily during the embryonic development
of an organism. In order to decode these highly specific
and extremely complex mechanisms, zebrafish research
1) Conventional, 2) ApoTome mode
Zebrafish (Danio rerio), triple fluorescence, Plan-APOCHROMAT 20x/0.75
Monika Marx and Martin Bastmeyer, University Karlsruhe, Germany
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also uses mutants. These are compared with wild type
individuals to identify the genes which are crucial for this
connection. These findings are of major significance for
clinical questions. This example shows the head region of
a zebrafish embryo (Danio rerio). In the ApoTome mode,
two different populations of neurons can be clearly distinguished
by the red and green marking. The nuclei are
marked using DAPI.
Additional applications, images and additional movies
are to be found at: www.zeiss.de/apotome
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The System Factor: Precise Interplay Reveals
Everything in Great Detail
Carl Zeiss offers a broad range of system components which all have one thing in
common: when used in combination with ApoTome, they fully utilize all of the
possibilities of Fluorescence Imaging. From research microscopes with brilliant
optics, via high-performance cameras and the comprehensive AxioVision software
platform, all system components are perfectly coordinated with one another.
Optimized for fluorescence:
Axio Observer and Axio Imager
The high-end systems Axio Imager und Axio Observer
combined with ApoTome are ideal research platforms for
3D Imaging.
• Fully motorized microscope for high-end research
• Apochromatically corrected fluorescence beam path
for brilliant image quality in Multichannel Image
Acquisition
Axio Imager system with ApoTome, AxioCam and AxioVision
• Z-focus with a resolution of 10 nm and high reproducibility
• Sophisticated ergonomics for comfortable microscopy
• TFT display for optimum ease of operation
Particularly appropriate for long-term experiments even
with living cells: Axio Observer with diverse, modular
and fully integrated incubation solutions. The hardwarebased
focus stablizer Definite Focus ensures stable focus
positions even under the most difficult conditions.

Powerful even in weak fluorescence:
the cameras
AxioCam is the leading generation of Peltier-cooled digital
cameras from Carl Zeiss. These cameras feature highest
sensitivity, a resolution of 1.4 megapixels in AxioCam MRm
or up to 13 megapixels in AxioCam HRm, and a dynamic
range of up to 1 : 2200 or 1 : 2500 in 12 or 14 bit, respectively,
as well as maximum image quality. Custom
tailored to your applications in fluorescence microscopy
naturally in monochrome version – and at a convincing
price-performance ratio.
Axio Observer system with ApoTome, AxioCam and AxioVision
Efficient in research and routine applications:
the microscope control
Using the Carl Zeiss AxioVision software platform, you
are able to control all motorized microscope systems
from Carl Zeiss interactively and fully automatically. The
advantage is simple and efficient operation and clarity of
the user interface. The microscope’s setting parameters
can be saved as required and retrieved again at any time
AxioVision determines magnifications automatically, and
complex acquisition modes are easy to configure. As a
consequence, your results can be reliably reproduced.
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The Intelligence Factor: AxioVision and ApoTome.
New Perspectives for Life Sciences.
ApoTome and AxioVision – an outstanding combination. AxioVision is the software
platform from Carl Zeiss for all requirements in Fluorescence Imaging – from twodimensional
imaging up to the sixth dimension. Any combination of x, y, z, lambda,
time lapse and different sample positions is possible for automatic image acquisition.
And with ApoTome in AxioVision, optical sections are available in every mode.
Multichannel Fluorescence
Using this module, in connection with ApoTome, you
can acquire multichannel images automatically with high
contrast and optimum quality. One channel is acquired
using the optimum exposure time for each excitation
wavelength. The grid is also focused automatically for
each fluorescence wavelength. Every channel of the optical
section is calculated online. The advantage is that you
see the results immediately, for example, colocalization
of fluorescence dyes in thick specimens.
User interface for multichannel images Gallery view of a Z-stack
Z-Stack
Using this module you can acquire and display different
focal planes of a specimen as an image stack – with high
precision and accurately reproducible. ApoTome controls
the acquisition of the three raw images. The optical section
is calculated online in every plane. Consequently, it
is possible for you to see the result quickly.
Time Lapse
Observing specimens, studying and recording changes
over time, documenting the results clearly – the Time
Lapse module enables you to control acquisition and the
microscope with precision.

Muscle-specific GFP labeling (yellow), nuclear labeling (blue), developmental stage of
10.5 days, double fluorescence Z-stack image in ApoTome mode,
Plan-NEOFLUAR 5x/0.15
Dr. Frederic Relaix and Emanuelle Perret, PFID, Pasteur Institute, Paris, France
MosaiX
Using this module and a motorized x-, y-stage, even large
specimens which extend beyond the field of view can
be displayed with high resolution and high contrast. The
specimen is scanned automatically in order to generate
a large composite image. This electronically generated
image of the specimen provides a complete representation
of the sample, and can be used as a basis for further
analysis.
Mark&Find
Mark&Find automatically acquires images at several
locations in a specimen, e.g. over time. With this module
you mark several locations in your specimen, which
you subsequently, repeatedly, and exactly relocate during
the experiment. In this manner you increase the data
throughput and efficiency in long experiments.
Above left: Conventional fluorescence image acquisition, lower right: optical section
with ApoTome, Platynereis dumerili, triple fluorescence (DAPI, Phalloidin-Alexa488,
alpha-Tubulin Alexa555), maximum intensity projection of a 3D image stack,
Plan-APOCHROMAT 20x/0.8
V. Wilkens, University of Osnabrück, Germany
HDR Image Acquisition
Sometimes even the high dynamic range of the best
cameras is inadequate. If you desire to simultaneously
examine very light and dark areas in your specimen,
HDR (High Dynamic Range) Image Acquisition provides
the solution. In HDR mode several images with different
exposure times are combined to form an image with increased
dynamic range. Thus, all the details in an image
are reproduced.
Inside4D
In combination with ApoTome, Inside4D makes multi-
dimensional visualization and presentation of cells and
tissue sections exceptionally easy. ApoTome generates
the optical sections online. You can then visualize the
data in Inside4D immediately. 3D reconstructions can be
viewed from any angle. With this module you can visualize
the structural context in the specimen. And you have
an excellent tool for the production of movies at your
disposal – for the best possible presentation of your 3D
images in the Internet, with PowerPoint TM , or with many
other media.
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Neuromuscular synapses in the sternomastoid muscle of a rat, triple fluorescence Z-stack
Dr. Le Tian, Dr. Wes Thompson, University of Texas, Austin, USA
Measurement and analysis
Measure your specimens in two or three dimensions,
manually or automatically. Various AxioVision modules
help you perform these tasks. Intensity or area measurements,
segmentations or colocalizations can be automatically
or interactively determined. Volumes, surfaces
or distances can also be measured in three-dimensional
image stacks. It is comfortable in its range of functions
and easy to operate.
Fast MosaiX
Dual Camera
Digital
High Speed
Recorder
HDR Imaging
Fast
Acquisition
Widefield
Multichannel
Unmixing
Image acquisition Image
Processing
Imaging Plus ASSAYbuilder
Panorama Mark&Find ApoTome 3D Measure TMA
MosaiX Time Lapse 3D
Deconvolution
Extended
Focus
Autofocus Multichannel
Fluorescence
Z-Stack 2D
Deconvolution
ELISPOT
Image Analysis
SFM
QuantiFISH Ratio Tracking
AutoMeasure AutoMeasure
Plus
Inside4D Interactive
Measurement
Combinations
All these modules can be freely combined with one another
and form system solutions which are designed to
address an extremely wide range of requirements. The
result is the cost-effective adaptation of the solution to
your application without unnecessary investments.
PowerPoint TM is a product of Microsoft ® .
Online
Measurement
2) 3D distance measurement from the surface on one yeast particle to
the surface of the next one. Cells: RAW 264.7 Macrophages with nuclear
staining (blue), actin staining (red) and zymosan yeast particle (green)
Dr. Birgit Kraus, University of Regensburg, Germany
Colocalization
Physiology Asset
Archive
Documen-
tation
VBA
Commander
Configuration
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Schematic of beam path
The fluorescence excitation light passes through two glass plates in the ApoTome slider.
There is a grid structure on the first glass plate; the grid pattern is “imprinted” in the
excitation light. The second glass plate is tilted by a scanning mechanism, thereby
slightly shifting the beam path with the imprinted grid pattern. The excitation light
(green) with the grid pattern is then projected onto the specimen via a conventional
fluorescence filter set. The emission light (red) is collected by the objective and imaged
onto the camera sensor.
Please visit our educational website www.zeiss.com/campus if you are interested to
see how ApoTome works and to view ApoTome application images.

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The Function Factor: Optical Sections by Means
of Structured Illumination.
ApoTome has continuously proven its leadership position in Fluorescence Imaging
and has received numerous awards from specialist groups. The functional principle
used by ApoTome to generate optical sections is structured illumination, which is so
well-engineered that it provides artifact-free images.
ApoTome: the grid pattern creates clarity
In the ApoTome mode, a grid structure is inserted into the
light field diaphragm plane of the reflected-light beam
path (page 17, Fig. 1A). The grid structure is precisely imaged
into the object plane; it is clearly visible through the
eyepieces and in the live image. In a second step, a highprecision
scanning mechanism moves the grid pattern in
defined steps across the sample plane. In the third step,
images are acquired at each grid position (Figs. 1A-C).
Finally, a single resulting image is calculated from the
three raw images using a fast mathematical algorithm
(Fig. 1D). The result is a precise optical section through
the specimen with no blurring, with elevated contrast,
and perfect for 3D reconstructions.
Precisely in focus: the calibration
A fundamental challenge in generating precise, deblurred
optical sections using structured illumination is to precisely
focus the grid for the selected objective and fluorescence
wavelength. This is very easy with ApoTome.
You start an interactive assistant in AxioVision, and it
An interactive dialog guides you once through the calibration
of the grid focus
guides you through the calibration process step by step.
And in the apochromatic fluorescence beam path of
Axio Imager and Axio Observer, the grid only has to be
shifted minimally or not at all for the different fluorescent
wavelengths.
Interior view of the ApoTome slider: iris diaphragm in position 1, grid and planparallel glass plate in position 2

Fig. 1.: Schematic depiction of the grid projection in a fluorescence specimen
A-C: Raw images with different positions of the grid
D: Optical section through the specimen
Background
In conventional fluorescence microscopy, the image
always consists of signal contributions coming from the
focused object plane and from the structures located
above and below it. The blurred structures from above
or below are either perceived as being out of focus or, if
they are clearly outside the focal plane, have the effect
of brightening the image background, which reduces
image contrast.
The “structured illumination” principle
In the “structured illumination” process used in ApoTome,
a grid is inserted into the field diaphragm plane of the
fluorescence beam path. Superimposing the projected
grid onto the fluorescent sample generates an image of
the specimen that has dark grid lines running through it
Section thicknesses with ApoTome when used with Axio Imager
(Fig. 1A). Outside the focal plane the grid is also blurred,
thus no grid projection is visible in defocused specimen
structures (Fig. 1A, arrow). In the context of image analysis,
this provides a criterion for differentiating in-focus
object structures from those which are defocused. The
determination of the grid contrast as a function of the
location (xy) can thus be used to calculate an optical section
(Fig. 1D). In order to reconstruct all the image information,
at least three raw images with different positions
of the grid projections must be acquired (Figs. 1 A-C: grid
shift marked by blue line).
Literature: Schäfer, L. et. al.: Structured Illumination Microscopy:
Artifact analysis and reduction utilizing a parameter optimization
approach. J. Microsc. 216 (2004) pp.165
Objective Magn. NA Immersion Recmd. grid Section thickness at 490 nm (RU/μm)
EC Plan-NEOFLUAR 10x/0.3 10 0.3 Air L1 1.01 / 11.04
EC Plan-NEOFLUAR 20x/0.5 20 0.5 Air M 1.03 / 4.05
EC Plan-NEOFLUAR 40x/0.75 40 0.75 Air H1 1.09 / 1.89
EC Plan-NEOFLUAR 40x/1.3 Oil 40 1.3 Oil M 1.07 / 0.94
EC Plan-NEOFLUAR 63x/0.95 Korr. 63 0.95 Oil H1 0.7 / 0.76
EC Plan-NEOFLUAR 63x/1.25 Oil 63 1.25 Oil H1 1.08 / 1.03
EC Plan-NEOFLUAR 100x/1.3 Oil 100 1.3 Oil H1 0.71 / 0.63
LCI Plan-NEOFLUAR 25x/0.8 Imm. Korr. 25 0.8 Oil, water or glycerin L1 1.01 / 2.26
LCI Plan-NEOFLUAR 63x/1.3 Imm. Korr. 63 1.3 Water or glycerin H1 1.05 / 0.88
Plan-APOCHROMAT 10x/0.45 10 0.45 Air L1 1.45 / 6.88
Plan-APOCHROMAT 20x/0.8 20 0.8 Air L1 1.08 / 1.65
Plan-APOCHROMAT 40x/0.95 Korr. 40 0.95 Air H1 1.07 / 1.16
Plan-APOCHROMAT 40x/1.0 Oil 40 1.0 Oil M 0.97 / 1.45
Plan-APOCHROMAT 40x/1.3 Oil 40 1.3 Oil M 1.07 / 0.94
Plan-APOCHROMAT 63x/1.0 W 63 1.0 Water H1 0.93 / 1.22
Plan-APOCHROMAT 63x/1.4 Oil 63 1.4 Oil H1 1.06 / 0.81
Plan-APOCHROMAT 100x/1.4 Oil 100 1.4 Oil H1 0.7 / 0.53
LD LCI Plan-APOCHROMAT 25x/0.8 25 0.8 Oil, water or glycerin L1 0.88 / 1.35
C-APOCHROMAT 10x/0.45 W 10 0.45 Water L1 1.46 / 9.40
C-APOCHROMAT 40x/1.2 W 40 1.2 Water M 0.94 / 0.85
C-APOCHROMAT 63x/1.2 W 63 1.2 Water H1 0.95 / 0.86
LD C-APOCHROMAT 40x/1.1 W 40 1.1 Water M 0.94 / 1.02
αPlan-FLUAR 100x/1.45 Oil 100 1.45 Oil H1 0.67 / 0.48
αPlan-APOCHROMAT 100x/1.46 Oil 100 1.46 Oil H1 0.67 / 0.47
*Analogously to the definition of the Rayleigh unit (distance from the PSF’s maximum to its first minimum)
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ApoTome is compatible with the following Carl Zeiss stands:
Axioplan 2 imaging (serial numbers: from 35 11 000001; from
35 10 000001; from 35 02 000001), Axio Imager.D1 and
Axio Imager.Z1, Axio Imager.A2, Axio Imager.M2, Axio Imager.D2
and Axio Imager.Z2, Axiovert 200M, Axio Observer.A1,
Axio Observer.D1 and Axio Observer.Z1

For optimum results we recommend the use of chromatically highly
corrected objectives, e.g. of the Plan-NEOFLUAR, Plan-APOCHROMAT,
or C-APOCHROMAT series.
Use of ApoTome in connection with Phase Contrast objectives is not
generally recommended, since the phase ring blocks spatial frequencies
in the image, which are important for the reconstruction of the
optical section.
You will find a detailed list of recommended objectives with recommendation
of appropriate grids in the AxioVision Online Help.
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Carl Zeiss MicroImaging GmbH
07740 Jena, Germany
BioSciences | Göttingen Location
change.
Phone : +49 551 5060 660
to
Telefax : +49 551 5060 464
subject
E-Mail : micro@zeiss.de
www.zeiss.de/apotome Information
Printed on environmentally friendly
bleached without cholorine.
60-2-0025/e – printed 01.09