Leica MACROFLUO User Manual

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Leica MacroFluo

The fluorescence macroscope

Precision from the total picture down to the smallest detail

Living up to Life

Studying Nature's Clues

In modern developmental, molecular and cell biology, fluorescence technologies play a critical
role in functional research of organisms and provide the researcher with enormous potential
for gaining insight into a world that is usually hidden to the human eye. Complex genetic studies
and cellular examinations of suitable model organisms are the prerequisites for understanding
the molecular basis and complex correlations of life.

In many respects, the development, cell biology and physiology of the mouse are very similar to
those of the human being. The mouse is therefore an ideal biological model for in-vivo studies
that explore the causes of disease in humans and develop new therapeutic approaches. Cell
and developmental biology are increasingly focused on understanding complex interrelation­ships in the organism. Therefore, a flexible microscope system is required that enables the
causes of disease to be examined on living animal models with maximum precision and resolu­tion in an intensely fluorescent image field and to be captured in digital images.

The Leica MacroFluo
large working distances, parallax-free and precise imaging – with fluorescence technology at
high resolution. The Leica MacroFluo™ provides brilliant images with maximum image precision
and depth of information over a wide magnification range to capture all aspects of the complete
image.

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concept combines the advantages of macroscopy – large object fields,

➊ Zebrafish embryo. GFP expression with control of Sonic hedgehog

gene-regulatory sequences. GFP expression in anterior baseplate
and notochord. Prof. Dr. Uwe Strahle, Research Center Karlsruhe,
Germany and Cédric Vonesch, Imaging Centre IGBMC, Illkirch,
France

➋ – ➐ Expression of green fluorescence in an EGFP (Enhanced Green

Fluorescent Protein) transgenic mouse, Dr. Massimo Pasqualetti,
Università di Pisa and Dr. Filippo Rijli, Imaging Centre IGBMC, Illkirch,
France

Special thanks to Jean Luc Vonesch and Didier Hentsch of the Insti-

tut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC),
Illkirch, France for their close cooperation in developing the Leica
MacroFluo

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➌ ➍➋

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When Excellence Meets

Brilliance

Parallax-free documentation

• Precise imaging of macroscopic

specimens from the total picture
down to the smallest detail

• Detail-accurate analysis and 3D view

From macro to micro

• Apochromatically corrected manual

or motorized 6:1 and 16:1 zoom sys­tems for optimum adaptability

• 6:1 zoom with high numerical aper-

ture for optimum light efficiency

• 16:1 zoom enables a wide range of

research tasks to be carried out with
just one microscope

• Optimal imaging of your specimens

with a wide selection of high-quality
objectives

With its consistently modular design, the
Leica MacroFluo
adaptation to the task for precise imaging of
all aspects of macroscopic specimens.

Parallax-free fluorescence macroscopy

While in stereomicroscopy, changing the Z
position of the observer is associated with a
parallax error (an apparent lateral shift of the
specimen when acquiring z-stacks), the Leica
MacroFluo™ combines macroscopic viewing
with a vertical beam path typical of micro­scopes. This enables parallax-free images
with simultaneously large working distances
and object fields with a maximum of precision.
The result is much greater accuracy in digi­tal image processing, analysis and measure­ments.

Leica MacroFluo™ Z6 APO (A) – high macro­resolution and powerful fluorescence

The Leica Z6 APO (A) is available as a fully
apochromatically corrected manual or motor­ized 6.3:1 zoom body, which, depending on the
objective equipment, can reach a macro reso­lution from 351 lp/mm (planapochromat 1×) up
to 1500 lp / mm (planapochromat 5× HR) and
thus can resolve specimens up to a structural
width of 330 nm. The wide range of planapo­chromatically corrected objectives creates
sufficient free room for specimen manipu­lation and, due to the high numerical aper­ture, enables high-performance fluorescence
documentation at low magnification.

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system enables individual

Motorization of the zoom, iris diaphragm and
built-in fine focus and the new motorized fo­cus make it easy to automate experiment pro­cedures. Results can be reproduced at any
time.

Leica MacroFluo™ Z16 APO (A) –
fluorescence analysis at the highest level

The Leica Z16 APO (A) is available as a manu­al and a motorized instrument. With the large
zoom range from 7.1× to 115× (planapochro­mat 1×), fluorescent specimens up to a struc­tural width of 330 nm (planapochromat 5×
HR) can be observed and documented. Thus
the Z16 APO (A) permits the study of clearly
contoured overviews of chicken embryos and
neural systems, but also selected details such
as the expression of proteins at high magnifi­cation and resolution.

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Brilliant fluorescence images,
rich in detail and contrast

• Coupling of illumination and zoom
optics – maximum light efficiency and
uniformly illuminated object fields

• Filters individually matched to your
specimens – maximum flexibility in
selecting fluorescent dyes

• Specimen-optimized transmission
properties and filters with Zero Pixel
Shift – for pin-sharp images

Adjustment-free light sources –
the best light for your specimens

• Leica EL6000: Uniformly illuminated
object fields with software-controlled
shutter control for documenting
image series

• Leica SFL100: Durable, cost-effective
and compact LED illuminator with
adjustable light intensity for optimum
image quality

• Leica SFL4000: 5 LED modules that
can be combined as desired for spe­cific excitation of fluorochromes –
high light efficiency for high-contrast
images with simultaneously low
phototoxicity

Powerful, Even with

Weak Fluorescence

Fluorescence without compromises

The Leica MacroFluo™ is equipped with a high-quality fluorescence axis that ensures an opti­mum light stream and thus the greatest brightness and homogeneity. Up to five filter blocks can
be inserted into the easy-to-rotate turret disk. The illumination is coupled into the zoom optics.
This guarantees precise illumination, maximum light efficiency, and homogenous object fields
at any zoom position.

Filter your specimens for best results

New fluorescent dyes and staining methods are continually opening new possibilities for the
study of development stages, reactions, signal transduction and metabolic pathways. For this
purpose, the wide range of Leica filter blocks can be expanded even further by adding indi­vidualized filter block combinations to ensure optimum transmission properties for almost any
application. With Zero Pixel Shift technology, when the filter blocks are switched, no undesir­able pixel shifts occur between the individual images of the different spectral ranges – an ideal
prerequisite for digital multicolor fluorescence experiments.

Let there be light

With the Leica EL6000, Leica Microsystems offers an external light source equipped with a
long-life metal halide lamp – a cost-effective and time-saving alternative to mercury vapor
lamps. Furthermore, the available Leica SFL100 is a compact, cost-effective LED fluorescence
illuminator that enables optimum excitation at 470 nm with adjustable light intensity. For easily
combining different wavelengths in multi-fluorescence experiments, the Leica SFL4000 offers
five high-performance fluorescence modules, which can be controlled via an intuitive user in­terface. All light sources listed here enable adjustment-free adaptation to the microscope and
thus guarantee uniformly illuminated, high-contrast fluorescence images: the best light for your
research.

➊ – ➌ Chicken with GFP expression in the wing. Institut Albert Bonniot,

Grenoble, France

➍ Transgenic mouse with GFP expression. Dr. Daniel Metzger,

Prof. Pierre Chambon, Imaging Centre IGBMC, Illkirch, France

➎ – ➏ Zebrafish. View of polarization contrast in false colors. Imaging

Centre IGBMC, Illkirch, France

➐ Zebrafish larva. CD41-GFP expression in thrombocytes. Time-lapse

image of thrombocyte circulation to illustrate the vascular system.
Dr. Philippe Herbomel, Institut Pasteur, Paris, France

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➍

➎

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Equipped for Future Tasks

Motorized focus and SmartTouch
focus made 100% reproducible by control using the
Leica LAS software

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: reliable, precise

Solid basis for your research

Advanced documentation, particularly of fine details, requires
a solid basis. The design of the new focusing column and the
carrier of the fluorescence axis reliably absorbs shocks and
vibrations. This prevents impairment of image quality even
when obeserving specimens in liquid medium.

The high-quality mechanical engineering of the coarse/fine
focus adjustment enables convenient, uniform focusing and
enables accurate adjustment of the focus position at all times,
even in the micrometer range. With the new Leica motorized
focus, you can focus on your specimen not only quickly and
precisely, but also reproducibly. The motorized focus of the Z
series makes parallax-free and accurate documentation as
easy as child's play.

Objective slide (here, with 2× and 5× objective): conve­nient extension of the range of applications by simply
toggling between the required magnifications

Supreme performance for your research

With the wide range of planapochromatically corrected objec­tives and the ability to adapt DM objectives, the Leica Macro­Fluo™ provides enormous flexibility with regard to magnifica­tion and working distance. In particular, the high-performance
5× planapochromatic objective enables brilliant, true-to-detail
reproduction of your research results with a numerical aper­ture of 0.5 and a resolution of 1500 lp/mm. The objective slide
for manual zooming allows you to combine the 2× and 5× pla­napochromat objectives to conveniently expand the range of
applications. From low magnification to fine details – the Leica
MacroFluo™ offers high-performance converging lenses for
brilliant fluorescence with sufficient free room for specimen
manipulation.

Binocular photo tube with image correction: easy han­dling and accurate adjustment of the image section for
documentation

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The basis for successful documentation

Leica Microsystems offers you a selection of powerful trans­mitted-light bases that always present your specimens in the
best light, with brightfield illumination with high or low diffu­sion, oblique transmitted-light illumination, and darkfield. The
Rottermann Relief contrast method also ensures excellent dis­play, even of unstained living cells.

The optional gliding stage ensures the necessary sensitivity
when positioning specimens. Furthermore, the integration of
the motorized Leica IsoPro™ stage enables you to carry out
multiposition experiments accurately, all the way to TileScan
for particularly large specimens. The Leica MATS Thermo­control System with a heated specimen stage made of optical
glass provides gentle, absolutely uniform temperature across
the entire stage surface. This temperature stability over long
periods lets you perform time-lapse experiments on tempera­ture-sensitive specimens with precision.

Investment for the future

In multiuser environments, adaptability is an important char­acteristic for satisfying all the requirements posed by various
fields of research. Particularly in this regard, the MacroFluo™
system is an investment that will pay off in the long run. Thanks
to the modular design, a complete range of accessories for
manual and motorized zoom systems is available for you to
choose from.

Leica TL bases: whether simple transmitted light, oblique
illumination or relief contrast – the Leica bases allow you
to get the best out of your specimens.

Automated specimen scans are child's play with the

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IsoPro

motorized cross-stage

Leica SmartTouch™: easy control and overview of the
motorized microscope functions via touch panel and
individually programmable control buttons

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Well-conceived and Customized

System Solutions

Integrated complete solution

In the Leica Application Suite (LAS), automated microscopes,
digital cameras and software are combined to create one
user-friendly, consistent imaging solution. Versatility and a
consistent, modular construction mean that you have enor­mous flexibility in building your microscope system, which in
turn is perfectly adapted to your applications. Thus LAS is an
intuitive solution that makes both routine and research analy­sis easier.

Leica MM AF

Leica MM AF powered by MetaMorph® is available as an
MM AF online version for image processing and analysis and
as a complete software package for image acquisition and
analysis. The latter offers the highest flexibility and versatil­ity in integrating the microscope, peripherals and camera for
multidimensional image acquisition, processing and analysis.

Optional modules such as 3D deconvolution or 3D measure­ments as well as various application modules for automatic
fluorescence analysis provide you with everything you need
to adapt the microscope system to the requirements of your
research.

Screenshots from the Leica LAS and MM AF powered by Metamorph®

(from top to bottom):
LAS Multifocus module: Drosophila melanogaster. Compound eye.
Dr. Tricoire, Institut Jacques Monod, Université de Paris, France
Leica MM AF 3D motion analysis: Trajectories of five identified
particles. Prof. Marianne Quiding-Järbrink, Department of Microbiology and
Immunology, The Sahlgrenska Academy, University of Gothenburg, Sweden
Leica MM AF 3D motion analysis: Graphic display of the analysis

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The expert in fluorescence applications

Leica Microsystems has cooperated with leading scientists
to develop the Leica AF6000, an exceptionally ergonomic fluo­rescence software program. The intuitive operating concept
guides you reliably and easily to brilliant results. In combina­tion with the motorized MacroFluo™ zoom modules, the Leica
AF6000 E is an entry-level software program that enables easy
documentation, image overlay and time-lapse images.

The modular design of the software means that you can add
to your system at any time as your needs grow and change.
Thus the Leica AF6000 fulfills additional requirements of fluo­rescence applications: The software-controlled EL6000 shut­ter control allows manual multichannel fluorescence with time
and Z series. The integration of the motorized Leica IsoPro™
cross-stage enables multi-position experiments and TileScan­ning as well as optional WellPlate acquisition.

A wide variety of functions for image documentation, quantifi­cation, optimization and analysis can be expanded by adding
software options such as 3D reconstruction of image informa­tion or deconvolution. Thus the MacroFluo™ becomes an inte­grated microscope system that is capable of growing along
with your research requirements.

Screenshots from the Leica AF6000 software

(from top to bottom)

AF6000: Image gallery in acquisition mode

AF6000: TileScan in configuration mode

AF6000: WellPlate acquisition module in configuration mode

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“With the user, for the user”

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