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Leica DMI6000 B with
Adaptive Focus Control
Explore Life in All Dimensions
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Adaptive Focus Control
Imaging dynamic processes in living cells is challenging, even for
the most precise and reliable microscopy systems. The goal is
clear – to record, analyze, and publish changes observed over
time, as accurately as possible, while keeping the specimen free
of side effects arising from the observation itself.
Improving the best:
The Leica DMI6000 B inverted microscope is the core component
of Leica Microsystems’ widefi eld and confocal systems for live cell
imaging, offering unsurpassed stability inherent to its design. It is
capable of effortlessly maintaining the focus under live cell imaging conditions over extended time periods. However, even the most
robust microscope is susceptible to sudden temperature fl uctuations, for instance if a climate chamber needs to be opened to add
a solution to the sample during the course of an experiment. This
can result in unsharp images in the time series, or even losing the
sample out of focus completely.
Explore Life
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Leica Microsystems has developed the DMI6000 B with Adaptive Focus Control (AFC) for researchers who demand consistent multidimensional imaging without loss of focus. Available for both widefi eld and confocal applications, the AFC dynamically regulates
the focus position, whenever or wherever the experiment requires it. Tested and approved in collaboration with scientifi c partners,
Leica’s Adaptive Focus Control ensures that the specimen remains in focus throughout the experiment.
Culture medium
Focus position
Δ
Detection sensor
with light spot
Δ
Objective
LED
850 nm
Cover glass
Immersion
medium
Leica Adaptive Focus Control is based on the refl ection of a
LED light beam at an appropriate surface such as the bottom
of a Petri dish or multi well plate. The light is projected through
the objective to the surface and refl ected onto a light-sensitive
sensor. The position of the light spot on the sensor is defi ned
by the position of the sample in relation to the objective.
Due to temperature drift, this distance changes and the light
spot on the sensor is shifted in relation to the original position.
A feedback mechanism records this shift in the light spot position and corrects the z-position accordingly.
0min
in All Dimensions
AFC switched on
0 s 30 s 60 s 90 s 120 s 150 s
COS cells were transfected with a Golgi specifi c GFP variant. The microscope system was placed in a climate chamber at 37°C.
Shortly after starting the acquisition, 1 ml of an ice-cold salt solution (PBS) was added (60 s). Throughout the entire acquisition sequence, the sample remains
in the desired focal plane. Courtesy of Prof. Dr. Ralf Jacob and Dr. Alexandra Elli, Institute of Cytobiology and Cytopathology, University of Marburg, Germany.
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When experience counts
Leica Microsystems’ experts designed the DMI series with great
emphasis on reliability. Now, with the addition of the AFC, the
microscope becomes the ultimate tool for live cell applications
that demand fast reactions to changing conditions. Based on pioneering technology, the AFC principle involves the refl ection of a
light beam at a suitable surface to keep the distance between the
objective and specimen constant. The process is established, robust, and remarkably fast.
Smoother integration
To be truly effective for a wide range of experiments, Adaptive
Focus Control is integrated into Leica Microsystems’ intuitive
software workfl ow. For full fl exibility, AFC can also be operated in
stand-alone mode without PC connection using the microscope’s
function keys. Remote control is possible via the Leica SmartMove
or Leica STP6000 control panel. No separate control devices are
needed. The underlying technique works for all selected contrast
methods and does not require taking additional images of the
specimen. This ensures that cells remain viable for longer, and
deliver reliable results over long periods of time.
The Intelligent Automation of the Leica DMI6000 B offers ultimate
ease of use. A single push of the ‘hold focus’ button is all it takes –
140min
TIRF imaging with simultaneous AFC
MDCK cells expressing GFP-p75
Courtesy of Prof. Dr. Ralf Jacob and Dr. Alexandra
Elli, Institute of Cytobiology and Cytopathology,
University of Marburg, Germany.
the system works automatically in the background, optimizing the
results for each objective. The system focuses on the specimen,
while you focus on getting results.
AFC switched off
0 s 30 s 60 s 90 s 120 s 150 s
COS cells were transfected with a Golgi specifi c GFP variant. The microscope system was placed in a climate chamber at 37°C.
Shortly after starting the acquisition, 1 ml of an ice-cold salt solution (PBS) was added. Focus drift can be clearly observed when the cold solution is added (60 s).
Courtesy of Prof. Dr. Ralf Jacob and Dr. Alexandra Elli, Institute of Cytobiology and Cytopathology, University of Marburg, Germany.
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The need for speed
When moving from position to position within a specimen, small differences in focus
are often observed as you look down the eyepieces. The impressive speed of the
AFC is clearly demonstrated here, as it automatically corrects for these small focus
differences in real time. This is of great benefi t when imaging multi well plates.
Naturally, AFC can be used for fast process monitoring. Imagine you want to monitor
vesicle movement in 3D over time. The challenge here is to keep the cells in focus
while acquiring high-speed z-stacks. This is achieved with the Leica Super Z Galvo
stage. You set up the time lapse experiment in combination with z-stacks, defi ning
when and where AFC will be activated. The experiment benefi ts from faster, reliable
results – no over-sampling, less stress to the specimen due to less light exposure,
and smaller data sets.
“AFC is such a powerful tool
for time lapse experiments.
I am impressed how fast and
Get to grips with multidimensional space
Multidimensional experiments are the backbone of live cell imaging – the ability to
visualize multiple locations in 3D over time is of paramount importance.
Focusing on change
Cells are dynamic structures – they change shape constantly and even round up
before entering mitosis. What is of interest and sharply in focus right now may be at
a completely different z position a couple of hours later. Here you need the fl exibility
to dynamically adapt to changing cell positions or morphology. The solution is simple: the combined action of AFC with the Leica digital autofocus for extra focusing
versatility. The possibility to defi ne exactly when and at which positions this focusing combination is activated ensures every event in the time lapse is captured.
Leica DMI6000 B with Adaptive Focus Control – your reliable system solution for live
cell imaging. Time saving and improving results.
reliable it is.”
Prof. Dr. R. Jacob
University Marburg,
Department of Clinical Cytobiology and
Cytopathology, Marburg, Germany
For best publishable results:
Building on the proven stability of the Leica
DMI6000 B, Adaptive Focus Control keeps
the specimen actively in focus – whenever
and wherever the experiment requires it.
■
Uncompromising speed
■
Utmost compatibility
■
Unrivaled ease of use
in All Dimensions
0 h 2 h 4 h 6 h 8 h 10 h
Time series with DIC contrast method
MDCK cells (Madin-Darby Canin Kidney) were imaged over a time period of 10 hours. AFC was switched on.
Courtesy of Prof. Dr. Ralf Jacob and Dr. Alexandra Elli, Institute of Cytobiology and Cytopathology, University of Marburg, Germany.
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Technical Specifications
Systems with Adaptive Focus Control Leica DMI6000 B with AFC, AM TIRF MC, TCS SP5 II with AFC, TCS SP5 II DS with AFC;
TCS SP5 MP with AFC
Suitable specimens Living cells in cell culture (embedded specimen only with dry objectives)
Suitable dishes, coverslips Glass bottom dishes and multi well plates, N 1.5 (0.15- 0.18 mm thick),
specifi ed plastic dishes*
Detection light 850 nm LED
Sensor CMOS sensor
Control via Function keys on the microscope; SmartMove, STP6000 and application software
Operation modes Stand alone (without software) and integrated in software workfl ows
Supported software Leica LAS AF as well as 3rd party software*
Supported objectives Please contact your local sales representative for the updated objective list
Memory Via software: unlimited AFC hold positions (in combination with LAS AF Mark & Find)
Focusing Via motorized z drive with travel range of 8 mm (2 mm below, 6 mm above the stage)
smallest increment: 15 nm/step; with parfocality manager,
Operating environment 15 – 42 °C
Relative humidity: 60%
Operating voltage 90 – 250 V, frequency 50/60 Hz
*Please contact your local sales representative for detailed information
by Leica Microsystems GmbH, Wetzlar, Germany, 2010
©
Order no.: English 914 724 • IX/10/???/???? • Copyright
LEICA and the Leica Logo are registered trademarks of Leica Microsystems IR GmbH.
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