Leica LMD6500Leica LMD7000 Laser Microdissection Systems

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SpecimenPreparation

Section and preparebiological specimens

For example: – Histological specimens

(formalin-fi xed/paraffi n-embedded or cryo sections)

– Live cells andcell cultures

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

forensic preparations

Staining

Visualize regions ofinterest

For brightfi eld,with for example: – HE (hematoxylin-eosin) – Cresyl violet – Toluidin blue – Thionin – Immunohistochemistry

For fl uorescence,with for example: – Secondary antibodies – Acridine-orange – FISH

Microdissection

Selectively dissectregions of interest

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

compartments of afew μm2 to 4.5 mm2

– Any shape – Up to a thickness of

300 μm and more

Extraction

Extract and prepare mol-ecules that are important for specifi c research

For example:– DNA – RNA – Proteins – Metabolites – Biomarkers

Analysis

Obtain relevant,reproducible, andspecifi c results

For example: – PCR – Quantitative real-time

PCR – Microarrays – Expression profi ling – Genetic fi ngerprinting – LOH – FISH – LC-MS/MS – 2-D PAGE – SELDI – MALDI

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Laser microdissection (LMD) is the ideal tool to be “specifi c.” LMD makes it possible to distinguish between relevant and non-relevant cells or tissues and enables the researcher to obtain homogeneous, ultrapure samples from heterogeneous start-ing material. A researcher can selectively and routinely analyze regions of interest down to single cells to obtain results that are relevant, reproducible, and specifi c.

When performing laser microdissection, a microscope is used to visualize small structures of interest. The individual cells or cell clusters are subsequently selected and removed from the sur-rounding tissue by a laser and transferred into a collection device for analysis. There has been enormous progress in the develop-ment of laser microdissection instrumentation in recent years. The driving force in this area is the development of laser microdis-section systems by Leica Microsystems.

Be Specific!

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At the leading edge of laser microdissectiontechnology, only the Leica LMD6500 andLeica LMD7000 offer:

• Specimen collection by gravity – contact-freeand contamination-free

• Movement of the laser beam via optics – for the highest possible precision and cutting speed

• Integration of a fl exible, adjustable laser –for the highest feasible power and thinnestcutting lines at the same time

• Dedicated objectives for laser microdissection – for the highest possible UV transmission

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Laser microdissection uses a laser to isolate specifi c microscopic regions from tissue samples. The way in which specimens of in-terest are transferred and collected infl uences the specifi city and quality of the end result. Leica Microsystems uses simple gravity for specimen collection.

Contact-free, contamination-free collection Gravity is the easiest method of specimen collection. Once the laser cuts the specimen of interest, it falls into the collection device for analysis. No additional steps or complicated methods are needed during the process, which makes specimen collection by gravity a fast and robust collection method. Most importantly, the specimen is not contacted or touched during the transport process. Therefore, specimen collection by gravity is also con-tamination-free.

Cut and transfer specimens of any shape or size Gravity does not take into account the shape or size of a specimen. It makes no difference if the specifi ed area is round and compact or long and thin – the specimen simply falls into the collection

Specimen Collection by Gravity

Step 1: Defi ne region of interest

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

Step 3: Specimen collection by gravity

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Benefi ts:

• Contact-free, contamination-freespecimen collection

• Fast and reliable specimen collection

• High recovery rates

• All specimen shapes and sizes are possible

• Collect directly into reaction buffer

• Unlimited amounts of specimencan be pooled

• Standard consumables can be usedfor collection

• No additional laser or laser pulsesare needed for collection

• Maximum fl exibility of dissection devicesfor best specimen preparation

device, which ensures high recovery rates. Also, it is possible to dissect large areas up to 4.5 mm2 in one step as well as cell com-partments of just a few μm2.

Collect directly into reaction buffer Gravity is the fastest way to progress from tissue section to reac-tion buffer. The collection device itself can be fi lled with a medium or the buffer of choice. Additionally, unlimited amounts of speci-men can be collected as needed for the experiment.

Most fl exible method Gravity is the most fl exible method of tissue dissection, and Leica Microsystems offers a range of slides and devices for the best specimen preparation. For example, foil-covered slides, which are widely used for laser microdissection, enable the foil to be cut together with the specimen to provide the best results. Specimen preparation must adapt to experimental needs, and gravity pro-vides this fl exibility – now and in the future.

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When a high-energy laser pulse hits the tissue, a fast reaction limited to the focus of the laser light called “cold ablation” takes place. This process is very fast, and surrounding tissue is not im-paired or heated. Single laser pulses, strung together, form the cutting line of the system. Only Leica Microsystems uses high-precision optics to steer the laser beam along the desired cut line on the tissue.

Highest precision Precision is the prerequisite for selective dissection of a specimen. The Leica LMD6500 and Leica LMD7000 provide the needed pre-cision by moving the laser beam like a pencil tracing on a fi xed piece of paper. The unique, patented* prism control moves the laser over the tissue – just by optics. The stage, and therefore the image, remain fi xed. The precision of this method of cutting, using a 150x objective of ± 0.07 μm, is unmatched and far superior to the precision achieved by using a fi xed laser and motorized stage. Highest speed Specimen preparation is a critical step in every experiment, and it is important to quickly and reliably collect the starting material for downstream analysis. By moving only small optical parts dur-ing the cutting process, the Leica LMD6500 and Leica LMD7000 ensure the highest possible speed for dissection. More speed is equivalent to less degradation of the specimen, which is the basis of the best results for analysis.

Direct, real-time laser cutting Because the specimen is fi xed, the researcher can use a smart software tool to cut directly, in real-time, the precise area where the mouse pointer is located. The user can perform live, on-the-fl y dissection and draw on the specimen like drawing on a piece of paper. This unique function is especially benefi cial for specimens that are thick or diffi cult to cut. This tool can also target real-time ablation of tissue, for example of neurons. A small but important effect of laser movement via optics is the ability to conveniently document the preparation with movies. The image is always fi xed and only the laser moves – just the way the eyes are used to seeing, which makes documentation of laser microdissection results easy.

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

Laser Movementvia Optics

Benefi ts:

• Highest possible precision

• Highest possible speed

• Direct, real-time laser cutting with“Move and Cut”

• The best view for convenient moviedocumentation

Compact design meets elegance: The laser and control elements are perfectly integrated, which ensures high-performance on the footprint of a typical microscope.

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The laser is the core part of a laser microdissection system. The two key features of a laser are the energy per pulse and the rep-etition rate. Up until now, these features have been contradictory: either high power or a high repetition rate within one system. Adapt the laser to the specimen The Leica LMD7000 is the fi rst laser microdissection system that integrates these contradicting approaches within one system. By controlling the repetition rate, the user can now adjust the laser according to the specimen and make narrow, powerful, and fast cuts with a single system. The UV offset corrects the focus of the UV laser with respect to the focus of the visible light, which is needed to adapt to the thickness of the specimen to be cut. Leica Microsystems’ LMD systems provide the fl exibility to adjust the UV offset for the most precise cutting performance, whether the specimens are thick or thin.

Control the thickness of the cutting line With the aperture control, the user can directly adjust the width of the cutting line. Together with the high repetition rate, it is possible to cut with the narrowest cutting lines. Unlike systems with fi xed laser apertures, the user can optimally adjust the laser aperture settings according to the objective in use. In addition, it is possible to integrate new objectives easily.

Longlife, solid state laser Leica Microsystems uses diode-pumped, solid state lasers. This state-of-the-art type of laser is maintenance-free and reliable, ensuring long lifetimes. Choose the laser according to specifi c needs: the Leica LMD6500 offers a pulse energy of 50 μJ and a fi xed repetition rate, whereas the Leica LMD7000 provides a pulse energy of 120 μJ and the unique fl exibility to adjust the repetition rate to the characteristics of a specimen.

Leading-edgeLaser Technology

Benefi ts:

• Fully control the repetition rate toadapt the laser to a specifi c specimen(for Leica LMD7000)

• High energy per pulse for thick orhard specimens

• High repetition rates for narrow cuttingsand high speed (for Leica LMD7000)

• Control the width of the cutting linewith the laser aperture

• Maintenance-free, longlife, solid state laser

• UV offset control for thicker samplesor laser ablation

LMD Laser Systems Leica LMD7000 Leica LMD6500

Max. pulse energy 120 μJ 50 μJ

Repetition rate Single pulse to

5000 Hz

80 Hz

Adjustable repetitionrate

Yes No

Wavelength 349 nm 355 nm

Laser aperturecontrol

Yes Yes

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All components of the Leica Microsystems LMD systems are fully integrated and act in concert, building a winning team that is more than the sum of the components.

High-end Leica DM6000 B Research Microscope The foundation of every Leica Microsystems LMD system is the high-end Leica DM6000 B upright research microscope. Excellent optical performance, intelligent automation, and user-friendly operation make this fully automated microscope the perfect tool for microscopic research. The system can be used in addition to laser microdissection for high-end applications such as FISH or advanced fl uorescence.

Specially designed optics for laser microdissection Leica Microsystems has developed dedicated objectives with the highest possible UV transmission and outstanding imaging performance – the Leica SmartCut series. Higher transmission is synonymous with more laser power on the tissue. With SmartCut objectives, the user can cut thicker tissue faster than with a stan-dard objective. Another example of perfect system integration is the 150x dry objective. This unique high magnifi cation objective enables dissection of even the smallest areas without having to use oil.

User-friendly software As a perfect tool for daily research, Leica Microsystems’ com-pletely new software is very easy to use, yet powerful without being complicated. A workfl ow guides the user through the dis-section process and intuitively provides all control elements where they are needed. A fully integrated two-screen solution provides a large amount of space for live microscope images and an over-view image that can be used for orientation and navigation. All elements are freely scalable and can be customized according to the user’s preferences. The software integrates all important features for dissection, such as autofocus, serial section cutting, an optional database, and fully integrated and automated cell recognition.

Leica Microsystems offers a wide range of objectives, from 5x to 150x, dedicated for laser microdissection.

System Integration

The excellent optics of the Leica DM6000 B form the basis for other high-end applications such as advanced fl uo-rescence or FISH.

The specially designed objectives for laser microdis-section provide a much higher UV transmission than standard objectives, and therefore, provide more laser power for cutting.

5x for LMD

5x Standard

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Application specifi c consumables for laser microdissection Perfect integration of the consumables used in the LMD work-fl ow is critical for obtaining superior, reproducible results. Gravity provides the fl exibility to use the widest range of consumables and to tailor them to a specifi c experimental design. Leica Micro-systems offers four different types of foils, metal frames, glass slides and petri dishes in different sizes. Whether the user needs slides that show no autofl uorescence because the areas of in-terest show only weak signals, or uses DIC contrast because no other method reveals the specimen areas – Leica Microsystems has the right slide for any application.

The full two-screen support of the Leica Microsystems LMD software offers new possibilities, such as a horizontal pen-screen for drawing, and a second vertical screen for viewing with all the necessary controls.

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Cancer research Cancer research requires visualization of morphologically altered cell populations and their subsequent isolation. This application will benefi t from: • Contamination-free, fast, and fl exible dissection by gravity

for reliable results • Fast dissection of areas of any size or shape • Dissection directly into buffer • Choose between at least four different collection devices • Optional holder for lab-on-a-chip devices for

single-cell PCR • Fully integrated database option for easy, automated

documentation of research results

Dissection of single cells The litmus test for every LMD system is its ability to dissect small areas. The fastest possible dissection and the highest precision, in combination with narrow cutting lines, are needed for expression analysis of single cells, for example. This application will benefi t from: • Laser movement via optics for fast and precise

laser cutting • Latest laser technology for the narrowest cutting lines • Pen-screens for the most accurate drawing of cutting lines,

directly on the screen • Leica SmartCut objectives with the highest UV transmission

for faster cutting • The unique 150x dry objective with the highest magnifi cation

available for laser cutting

Proteomics For proteomics there is no method of target molecule amplifi cation, such as the PCR technique for nucleic acids. Therefore, either very sensitive analysis methods or large amounts of protein are needed for analysis. This application will benefi t from: • Fast dissection of large areas of any shape • Auto vision control (AVC) for fast and fully automated

cell recognition and dissection • Semi-automated cell recognition for assisted dissection • Fast autofocus, fully integrated with the overall system • Unlimited pooling of collected tissue

Solutions forAdvanced Applications

Section of muscle (human) following dual histochemistry for cytochrome c oxidase (COX) and succinate dehydro-genase, both mitochondrial enzymes. The blue muscle fi ber is defi cient in COX. Courtesy of Dr. G. Borthwick, In-stitute of Human Genetics, University of Newcastle.

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Live cell cutting Laser microdissection of live cells enables the separation of single cells and cell clusters for recultivation or analysis. Integrated con-sumables and dissection hardware are essential for success. This application will benefi t from: • Unique, integrated consumables for the sterile dissection

of single live cells or cell clusters • Dissection of non-adherent cells or bacteria using

gravity based laser microdissection • Optional incubator (when temperature control is needed) • Sputtered GFP cube for the sensitive detection of fl uorescence

signals while laser cutting

Ablation and multi-dimensional imaging The laser can also be used for selective destruction of areas, struc-tures, etc. without dissecting them. Directly after the destruction of a neuronal structure of an embryo, for example, imaging of the induced processes is needed. This application will benefi t from: • Time lapse series with different fl uorescence channels, using

Leica AF6000 (Advanced Fluorescence) software and laser microdissection simultaneously

• Fine adjustment of laser for targeted destruction • Laser movement via optics for a fi xed image during

movie documentation • “Move and Cut” mode for direct online ablation • Special fl uo-cubes for simultaneous multi-dimensional

imaging and ablation • Fully automated fl uo axis with 8x fl uo-cube changer

for the fl exibility to use different cubes

Frozen section (10 μm) of a mouse aorta (whole vessel) stained with cresyl violet on a POL frame slide.

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, Frank-furt, Germany.

200 μm 200 μm 200 μm

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Specimen collection by gravity • Contact-free, contamination-free specimen collection • Fast, reliable specimen collection • High recovery rates • Dissect all specimen shapes and sizes • Collect directly into reaction buffer • Unlimited amounts of specimen can be pooled • Standard consumables can be used for collection • No additional laser or laser pulses are needed for collection • Flexibility to use a variety of dissection devices

for best specimen preparation

Laser movement via optics • Highest possible precision • Highest possible speed • Direct, real-time laser cutting with “Move and Cut” • The best view for convenient movie documentation

Leading-edge laser technology • Fully control the repetition rate to adapt the laser

to a specifi c specimen • High energy per pulse for thick or hard specimens • High repetition rate for narrow cuttings and high speed • Control the width of the cutting line with the laser aperture • Maintenance-free, longlife, solid state laser • UV offset control for thicker samples or laser ablation

Highlights of theLeica LMD Systems

Dissection of mouse hippocampus, 12 μm thick section, stained with Toluidin blue.

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Fully automated high-end Leica DM6000 B research microscope • Excellent optics for brilliant images • Intelligent automation for fast, reproducible results • Use for other high-end applications like advanced

fl uorescence or FISH • Designed for multi-user environments • Constant color intensity control – no need to adjust

the white balance after changing the light intensity

Optics are specifi cally designed for laser microdissection • Wide range of objectives, from 5x to 150x, dedicated

for laser microdissection • Much higher UV transmission than standard objectives • More laser power on the tissue – cut thicker tissue

and cut faster • Use the unique 150x dry objective for the fi nest dissection

Simultaneous cutting within fl uorescence • See three fl uorescence channels (such as DAPI, FITC, TxRed)

at a glance through the eyepieces • No need to prepare an overlay of the single channels

via software • Use the internal fi lter wheel to visualize a single fl uorochrome

Easy-to-use software • Workfl ow based • Fully integrated database • Create overview images and use them for navigation • Fast built-in autofocus • Optional automated cell recognition

Flexibility to use a variety of dissection devices • Adapt the collection device to specifi c research needs • Use metal frames or glass slides, without membrane or

with membrane (PEN, PET, POL, FLUO) • Dissect sterile live cells out of Petri dishes, stackable

membrane rings or 18-well membrane slides • Use a sandwich of two metal frames for dissecting

non-adherent cells or bacteria, for example • Use fl exible holder for lab-on-a-chip devices

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

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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 specifi c objective saved Yes Yes

Laser beam movement Via optics

Cutting precision From ± 2 μm (5x objective) to 0.07 μm (150x objective)

Features and Specifications

Microscope

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

Illumination 12 V 100 W halogen lamp

Automation Automated illumination managerAutomated contrast managerConstant color intensity control (CCIC)

Condenser Condenser head S28, 0.55 NAMotorized 7x condenser diskMotorized polarizer

Fluorescence axis Filter cube turret Motorized 5x or 8x

Automation Fluorescence intensity manager (FIM) for brightness adjustment Circular and rectangular fi eld diaphragms for eyepiece or camera viewing Internal fi lter wheel and motorized Excitation Manager

Illumination Leica EL6000 (120 W metal halide) or 100 W HBO

Cubes All cubes size kSpecial cubes for simultaneous fl uorescence and cutting e.g.– LMD-BGR– LMD-GFP

Operation Focus Motorized:– 5 electronic ratios– Includes parfocal functionMemory 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 fi ne focus) and x, y (stage) movement 11 programmable function buttons Touchscreen with information and control panels

Stand Display With integrated touchscreen Leica SmartTouch

Interfaces 2x USB 2.0, 2x I2C

Dimensions With high precision and collection unit: 649.6 mm height, 512.0 mm width, 596.5 mm depth

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

Specimen Collection Contact free by gravity

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

Serial section cutting Yes No

Type of stage Scanning stage Motorized stage

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

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

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 Optional lab-on-a-chip devices(25 mm x 76 mm, height-adjustable from 1 mm to 7.6 mm thickness)

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

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 restore of drawn shapes

User guidance Workfl ow based graphical user interface Free scaling of live images and drawn shapes Saving user profi les Overview images in BF and Fluorescence Full two-screen support

Control Full laser control Control software for the microscope Laser and illumination settings are linked to objective Autofocus

Interfaces Export of shape list data for Microsoft Excel or OpenOffi ce 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 fi eld of view (standard version) or fully automated or semi automated over freely defi ned area (professional version) Database Leica IM500 or Leica IM1000

Camera Leica DFC400 Leica DFC310 FX Hitachi HV-D20P

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

Cooled No Yes, -20°K to ambient No

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

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

Speed 20 fps at 1392 x 104039 fps at 696 x 520

20 fps at 1392 x 104071 fps at 348 x 260

25 fps

Electronic interface Single FireWire b cable (IEEE1394b) Single FireWire b cable (IEEE1394b) PCI board

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

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

• Industry DivisionThe 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 micro-structures in routine and research industrial applications, materials science, quality control, forensic science inves-tigation, and educational applications.

• Biosystems DivisionThe Leica Microsystems Biosystems Division brings his-topathology labs and researchers the highest-quality, most comprehensive product range. From patient to pa-thologist, the range includes the ideal product for each histology step and high-productivity workfl ow solutions for the entire lab. With complete histology systems fea-turing innovative automation and Novocastra™ reagents, Leica Microsystems creates better patient care through rapid turnaround, diagnostic confi dence, and close cus-tomer collaboration.

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

“With the user, for the user”Leica Microsystems

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 fi ve 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 Australia: North Ryde Tel. +61 2 8870 3500 Fax +61 2 9878 1055

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www.leica-microsystems.com

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