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Powerful Instrumentation and Intuitive Software

3D imaging at its best: the outstanding sensitivity of the

LSM 710 permits the detection of even faint signals. The precise

motorized Z-drive of the microscope stand enables accurate

Z-stack acquisition. Piezo driven objectives and stages are

available for fast acquisition of Z-stacks in live cell imaging.

The heart of the LSM 710: the design of the QUASAR

detection unit gives users maximal freedom in multi-color 3D

imaging. Up to 10 different fluorescent dyes can be imaged

simultaneously and complete emission spectra recorded as

lambda stacks with up to 34 detector elements. Such parallel

acquisition of spectral information is ideal in 3D imaging as

it enables fast data acquisition and minimizes laser exposure

to the sample.

ZEN Software for 3D imaging: the graphical interface helps

users to navigate through Z-stack settings intuitively.

In BASIC mode, only essential tools are visible to the user.

If PRO mode is activated, additional advanced options are

accessible.

LSM 710 on the Axio Imager.Z1

LITERATURE Herr, J. M. jr. (1993). Clearing techniques for the study of vascular plant tissues inwhole structures and thick sections. Pages 63-84, in Tested studies for laboratory teaching, Volume 5 (C.A. Goldman, P.L. Hauta, M.A. O’Donnell, S.E. Andrews, and R. van der Heiden, Editors).

Liu, Y. C., and Chiang, A. S. (2003). High-resolution confocal imaging and three-dimensional rendering. Methods 30: 86-93.

Lin HH, Lai JS, Chin AL, Chen YC, Chiang AS. (2007). A map of olfactory representation in the Drosophila mushroom body. Cell 128:1205-17.

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Carl Zeiss MicroImaging GmbH07740 Jena, Germany

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

www.zeiss.de/micro

Dr. Ann-Shyn ChiangNational Tsing Hua University, TaiwanTsing Hua Chair Professor

“The integration of FocusClear tissue-clearing technology with ZEISS LSM confocal microscopy provides an unparalleled opportunity for penetrative high-resolution 3D imaging of tissues and biomaterials.”

FocusClear is available from CelExplorer Labs Co.P.O. BOX 2-238 Hsinchu, 30099 Taiwanwww.celexplorer.com

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

3D Imaging with Laser Scanning Microscopes

Whole brain 3D imaging: a single gustatory neuron (green) extends across the entire brain of the fruit fly Drosophila.

Visualizing the Architecture of Cells and Tissues

Laser Scanning Microscopes from Carl Zeiss have proven extremely powerful in 3D imaging.

Built-in features permit accurate recording of 3D information even if certain characteristics

of the specimen would otherwise compromise data acquisition. Upon sample preparation,

optical clearing techniques can be employed to optimize 3D imaging of fi xed material.

Laser Scanning Microscopes (LSMs) from Carl Zeiss offer

a tremendous amount of fl exibility for non-destructive,

high-resolution 3D imaging of samples labeled with

fl uorescent dyes. Their design enables optical sectioning by

preventing out-of-focus signals from being detected. For

visualizing three-dimensional structures, Z-stacks are

recorded that represent series of successive optical sections

each of which were acquired at a different Z-position.

LSMs from Carl Zeiss employ spectral imaging techniques for

multi-color visualization that provide cross-talk-free images

even if fl uorescent labels exhibit overlapping spectra.

Most diffi culties encountered in biological 3D imaging are

caused by intrinsic sample characteristics that compromise

3D imaging as they limit the penetration depth and cause

image distortions. Discriminating fl uorescent labels from

autofl uorescence is another common challenge in tissues of

plants and animals. Many of these problems can be overcome

either by sample preparation with optical clearing techniques

or with built-in tools of LSMs from Carl Zeiss.

2

Confocal 3D Imaging for Mapping Neural Connections in Drosophila Brains

Drosophila melanogaster has become a well-established model for

studying neural connectivity: Since structure and function are closely

linked in neural signal processing, detailed information on how the brain

is wired will shed new light on how it actually functions. Dr. Ann-Shyn

Chiang at the National Tsing Hua University in Hsinchu (Taiwain) aims to

generate comprehensive topographic maps of neural projections at the

level of individual neurons.

From Z-stacks to high resolution maps of neural connectivity

Structural studies of brain tissue are challenging because neurons are

extremely densely packed. Dr. Chiang’s group overcomes this problem

by imaging large numbers of brains taken from transgenic flies. Each

brain, due to genetic mosaic generation, expresses a GFP reporter in a

different subset of only a few neurons. With optical clearing using

FocusClear solution (see page 3), it is possible to acquire high-resolution

Z-stacks of entire Drosophila brains. After scanning, Z-stacks are subjected

to segmentation and surface rendering to extract 3D data of individual

neurons from the acquired raw data. Next, extracted 3D data derived

from several individual fl ies is fi t into a volume model using customized

software tools. This procedure of neuronal modelling is possible in

Drosophila since there are only minor variations from one individual to

the next. A 3D image database containing raw Z-stacks together with

3D data of classifi edneurons enables Dr. Chiang to analyze the likelihood

of connectivity by direct 3D visualization.

3D imaging of Drosophila’s brain reveals

universal aspects of brain function

This approach enabled Dr. Chiang to publish the first description of a

topographic olfactory map in a secondary olfactory center for any

species, from insects to mammals. This anatomical study based on 3D

confocal imaging, performed with LSMs from Carl Zeiss, provides new

insights into mechanisms of olfactory coding in Drosophila. Moreover,

mutant fl ies which exhibit an altered response to distinct olfactory stimuli

were found to differ also structurally from wild-type fl ies at the level of

neural connectivity. Insects exhibit many complex behaviours controlled

by an olfactory system that is genetically and anatomically similar to those

in vertebrates – including humans. High resolution confocal 3D imaging may

therefore provide clues to human neural disorders as well.

Model of an entire Drosophila head. Different sub-structures of the brain and nevoussystem are colour-coded. The raw data for rendering this view was acquired with LSMs.

3D model of an antenna in Drosophila.

Volume model showing olfactory circuits in the brain of Drosophila.

Volume model combining 3D data of several identivifi ed projecting neurons.

3

Projection of a Z-stack of the optical lobes in a Drosophila brain. The sample was subjected to the optical clearing procedure prior to image acquisitions.

Sample: 3D-rendered views of a transgenic zebrafish embryoexpressing GFP in glia cells. The GFP signal and tissue autofluorescence exhibit extensive overlap and cannot be distinguished without spectral imaging techniques (left image). Emission Fingerprinting enables a clear discrimination of GFP and autofluorescence (right).

Optical ClearingMaking Fixed Samples Transparent

Emission FingerprintingDiscriminating Autofluorescence from Fluorescent Labels

Since light carries all information on the sample’s structure to the detectors,

LSMs are carefully designed to enable the best possible 3D visualization of

a sample. Unfortunately, many samples are not transparent but instead

contain opaque components: they transmit some light, but also reflect,

scatter, or absorb some of it. As light has to pass through the sample, such

circumstances severely compromise microscopic 3D imaging, limit the

penetration depth and cause image distortions. A number of published

approaches, collectively called optical clearing, aim to achieve

refractive uniformity by making fixed biological samples transparent.

Some of these use agents, such as xylene, clove oil, cedar oil, and chloral

hydrate that have a refractive index similar to that of glass for imaging use

with oil-immersion lenses. Dr. Chiang developed the clearing reagent

FocusClear, a ready-to-use solution into which fixed samples labeled with

dyes (including lipophilic dyes, such as DiI, DiD and NBD-ceramide)

can be directly transferred from water, buffer solutions, alcohol, DMSO,

DMF and glycerin. Dr. Chiang routinely applies FocusClear to his samples

to obtain a sample refractive index that is similar to that or glycerol and

then views these samples with glycerine immersion objectives.

Model of an entire Drosophila head. Different sub-structures of the brain and nevous system are colour-coded. The raw data for rendering this view was acquired with LSMs.

3D model of an antenna in Drosophila.

Volume model showing olfactory circuits in the brain of Drosophila.

Volume model combining 3D data of several identivified projecting neurons.Sample: Sok-Keng Tong and Bon-Chu Chung, Institute of Molecular Biology, Academia Sinica, Taiwan

The quality of biological samples sometimes suffers from intrinsic

fluorescence signals, a phenomenon known as autofluorescence. This

unwanted signal compromises fluorescent dye detection because of its

non-uniform distribution as well as emission spectra that can extend

across the entire visible spectrum. With laser scanning microscopes from

Carl Zeiss, Emission Fingerprinting solves this problem: stored reference

spectra of fluorescent dyes and autofluorescence signals are recorded

from control samples and provide the information needed to achieve a clear

discrimination of fluorescent labels from autofluorescence using linear

unmixing.

Optical clearing: incubation with the clearing solution FocusClear gradually turns an insect brain into transparent tissue. Optical clearing increases the penetration depth and greatly improves deep imaging of fluorescent signals in the insect brain.

63

Since light carries all information on the sample’s structure to the detectors,

LSMs are carefully designed to enable the best possible 3D visualization of

a sample. Unfortunately, many samples are not transparent but instead

contain opaque components: they transmit some light, but also refl ect,

scatter, or absorb some of it. As light has to pass through the sample, such

circumstances severely compromise microscopic 3D imaging, limit the

penetration depth and cause image distortions. A number of published

approaches, collectively called optical clearing, aim to achieve

refractive uniformity by making fi xed biological samples transparent.

Some of these use agents, such as xylene, clove oil, cedar oil, and chloral

hydrate that have a refractive index similar to that of glass for imaging use

with oil-immersion lenses. Dr. Chiang developed the clearing reagent

FocusClear, a ready-to-use solution into which fi xed samples labeled with

dyes (including lipophilic dyes, such as DiI, DiD and NBD-ceramide)

can be directly transferred from water, buffer solutions, alcohol, DMSO,

DMF and glycerin. Dr. Chiang routinely applies FocusClear to his samples

to obtain a sample refractive index that is similar to that or glycerol and

then views these samples with glycerine immersion objectives.

Selecting the Perfect Lens for 3D Imaging

ZEN Software

Using Auto Z BrightnessCorrection to Compensate for Signal Loss in Deep Imaging

Refractive Index Correction

Since refractive index mismatch will compromise 3D imaging

due to spherical aberration, the right lens and immersion

medium will have to be chosen to exploit the Laser Scanning

Microscope’s capabilities to the fullest. No matter which

mounting medium or clearing method is chosen, Carl Zeiss

provides the right objective lens. For 3D imaging, LD lenses

with long work ing distances are

available. The Objectives Database

holds all lens specifi cations to make

this selection straightforward:

www.zeiss.de/objectives

Biological samples absorb and scatter photons thereby causing

gradual reduction of signal intensity in Z-stacks planes deep

inside the sample. For an accurate visualization of three-

dimensional structures, Auto Z Brightness Correction compen-

sates for such signal losses by continuously adjust ing settings

for laser power and detector gain when acquiring Z-stacks.

For maximal accuracy in microscopic 3D imaging, the

refractive indices of the immersion medium, the embedding

medium, and of the sample itself should match perfectly. In

practice, this match may not always be achieved. Because

refractive index mismatch causes spherical aberrations,

volumes will not be imaged accurately along the Z-axis. The

Refractive Index Correction in ZEN compensates for the differen-

ces in refractive indices between the immersion medium of the

objective (n‘) and the embedding medium of the specimen (n),

which can be adjusted between 0.5 and 3 (Ratio = n / n’).

Gallery view of Z-stacks acquired from the same sample. Auto Z Bright-ness Correction, a feature in ZEN, was activated (right panel)to compensate forsignal loss.

Effects of refractive index mismatch: depending on the refractiveindex mismatch between the immersion medium and the sample,a perfectly spherical object (middle) may appear compressed (left)or elongated (right) along the Z-axis upon Z-stack acquisition.ZEN features a refractive index correction that compensatesfor spherical aberrations.

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