Confocal Fixed Stage System: Leica DM6000 CFS


Confocal Fixed Stage Microscope System

Excellent Results under All Conditions

The Confocal Fixed Stage System Leica DM6000 CFS sets a new standard in physiology and electrophysiology.

The system integrates the Leica DM6000 CFS fixed stage microscope into the Leica TCS SP5 confocal platform.

A whole set of new, innovative features helps to realize the highly sophistcated experimental setups in physiology and electrophysiology from single cells to whole organisms.

Features at a glance:


Leica objective HCX APO L 20x1.0

Variable Field at High Resolution

The new and highest aperture objective HCX APO L20x1.0 NA offers both a large field and high resolution and makes the change of objectives for overview and detail imaging unnecessary. It is corrosion-free, chemically neutral and avoids diffusion of metal ions. The thermal conductivity is minmal and magnetic fields at the front of the objective have been eliminated. A crucial advantage is the access angle of the objective. Widened to 39 degrees it has almost reached the limit of what is theoretically possible, In combintation with a free working distance of 2 mm, the objective is ideal for many applications.

Features at a glance:


Leica DFC350 FX Camera with parfocal magnification changer

Free Choice of Magnification

To cover the demand for large scale sample screening and fine needle approaches for electrophysiology, the Leica DM6000 CFS has a special observation tube with 3 magnification positions (0.35x, 1x and 4x). This parfocal magnification changer allows a total change in magnification up to a factor 12 without changing the objective


exchangeable nosepiece

Maximum Flexibility for Multi-user Environments

Besides the new Leica HCX APO L20x1.0 objective for electrophysiology the system has a high precision adapter, allowing to repace the single objective with an electronic 6x objective nosepiece. With this additional nosepiece the Confocal Fixed Stage System covers also the requirements in developmental biology which recently tends towards the imaging of larger organs and organisms. A high precision adapter is available, allowing to replace the single objective with an electronic 6x objective nosepiece. This change of objectives works vibration-free, with automatic power switch-off to avoid disturbing measurements. For each objective, the focus position can be programmed. Thus, by simply pushing the button, a quick change between the magnification is possible even in the near infrared without losing the area of interest, implementing automatically parfocality.

Features at a glance:


Dodt gradiant contrast tube

Orientation and Contrast

A specially designed infrared illumination filter in combination with an IR polarizer and the infrared differential interference contrast (DIC) prisms give extremely good resolution even in the thickest specimen.

Used in combination or separately, fluorescence and DIC are great techniques for patch clamping. However, to avoid having any optical components in the fluorescent light path and to ensure the highest photon collection efficiency for two-photon excitation fluorescence microscopy, the Dodt gradient contrast technique is of advantage. It converts the phase information into an amplitude contrast. Images of neurons look similar to images obtained with DIC.

To study the fundamental properties of basal dentrites via patch clamp recordings, it is now possible to combine two-photon excitation fluorescence microscopy with a scanning version of this technique, called infrared-scanning gradient contrast (IR-SCG). The infrared excitation laser light and the fluorescent light are seaparated by a dichroic mirror, underneath a high NA condensor. The fluorescent light is detected by Non-Descanned Detectores (NDD) and the IR-scanning gradient contrast images are detected by spatially filtering the forward scatterd infrared laser light with a Dodt tube and subsequent detection by a photomultiplier. This allows the online-overlay of a highly contrasted IR image of a brain slice with the fluorescence image of the neuron system. This detection method is patented by Leica Microsytems)

Features at a glance:


reflected ligth detector and transmitted light detector

Detection Efficiency

As with multi-photon excitation the fluorescence is only generated in the diffraction limited focal volume, the detectors can be placed directly behind the objective (reflected light detectors, RLD) as well as directly behind the condenser (transmitted light detectors, TLD) without losing spatial resolution. This close-coupling detection method results in the highest photon collection efficiency, as scattered fluorescent photons can also be collected over a large detection angle due to the high numerical aperture of the objective and the condenser. Two-channel detection on both sides add a maximum of detection flexibility.

Apart from its high NA, the new Leica DM6000 CFS pateted turret condenser for brightfield and interference contrast provides a number of other advantages. The system allows the exchange between dry and oil condensers. the condenser base with condenser head 1.4 NA oil S1 stands for hightst collection efficiency, while the patented condenser base provides a watertight seal with an outlet pipe for liquid leaking fom the sample.

Features at a glance:


Double labeled neurons of brain slices. Courtesy of Thomas Nevian, Institute of Physiology, Bern, Switzerland

New XYTZ-Scan Mode for 3D Visualization of Calcium Tranisients

The dentritic tree has a complex three dimensional structure. Repeatedly acquiring complete 3D stacks gives a temporal resolution much too low for imaging the fast calcium transients in dentrites. To circumvent this problem, optical sectioning, the fast scanning of the resonant system and the triggering capability are combined for the new XYTZ-Scan Mode.

individual time series are taken at different focal depths and combined into a 4D image stack. A stimulus is always delivered efore the same of each time series, synchronized by a trigger out event. After a complete focal series, the image data isproected into a 3D data stack over time. For each structure in the sampled 3D volume the fluorescence transients can be analyzed. The time course of fluorescence in all parts of the dentritic tree is clearly visible.


Confocal interface

Scanner Patching and Optimized Workflow

In combination with the IR-SGC, which makes electrophysiology needles visible in the scanning mode, the extreme speed of the resonant scanner allows electrode patching while imaging at video rates, without ever switching to the camera mode. Together with the one-for-all electrophysiology 20x.1.0 objective, scanner patching minimizes the number of the steps between experiment setup and data collection, optimizing your workflow.

so even though you might never need it, the workflow oriented user interface of the Leica TCS SP5 with Leica DM6000 CFS includes the camera as well. Wether your are doing CCD-camera of confocal / multiphoton imaging, the other option is always just one fast click away. Integrating both operation modes in the same software allows you to concentrate on what's in your images, not on where they came from.


Data correlation

Data Correlation

Recording of electrical data is typically related to stimulation. Current or voltage is recorded briefly before, during and after stimulation. The time frame for recording data after stimulation depends on effect-relaxation.

To synchronize the image acquisition and the electrophysiological recording, precise triggers are necessary. The Leica TCS SP5 hardware provides different types or outbound triggers: frame, line, pixel. They can be used to synchronize the application of stimuli or external recording devices. Input triggers can also be used to start or continue image scanning in response to arbitrary external events, thus increasing the flexibility in data acquisition could be performed by a special input trigger to minimize the influence of heart activity on the image data.

With the Leica software LAS AF, data evaluation of the electrophysiological signals can be performed online providing precise correlation with imaging data. For the analysis of electrical and optical data, the relevant basic functions are implemented. Online data evaluation enables the validation of the recorded data and is important in helping the researcher quickly decide how the external parameters should be modified for the best results.

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