Leica LMD6500, LMD7000 User Manual

Leica LMD6500
Leica LMD7000

Laser Microdissection Systems

Ultimate Perfection!

Laser microdissection (LMD) is the ultimate tool for perfection.
LMD makes it possible to obtain homogenous, ultrapure samples
from heterogenous starting material. A researcher can selectively
and routinely analyze regions of interest down to single cells to
obtain results that are reproducible, and specific.

Laser microdissection uses a microscope to visualize individual
cells or cell clusters. Regions of interest are selected by software,

excised from the surrounding tissue by a laser, and collected by
gravity into specialized devices for analysis.

There has been enormous progress in the development of laser
microdissection instrumentation in recent years. The driving force
in this area is the development of laser microdissection systems
by Leica Microsystems.

2

At the forefront of laser microdissection technology,
only the Leica LMD6500 and Leica LMD7000 offer:

t Laser beam movement via optics – fast and precise cuts

t Specimen collection by gravity – contact- and contamination-free

t Dedicated objectives for LMD – highest possible laser power

t Adjustable laser – for thick, thin, soft, and hard tissue

3

Specimen Collection
by Gravity

Benefits:

t Contact- and contamination-free collection

t Fast and reliable specimen collection

t Dissect all specimen shapes and sizes

t Pool unlimited amounts of specimen

t Use standard consumables

The three slide holder allows simultaneous work on up
to three slides. Several holders for different applications
are available.

LMD uses a UV laser to isolate microscopic regions from samples.
Leica Microsystems applies the most gentle technique for speci­men collection – gravity.

Contact- and contamination-free

Gravity is the most sensible method of specimen collection. Once
the region of interest is excised, it gently falls into the collection
vessel. No additional steps or complicated methods are neces­sary. Most importantly, the specimens are not touched. This makes
specimen collection by gravity a contamination-free procedure.

Transfer dissectates of any size or shape

Gravity is the ideal tool to collect dissectates of any size or shape –
round and compact, or long and thin – it simply falls into the col­lection device. Large areas up to several mm2 are excised in one
step. No subdivision into smaller transportable sections is re­quired. This ensures the complete recovery of the sample without
the loss of tissue due to additional cut lines.

Collect directly into reaction buffer

Collection by gravity is the fastest way to progress from tissue
section to reaction buffer. The collection vessel may contain
medium or buffer. Additionally, unlimited amounts of specimen
can be pooled.

Most comprehensive method

Leica Microsystems offers a wide range of specially designed
membrane systems for the best specimen preparations from tis­sue sections, over cell cultures, bacteria suspensions, plant ma­terials, and many more. Samples and membranes are simultane­ously cut by the laser. Specimen preparation has to be adapted to
the experimental needs, and with gravity as the motor for collec­tion, the indispensable flexibility is provided.

Convenient handling of the collectors due to the automat­ically movable tray. The tray takes customized devices as
well.

4

Laser Beam Movement
via Optics

When a high-energy laser pulse hits the sample a fast reaction
limited to the focus of the laser light called “cold ablation” occurs.
Surrounding tissue is not impaired or heated.

Only Leica Microsystems uses high-precision optics to direct the
laser beam along the desired cutting line.

Highest precision

Precision is the prerequisite to obtain homogenous material for
downstream analysis and reliable results. The unique laser beam
movement of the Leica LMD7000 and LMD6500 is controlled by
patented* high precision optics, while the microscope stage and
the sample are stationary. The cutting precision is unmatched and
far superior to using a fixed laser beam and moving the sample.

Maximal speed

Quick and reliable material collection for downstream analysis is
of highest importance. By moving only small optical parts during
the cutting process, Leica Microsystems’ approach allows pre­cise cutting at high magnifications, and fast cutting speeds at low
magnifications. This technique ensures rapid capture, minimizing
degradation of the specimen.

Real-time laser cutting

A smart software tool allows live, on-the-fly dissections. The
unique function is especially beneficial for specimens that are dif­ficult to cut. This tool can also be used for real-time ablation of
tissue. An important effect of laser beam movement is the ability
to conveniently document the dissection with movies. The image
remains fixed and only the laser beam moves – just the way our
eyes are used to.

Benefits:

t Highest possible precision

t Maximal speed

t Real-time laser cutting

t Convenient movie documentation

t Maintenance free

Step 1: Define region of interest

➟

Step 2: Laser beam steered by optics along the cut line

* Patented EP 1276586 B1, US 7035004 B2, JP 3996773 B2, TW 486566 B

➟

Step 3: Specimen collection by gravity

5

➊

➋

6

➌

➍

Leading-edge
Laser Technology

The laser is the core part of a laser microdissection system. The
two key features of a laser are pulse energy and pulse frequency.
The combination of these features within a LMD system was real­ized by Leica Microsystems for the first time.

Adapt the laser to the specimen

The Leica LMD7000 allows the control of laser pulse energy and
frequency. Adjust the laser according to the specimen and make
narrow, powerful, and fast cuts within a single system. Leica
Microsystems’ LMD systems provide the flexibility to align the UV
offset with respect to the focus of the visible light for the most pre­cise cutting performance, for thick or thin samples, and ablation.

Control the thickness of the cutting line

The aperture control enables the adjustment of the width of the
cutting line. Combined with the high repetition rate, the narrow­est cuts are possible. The laser aperture settings can be stored
individually for each objective.

Longlife, solid state laser

Leica Microsystems uses diode-pumped, solid state lasers. These
state-of-the-art lasers are maintenance free and reliable, ensuring
long lifetimes.

Choose the laser according to your needs: the Leica LMD6500 of­fers a pulse energy of 50 μJ and a fixed repetition rate, whereas
the Leica LMD7000 provides a pulse energy of up to 120 μJ and the
unique flexibility to adjust power and frequency.

Benefits:

t Control the pulse energy and frequency

(for Leica LMD7000)

t Adjust the width of the cutting line

t Control the UV offset

t Maintenance free solid state laser

LMD Laser Systems Leica LMD7000 Leica LMD6500

Max. pulse energy 120 μJ 50 μJ

Repetition rate Single pulse to

5000 Hz

Adjustable repetition
rate

Wavelength 349 nm 355 nm

Laser aperture
control

Yes No

Yes Yes

80 Hz

Please visit www.leica-microsystems.com/lmd
for more images and movies, application letters, etc.

Fig. 1: Brain, frozen section of GFAP-immunopositive astrocytes. Courtesy of G.J.
Burbach, MD, and T. Deller, MD, Institute of Clinical Neuroanatomy, J.W. Goethe
University, Frankfurt, Germany.

Fig. 2: Primary cell culture from pancreatic adenocarcinoma (PDAC) stained with
hematoxylin, objective 10x (before cutting, after cutting and inspection). Courtesy
of N. Funel, Department of Oncology, University of Pisa, Italy

Fig. 3: Maize root meristem. Courtesy of L. Feldman, University of California,
Berkeley, USA

Fig. 4: Brain section, stained with thionin. Courtesy of Axaron Bioscience AG,
Heidelberg, Germany

7

System Integration

Benefi ts:

t Fully automated DIC

t Motorized fl uorescence axis with

5- or 8-position fi lter cube turret

t Fluorescence intensity manager (FIM)

t Motorized Excitation Manager and fast

Internal Filter Wheel (IFW)

t Convenient Leica SmartTouch touchscreen

for controlling the automated modules

Leica Microsystems offers a wide range of objectives
(from 5x–150x) dedicated for laser microdissection.

All components of the Leica LMD systems are fully integrated and
act in concert, building a winning team greater than the sum of its
components.

High-end Leica DM6000 B research microscope

The foundation of every Leica LMD system is the high-end Leica
DM6000 B upright research microscope, which is suitable for all
types of life science research. Excellent optical performance, in­telligent automation, and user-friendly operation make this fully
automated microscope the perfect tool for microscopic research.

Specially designed optics for laser microdissection

Leica Microsystems’ dedicated LMD objectives feature the high­est possible UV transmission and outstanding imaging perform­ance – the Leica SmartCut series. With SmartCut objectives,
thicker tissue can be cut faster. The unique high magnifi cation
150x dry objective enables the dissection of even the smallest ar­eas without using oil.

User-friendly software

Leica Microsystems’ LMD software is very easy to use, yet pow­erful without being complicated. A workfl ow guides the user
through the dissection process and intuitively provides all con­trol elements where they are needed. The software contains all
features for dissection, such as serial section cutting, an optional
database, and fully integrated and automated cell recognition.

5x obj. for LMD

5x obj. Standard

The specially designed objectives for laser microdis­section provide a much higher UV transmission than
standard objectives, and therefore, provide more laser
power on the sample.

8

With the Leica SmartTouch all automated functions are easily controlled.

Application specifi c consumables for laser microdissection

Leica Microsystems offers four different types of metal frames,
glass slides and Petri dishes in different sizes. Whether the user
needs slides that show no autofl uorescence, or uses DIC contrast
to reveal the specimen areas – Leica Microsystems has the right
solution for any application.

a)

Benefi ts:

t High-end Leica DM6000 B

t Specially designed optics

t User-friendly software

t Application specifi c consumables

b)

d)

c)

a) 8-well strips building up a 96 well plate
b) Petri dishes
c) Big slides (50 mm x 76 mm)
d) Frame support
e) Membrane slides (PEN)
f) Frame slides (PET, POL, FLUO)
g) Microcentrifuge tubes

b)

e)

e)

f)

e)

g)

9

200 μm

Solutions for
Advanced Applications

Cancer research

Cancer research requires visualization of morphologically altered
cell populations and their subsequent isolation.

t Contamination-free dissection by gravity
t Fast dissection of areas of any size or shape
t Collect directly into buffer
t Fully integrated database option for automated documentation

Single cells

The fastest possible dissection, the highest precision combined
with narrow cutting lines, are the prerequisite for expression
analysis of single cells.

t Laser movement via optics for fast and precise lasing
t Pen-screens for the most accurate drawing of cutting lines
t Leica SmartCut objectives with the highest UV transmission
t The unique 150x dry objective with the highest magnification

available for laser microdissection

200 μm

200 μm

Frozen section (10 μm) of a mouse aorta (whole vessel)
stained with cresyl violet on a POL frame slide. Courtesy
of K. Beuerlein, Rudolf-Buchheim-Institut für Pharmako­logie, Justus-Liebig-Universität Giessen.

Proteomics

For proteomics analysis, either very sensitive analysis methods or
large amounts of protein are necessary for analysis.

t Auto vision control (AVC) for fast and fully automated

cell recognition and dissection

t Semi-automated cell recognition for assisted dissection
t Pool unlimited quantities of dissectates

Live cell cutting

Laser microdissection of living cells enables the separation of
single cells and cell clusters for recultivation or analysis.

t Unique, integrated consumables for sterile dissections
t Dissect non-adherent cells or bacteria
t Optional incubator
t Special sputtered GFP and BGR cubes for lasing in fluorescence
t Fully automated fluorescence axis with 5- or 8-position filter

cube turret

10

Ablation and multi-dimensional imaging

Directly after the targeted ablation in a living structure, the imag­ing of the induced processes is important.

t Time lapse series with different fluorescence channels,

using Leica AF6000 (Advanced Fluorescence) software
and LMD at the same time

t Convenient movie documentation
t Real-time lasing for direct ablation

Specimen

Preparation

➟➟➟➟

Section and prepare
biological specimens

– Histological specimens

(formalin-fixed/
paraffin-embedded or
cryo sections)

– Living cells and

cell cultures
– Chromosome spreads
– Smears
– Cytospins
– Plant material
– Sperm and other

forensic preparations

Staining

Visualize regions of
interest

Brightfield:
– HE (hematoxylin-eosin)
– Cresyl violet
– Toluidin blue
– Thionin
– Immunohistochemistry

Fluorescence:
– Secondary antibodies
– Acridine-orange
– FISH

Microdissection

Selectively dissect
regions of interest

– Contact-free
– Contamination-free
– Any size from cell

compartments of a

2

few μm

to several mm

– Any shape

2

Extraction

Extract and prepare
important molecules

– DNA
– RNA
– Proteins
– Metabolites
– Biomarkers

Analysis

Obtain reproducible
and specific results

– PCR
– Quantitative real-time

PCR
– Microarrays
– Expression profiling
– Genetic fingerprinting
– LOH
– FISH
– LC-MS/MS
– 2-D PAGE
– SELDI
– MALDI

11

4 5

3

11504133
HBO Lamp Adapter

11504117
1” fiber-optics
adapter

11888829
Condenser BF
mot. condenser head

11888830
Condenser DIC (suitable for BF, PH, DF, ICT)
mot. condenser head,
with mot. condenser disk (7 pos.),
with mot. polarizer

11504070
Lamp housing LH 106z
12 V 100 W halogen lamp
4-lens collector

0.55 m connection

11504114
Lamp housing
LH 106z Hg 100 W,
1” 6-lens collector

11504116
Liquid light guide, 2 m

ICT condenser prisms
(K1–K15)

11521505
Light ring set

11500325
Supply unit
Hg 100 W

11504115
External light
source EL6000

11505161
Tube attachment
with 1 camera port

C-Mount
HC 0.70x

11505223
Tube attachment
with 2 camera ports
selectable 100:100, manual

(LMD6500 only)

11505146
BDT 25+ V 100/50/0
Documentation tube
with documentation port
with variable beam splitting

12730203
Leica DFC400 FX

11547000
Leica DFC360 FX

11547002
Leica DFC310 FX

11501450
Hitachi HV-D20P

11505202
Imaging module
beam splitting: 100%:0%
with integrated and centrable
C-mount (0.7x)
(LMD7000 only)

11518146
Objective 5x/0.12
Microdissection

11518145
Objective 6.3x/0.13
Microdissection

11506208
Objective 40x/0.60 XT
Microdissection

11506222
Objective 63x/0.70 XT
Microdissection

11506214
Objective 150x/0.90 XT
Microdissection

Objective series BF

33

11888828
Stand top without fluorescence axis,
with 7-position objective turret M25, mot.

1

1

11600184
Holder for 1 slide
25 x 76 mm

11505214
Holder for big slide
50 x 76 mm

11505257
Holder for Petri dish

11505131
Lever for
4 x 0.2 ml PCR-tube

11505130
Lever for
4 x 0.5 ml PCR-tube

11505258
Lever for
1 x 8-well strips

11888826
Motorized Stage

1

4 4

11501255
Leica STP6000
SmartTouch Panel

11888831
Stand top with fluorescence axis,
with 5-position filter turret, mot.,
with 7-position objective turret M25, mot.

11888827
Scanning Stage

2

11505180
Remote Control
Smart Move

55 5

11888832
Stand top with fluorescence axis,
with 8-position filter turret, mot.,
with 7-position objective turret M25, mot.

2

2

11505226
Holder for 3 slides
25 x 76 mm

11505227
Holder for Petri dish and
big slide 50 x 76 mm

11505255
Holder for 18-well
double stack ibidi slide

11505229
Collector for
4 x 0.2 ml PCR-tube

11505228
Collector for
4 x 0.5 ml PCR-tube

11505230
Collector for
2 x 8-well strips

11888825

Leica LMD6500

max. 50 μJ laser

12

Basic stand without
stage and transmitted light axis

11888834

Leica LMD7000

max. 120 μJ laser
Basic stand without
stage and transmitted light axis

Highlights of the Leica LMD Systems

Specimen collection by gravity

t Contact- and contamination-free specimen collection
t Collect all specimen shapes and sizes directly

into reaction buffer

t Pool unlimited amounts of specimen
t Use standard consumables for collection
t No additional laser pulses are needed for collection

Laser movement via optics

t Highest possible precision and speed
t Real-time laser cutting
t Convenient movie documentation

Leading-edge laser technology

t Control the pulse energy and frequency for thick, thin,

hard and soft tissues

t Control the width of the cutting line using the laser aperture
t Maintenance-free, longlife, solid state laser
t Control the UV offset for thicker samples or ablation

Specially designed optics for laser microdissection

t Wide range of LMD objectives, from 5x to 150x
t Higher UV transmission than standard objectives
t Use the unique 150x dry objective for the most

precise dissection

Lasing within fluorescence

t Special GFP and BGR cubes
t See three fluorescence channels (such as DAPI, FITC, TxRed)

at a glance through the eyepieces

t Use the internal filter wheel to visualize a single fluorochrome
t Non-fluorescent FLUO-frame slides

Intuitive software

t Workflow based and time saving
t Fully integrated database
t Overview images for navigation
t Serial section cutting
t Automated cell recognition

Fully automated high-end Leica DM6000 B
research microscope

t Fully automated DIC
t Unique Fluorescence Intensity Manager (FIM)
t Motorized excitation manager and fast internal filterwheel
t Constant color intensity control

Flexibility to use a variety of dissection devices

t Adapt the collection devices to specific research needs
t Use metal frames or glass slides, with or

without membrane (PEN, PET, POL, FLUO)

t Sterile dissection of living cells out of Petri dishes, stackable

membrane rings or 18-well membrane slides

t Use a sandwich of two metal frames for dissecting

non-adherent cells or bacteria

13

Features and Specifications

Laser Leica LMD7000 Leica LMD6500

Type Diode pumped, solid state Diode pumped, solid state

Wavelength 349 nm 355 nm

Maximum pulse energy 120 μJ 50 μJ

Repetition rate Single pulse to 5000 Hz 80 Hz

Adjustable repetition rate Yes No

Laser aperture control Yes, continuously adjustable Yes, continuously adjustable

Free intensity control 1-100% 1-100%

UV offset freely adjustable and specific objective saved Yes Yes

Laser beam movement Via optics

Microscope

Transmitted light axis Contrast methods BF, optional PH, DF, POL, DIC (fully automated)

Illumination 12 V 100 W halogen lamp

Automation Automated illumination manager

Automated contrast manager
Constant color intensity control (CCIC)

Condenser Condenser head S28, 0.55 NA

Motorized 7x condenser disk
Motorized polarizer

Fluorescence axis Filter cube turret Motorized 5x or 8x

Automation Fluorescence intensity manager (FIM) for brightness adjustment

Circular and rectangular field diaphragms for eyepiece or camera viewing
Internal filter wheel and motorized Excitation Manager

Illumination Leica EL6000 (120 W metal halide) or

100 W HBO

Cubes All cubes size k

Special cubes for simultaneous fluorescence and cutting e.g.
– LMD-BGR
– LMD-GFP

Operation Focus Motorized:

– 5 electronic ratios
– Includes parfocal function
Memory function for two z-positions

Objective turret Motorized 7x M25 thread including dry and immersion modes

Controls 6 programmable function buttons

Leica SmartMove
Controls for z (focus) movement and x, y (stage) movement
4 programmable function buttons

Optional Leica STP6000
Controls for z (coarse and fine focus) and x, y (stage) movement
11 programmable function buttons
Touchscreen with information and control panels

Stand Display With integrated touchscreen Leica SmartTouch

2

Interfaces 2 x USB 2.0, 2 x I

Dimensions With scanning stage:

649.6 mm height, 512.0 mm width, 596.5 mm depth

C

14

Dissection Dissection and Collection Unit Based on Scanning Stage Dissection and Collection Unit Based on Motorized Stage

Specimen Collection Contact- and contamination-free

Dissection modes Draw & Cut

Move & Cut (direct online cutting)
Draw & Scan (dot dissection scan)

Serial section cutting Ye s No

Stage precision ± 2 μm > ± 5 μm

Holding devices 3x standard slides (25 mm x 76 mm)

Optional 1x big slide (50 mm x 76 mm)
Optional Petri dish (50 mm)
Optional 18-well slide stack

Collection devices 4x 0.2 ml standard PCR tubes

4x 0.5 ml standard PCR tubes
Petri dish (50 mm)
Optional 2x 8-well strips building up a 96-well plate

Power supply CTR6500 CTR6000

System software

Package includes Dissection Automated collection devices and positioning of the PCR tubes

Fully automated inspection mode
Multi-cutting over the entire slide
Save and load drawn shapes

User guidance Workflow based graphical user interface

Free scaling drawn shapes
Saving user profiles
Overview images in BF and Fluorescence

Control Full laser control

Control software for the microscope
Laser and illumination settings are linked to objectives

Interfaces Export of shape list data for Microsoft Excel or OpenOffice

Integrated database interface to transfer all relevant data (laser, microscope and camera; database itself as option)

Optional software packages Automated vision control (AVC) for automated cell recognition within field of view (standard version) or

fully automated or semi automated over freely defined area (professional version)
Database Leica IM500 or Leica IM1000

1x standard slide (25 mm x 76 mm)
Optional 1x big slide
Optional Petri dish (50 mm)

4x 0.2 ml standard PCR tubes
4x 0.5 ml standard PCR tubes
Optional 1x 8-well strips building up a 96-well plate

Camera Leica DFC310 FX Leica DFC360 FX Leica DFC400 Hitachi HV-D20P

Type High sensitivity digital color High sensitivity digital monochrome High sensitivity digital color High sensitivity analog 3 CCD color

Cooled Yes, -20°K to ambient Yes, -20°K to ambient No No

Resolution 1392 x 1040 1392 x 1040 1392 x 1040 795 x 596 (x 3)

Pixel size 6.45 μm x 6.45 μm 6.45 μm x 6.45 μm 4.65 μm x 4.65 μm 8.00 μm x 8.00 μm

Speed 20 fps at 1392 x 1040

71 fps at 348 x 260

Electronic interface Single FireWire b cable

(IEEE1394b)

20 fps at 1392 x 1040
39 fps at 696 x 520

Single FireWire b cable
(IEEE1394b)

20 fps at 1392 x 1040
39 fps at 696 x 520

Single FireWire b cable
(IEEE1394b)

25 fps

PCI board

15

“With the user, for the user”
Leica Microsystems

Leica Microsystems operates globally in four divi sions,
where we rank with the market leaders.

Life Science Division

•

The Leica Microsystems Life Science Division supports the
imaging needs of the scientific community with advanced
innovation and technical expertise for the visualization,
measurement, and analysis of microstructures. Our strong
focus on understanding scientific applications puts Leica
Microsystems’ customers at the leading edge of science.

Industry Division

•

The Leica Microsystems Industry Division’s focus is to
support customers’ pursuit of the highest quality end result.
Leica Microsystems provide the best and most innovative
imaging systems to see, measure, and analyze the micro­structures in routine and research industrial applications,
materials science, quality control, forensic science inves­tigation, and educational applications.

Biosystems Division

•

The Leica Microsystems Biosystems Division brings his­topathology labs and researchers the highest-quality,
most comprehensive product range. From patient to pa­thologist, the range includes the ideal product for each
histology step and high-productivity workflow solutions
for the entire lab. With complete histology systems fea­turing innovative automation and Novocastra™ reagents,
Leica Microsystems creates better patient care through
rapid turnaround, diagnostic confidence, and close cus­tomer collaboration.

Surgical Division

•

The Leica Microsystems Surgical Division’s focus is to
partner with and support surgeons and their care of pa­tients with the highest-quality, most innovative surgi cal
microscope technology today and into the future.

The statement by Ernst Leitz in 1907, “with the user, for the user,” describes the fruitful collaboration
with end users and driving force of innovation at Leica Microsystems. We have developed five
brand values to live up to this tradition: Pioneering, High-end Quality, Team Spirit, Dedication to
Science, and Continuous Improvement. For us, living up to these values means: Living up to Life.

Active worldwide

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