Leica DM6000 CFS User Manual

Leica DM6000 CFS

Confocal Fixed Stage System

The solution for applications in live cell research, from physiology
to electrophysiology – from single cells to whole organisms

Leica TCS SP5 with integrated fixed stage micro­scope Leica DM6000 CFS

Excellent Results
Under All Conditions

Leica Microsystems sets a new standard with the integration of the
Leica DM6000 CFS fixed stage microscope into the Leica TCS SP5
confocal platform.

Neurobiological research has a long history not only in measure­ments of single cells, but also of thick brain slices. Here more
realistic results can be achieved due to the intact network. Ulti­mately, accessing the brain directly in a whole animal provides
the most intact environment for conducting measurements on
individual cells. So for optimal results, space for whole animal
samples is an absolute must.

Electrophysiological research employs micropipette systems for
recording electrical signals (patch clamp, whole cell recordings),
electrical stimulation (intracellular stimulation, synaptic stimulation,
soma-stimulation), dye injection and intracellular perfusion. To make
such measurements effective, the microscope needs to have as
much space as possible for micropipettes and other manipulators.

Features and Benefits

Maximum workspace for mani -

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pulators and attached pipettes

High collection efficiency due to

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short collection path before

detectors

Integrated change of magnification

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(0.35x, 1x, 4x) of camera port

Remote control of all microscope

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functions via touch panel

Special booster optic to fill the

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entrance pupil of the 20x1.0

objective

“Dip-in” feature for immersing the

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objective

Patented condenser drainage

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system

High microscope and sample

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stability

Searching for the perfect spot in the sample requires a large field
of view, whilst the precise positioning of the micropipettes requires
high magnification. Furthermore, high quality confocal image scan­ning requires a large numerical aperture for best performance. To
fulfill these experimental prerequisites, a camera with an
adjustable magnification and specially adapted objectives are
needed. Any direct manipulation of the system potentially disturbs
the delicate positioning of the micropipettes within the sample.
This emphasizes the importance of a remote control for the imag­ing setup, providing convenient access to all relevant functions.

Finally, recording electrophysiological and imaging data with per­fect synchrony is paramount for correct interpretation. Triggering
image recording by external events and synchronizing the appli­cation of stimuli with image scanning down to the single line lev­el helps realize sophisticated experimental setups. Online analy­sis of the resulting data allows on-the-fly adjustment of external
parameters, helping the researcher to get everything just right.

The Leica TCS SP5 with the integrated fixed stage microscope
Leica DM6000 CFS gives excellent experimental results under all
conditions.

2

Variable Field
at High Resolution

With the highest aperture objective 20x 1.0 NA, with the innova­tive, ergonomic and stable microscope stand Leica DM6000 CFS,
with short coupled non-descanned detectors and with the fastest,
most sensitive scanning system SP5, we enter a new dimension
in imaging, from single cells to whole organisms.

The objective

The new Leica HCX APO L 20x 1.0 water immersion objective
offers both a large field and a high resolution with one single
objective lens, making it unnecessary to change objectives
between overview imaging for sample preparation and detail
imaging for data recording.

In addition to optimizing the optical parameters, a number of
physical, chemical and mechanical improvements have been
integrated into the new HCX APO L 20x1.0 application objective: It
is corrosion-free, chemically neutral and avoids diffusion of met­al ions. Wettability is outstanding, thermal conductivity is minimal
and magnetic fields at the front of the objective have been elimi­nated.

Leica objective HCX APO L 20x1.0

Transmission HCX Apo L 20x/1.00 W

These new objective characteristics are possible by employing a
special, extremely hard ceramic material for the whole front lens
area. This material is resistant to mechanical damage and much
better suited to the requirements of electrophysiology than typical
objective metal sleeves.

A further advantage is the access angle of the objective, which is
a measure of how easily manipulators can be fitted. This has been
widened to 39 degrees, which is almost the limit of what is theo­retically possible. In combination with a free working distance of
2 mm, the 20x objective is ideal for many applications.

Features and Benefits

20x water immersion objective

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High numerical aperture (NA) of 1.0

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Free working distance (FWD) of

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2 mm

Optimized access angle of 39°

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Large objective focusing range of

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13 mm

Insulated ceramic tip

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Minimum conductivity

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High transmission in VIS and IR

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(see graph)

Best DIC and Dodt contrast

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3

Leica DFC350 FX Camera with parfocal magnifi­cation changer

The microscope stand

But an objective alone doesn’t make an electrophysiology setup.
The Leica DM6000 CFS combines the specific needs of electro­physiological experiments with the optimized imaging perfor­mance of the DM series. With its focusing objective or nosepiece,
it offers a highly stable and convenient optical platform for all
fixed stage applications.

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

The specimen is completely decoupled from the microscope, both
mechanically and electrically. The microscope provides a high
degree of free space for application-specific sample holders,
bath chambers and several manipulators at a time. In this way,
the sample and the patch electrodes can be moved below the
fixed optical axis of the microscope. This allows the researcher to
scan the entire dendritic tree and follow the axons of fluores­cently labeled cells. The DM6000 CFS is also designed for use
with a separate third-party fixed platform for holding micromanip­ulators and other devices, as well as an x,y-translation stage.

Features and Benefits

of the Leica STP6000

Intuitive user interface

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Control of all microscope functions

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Z-focusing wheel for coarse and

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fine z-movements

Freely programmable function keys

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Helps to avoid vibrations caused by

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manual operation

To avoid the need for direct manipulation of the microscope stand
which could cause accidental vibrations, the external control panel
Leica STP6000 (Smart Touch Panel) controls all the microscope
functions. A touch screen and freely programmable function keys
allow quick and easy operation without adversely affecting the
measurements.

4

Large range of applications

Even though the new 20x 1.0 objective makes electrophysiology
with one single objective possible, sometimes this isn’t enough. In
developmental biology, there is a clear trend towards imaging
even larger organs and organisms, calling for different special
application objectives. The Leica DM6000 CFS offers maximum
flexibility in a multi-user environment to cater to everyone’s
needs. A high precision adapter is available, allowing to replace
the single objective with an electronic 6x objective nosepiece.
The patented 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 a button, a quick change between the magnifi­cations can be achieved even in the near infrared without losing
the area of interest, implementing automatic parfocality.

Exchangeable nosepiece

Top: Zebrafish eye (courtesy of: Carl Neumann, EMBL).
Right (from top to bottom):
Raju Tomer, EMBL, Heidelberg, Germany). Neurons in
brain slice (courtesy of: Thomas Nevian, Institute of
Physiology, Bern, Switzerland). Mouse embryo, detail of
the heart (courtesy of: Dr. Elisabeth Ehler, King’s College,
London, UK).

Platynereis larva

(courtesy of:

Features and Benefits

High precision adapter for switching

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between single objective and

objective revolver

6 objective nosepiece positions

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Patented electronic nosepiece

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turret

Automatic power switch-off after

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objective change

Automatic electronic parfocality

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5

Features and Benefits

of Gradient Contrast

Usable both with camera (IR-video

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microscopy) and scanner (IR-

scanning gradient contrast, IR-SGC)

Optical elements are outside the flu-

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orescent light path, allowing highest

possible photon collection efficiency

of two-photon microscopy

Alignment-free overlay of IR-SGC

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and fluorescence images

Scanner patching with IR-SGC –

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no need to patch in camera mode

Orientation and Contrast

Tissue or brain slices up to a thickness of several hundreds of
microns can be optimally imaged with infrared illumination. A spe­cially designed infrared illumination filter in combination with an
IR polarizer, IR analyzer and the infrared differential interference
contrast (DIC) prisms give extremely good resolution even in the
thickest specimens.

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 ensure
the highest photon collection efficiency for two-photon excitation
fluorescence microscopy, the Dodt gradient contrast technique
can also be used. This gradient contrast 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 dendrites via patch­clamp 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-SGC).
The infrared excitation laser light and the fluorescent light are
separated by a dichroic mirror, underneath a high NA condenser.
The fluorescence light is detected by Non-Descanned Detectors
(NDD) and the IR-scanning gradient contrast images are detected
by spatially filtering the forward scattered infrared laser light with
a Dodt tube and subsequent detection by a photomultiplier tube.
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 a Leica patent: US 6,831,780 B2.

Optical light path of Dodt gradiant contrast

6

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 scheme results in the highest possible 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 detec­tors on both sides add a maximum of detection flexibility.

Apart from its high NA, the new DM6000 CFS patented 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 highest collection efficiency,
while the patented condenser base provides a watertight seal
with an outlet pipe for liquid leaking from the sample.

BA

Features and Benefits

Close-coupled two-channel NDDs

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both in reflected and transmitted

C

Gradient contrast with camera,
magnification 0.35x (A), 1x (B), 4x (C).
Online overlay of IR-SGC and
fluorescence image (D).

D

light paths

Electrophysiology condenser system

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with outlet pipe for draining

Condenser base with condenser

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head 0.9 NA S1

Condenser base with condenser

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head 1.4 NA oil S1 for highest

collection efficiency

7

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

Two worlds in one

Two completely different experimental requirements can be satis­fied with one single system. The Leica TCS SP5 with the Tandem
Scanner provides classical morphology on large samples, where
high spatial resolution is required, e.g. research on structures of
cytoskeleton, organelles or tissues, as well as physiology and bio­physics imaging, where temporal resolution becomes very impor­tant.

Calcium is an important second messenger to trigger many sig­nalling cascades in neurons. Functional imaging of calcium influx
is possible with calcium ion sensitive indicator dyes, but calcium
transients in neurons are very fast. Therefore, they are typically
recorded in line scan mode, also known as xt-scan mode. The
laser beam is continuously scanned back and forth along the
same line and fluorescence over time is recorded. The resulting
image consists of one spatial and one time axis. The resonant
scanning system of the Leica TCS SP5 oscillates at 8000 Hz,
enabling a line rate of 16,000 lines per second. Fast dynamics of
initial calcium inflow can now be investigated.

Even in the xy-scan mode, the scan rate can be as high as 180
frames per second in the frame size 512 x 64. This frame rate
enables imaging of extended regions of the dendritic tree and
multiple spines at the same time.

Confocal interface

8

New XYTZ-Scan Mode for 3D visualization of calcium transients

The dendritic 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 den­drites. 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 before
the same frame of each time series, synchronized by a trigger out
event. After a complete focal series, the image data is projected
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 dendritic tree can be
seen clearly.

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 steps between experiment
setup and data collection, optimizing your workflow.

XYTZ-Scan Module:Time series at different focal
positions, stimulus (t0) is always delivered before
the same frame of each time series

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. Whether you are doing CCD-camera
or 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.

Camera interface

Features and Benefits

Software-integration of CCD-

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camera and confocal scanner

Single click switching between

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camera and confocal

Workflow oriented user interface

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9

Data Correlation ( /F)

Recording of electrical (e.g. patch-clamp) data is typically related
to stimulation (electrical or chemical sensory stimulation of the
animal). 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
recordings, precise triggers are necessary. The Leica TCS SP5
hardware provides different types of outbound triggers, such as
frame, line and pixel triggers. They can be used to synchronize the
application of stimuli or external recording devices. On the other
hand, input triggers can be used to start or continue image scan­ning in response to arbitrary external events, thus increasing the
flexibility in data acquisition. For example, the synchronization of
heart beat and image acquisition could be performed by a special
input trigger to minimize the influence of heart activity on the
image data.

Options for data correlation

Correlation of optical and electrical

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data

Input triggering to start image

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acquisition

Output triggering to control stimuli

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Synchronization of scanning with

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external devices

Control of environmental conditions

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by online data analysis

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.

10

System Components

Leica Confocal Fixed Stage System DM60000 CFS

11

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