Leica AS LMD. Laser Microdissection System

Transcription

1 Leica AS LMD Laser Microdissection System

2 Leica AS LMD Laser Microdissection System Molecular techniques are transforming our understanding of cellular function. The sensitivity and specificity of these methodologies, however, is best realized when applied to homogeneous samples. In the case of tissues and other heterogeneous samples this has often been a limitation to their most effective use. Laser microdissection is an innovative technique which provides a simple and rapid method for the contaminationfree selection of single cells or groups of cells from a wide variety of samples for DNA, RNA and protein analysis. Leica Design by Christophe Apothéloz 2

3 Applications The recent sequencing of the human genome and other model organisms has provided researchers with the foundation for the more critical studies of how genes are regulated and expressed. These studies, in conjunction with the direct study of proteins, the molecules which ultimately control cellular processes, are bringing us ever closer to being able to understand the molecular basis of cellular function and disease. Laser microdissection has proved to be an invaluable technique in such studies. Specifically for cancer research it provides the researcher with the unique ability to combine traditional diagnostic methods based on morphology with molecular techniques on the same sample, even the same single cell. Microdissection screenshot PCR product from T cells after microdissection approx. 400 bp long amplification product of a receptor gene Proteomics: MALDI NEPHGE Immunoblots Edman sequencing Isoelectric focusing Two-dimensional gel electrophoresis 3

4 In the present "post-genomics" age, more and more research scientists are concentrating on protein functions, molecular mechanisms and their organized interaction within intracellular transduction paths. Knowing the base sequence of genes and the sequence of the amino acids of gene products also helps to answer questions in neurobiology and immunology, and in developmental and cell biology of animal and vegetable organisms. Illeum biopsy with hyperplastic lymph follicles Leica Microsystems' third generation of laser microdissection systems is now making a significant contribution towards gaining a deeper insight into the function of genes or proteins. The AS LMD is a tool for elegant and most importantly precise and contamination-free isolation and selection of single cells or cell groups. Tissue samples can be embedded, sectioned and stained according to conventional protocols. The sample can be a standard histological section, a cryo section, smear, metaphase spread or cultured cells. The cells of interest are simply circumscribed on the screen image using a mouse and then cut out automatically, upon which they fall directly into a PCR tube provided by the user for further investigation. The dissected tissue can now be examined with nearly any method in molecular biology and biochemistry, for example: DNA analysis: DNA isolation Cloning PCR, real-time PCR (quantitative PCR) Construction of genomic libraries Southern blots DNA sequencing RNA analysis: Total and polya + RNA isolation Reverse transcription PCR cdna libraries Northern blots Protein analysis: 2D SDS PAGE LC MS B-cell monoclonality test: ST = Standard; PC = polyclonal test; MC = monoclonal test From Virchows Archive, Vol. 439 no. 4, courtesy of Springer publishing 4

5 Fluorescence Ready The AS LMD is easily upgraded for fluorescence applications. All standard wavelengths can be used, and a single mouse click switches between brightfield and fluorescence views of the sample. The exposure times of the camera are program-controlled. To prevent unnecessary fading of the sample, the user can define the cutting line in fluorescence mode and then return to brightfield mode to make the cut. The dissected material can then be inspected in fluorescence mode. Chromosome cutting To allow applications that require extremely high precision such as the cutting out of individual cells and the dissection of chromosomes, the laser focus is linked to the objective magnification. A special high-magnification objective with high UV transmission provides the precision for dissecting single metaphase chromosomes, which can then be subjected to further analysis. Photos: I. M. Janssen, University Medical Centre Nijmegen, the Netherlands, Department of Human Genetics and Pathology Specimen with metaphase chromosomes before microdissection Specimen with metaphase chromosomes after microdissection. Isolation of human chromosome Metaphase chromosome specimen with fluorescence-labelled chromosomes. A chromosome fragment was selected for laser microdissection and subjected to PCR (see right-hand illustration). DOP-PCR products 1 Microdissection product after 2 rounds of PCR 2 Negative control 3 100bp ladder 5

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8 The Principle The cutting technology, known as laser ablation, is based on a pulsed UV laser which melts or "cuts" the plastic film. The cut is made by tracing round the area of interest, which may even be a single cell. This technique avoids direct UV irradiation or mechanical contact which could result in contamination. Mounting the histological section on a thin plastic film improves the cutting results and facilitates transfer to the PCR tube. Ergonomic controls for stage movement (xy), focus (z), objective change and illumination intensity. Leica specials The AS LMD is based on an automated laboratory microscope integrated with a UV laser. The material cut out by the laser simply falls by the force of gravity (i.e. without any mechanical contact and without the application of additional physical forces) into a capture PCR tube which is located directly underneath the slide. There is an inspection mode to visually confirm that the dissected cells have been deposited in the tube. A longworking distance objective is used so that the specimen slide need not be removed from the stage. The process is automated by motorized focus and objective change functions. Multi-well trays and holders for various types of PCR tube can be positioned by motor control. The laser beam moves over the specimen to make the cut. The specimen remains stationary so that it can be clearly observed during the cutting process. Cutting precision is optically coupled to the chosen magnification if a higher viewing magnification is selected, the cutting movement automatically becomes finer, as the laser beam and its movement are reduced on the same scale. Specimen stage with removable slide holder Lower part of stage (cover removed); motor-driven positioning of collection vessels (here PCR tubes) 8

9 Easy Operation All components of the AS LMD such as laser activation, laser motion, stage movement, focus, changing objectives and illumination aperture are motorized and under direct computer control. The device collecting the dissected material is also positioned by computer control for an efficient workflow and clear assignment of the specimen and dissected cells in the PCR tube. The system operates under a very intuitive MS Windows environment. Viewing and Image Management The microscope image is monitored with a high performance 3 chip color camera and displayed live on the computer monitor. The images can be easily stored in digital format with all key data such as specimen name, slide position (X,Y coordinates), PCR tube, etc. Inserting the PCR tube into the holder Laser Safety The laser has been carefully integrated into the microscope to prevent any exposure to UV light. The eyepieces and camera ports are equipped with UV barrier filters and a UV blocking shield surrounds the sample area. The laser can only be operated after releasing various safety devices. Removable slide holder for preventing contamination Screenshot of user software easy-to-follow operation of main functions 9

10 Specimen before microdissection The Inspection mode Besides effective and user-friendly menu guidance during selection of the cells of interest, the inspection mode offers another important advantage for laser dissection. Dissected cells can be inspected in the collection tube itself simply by switching from cutting to inspection mode. By pressing a single button, the slide is moved aside, the objective is changed and automatically refocused into the collection tube. When the button is pressed again, the slide is returned to its original, in focus position. This procedure permits 100% checking of the quality of the experiment and the result of the dissection. Archiving The IM1000 (Image Management Software 1000) implemented in the AS LMD offers professional level image archiving and image processing. For the user this means convenient archiving and handling of complex routines. Images are readily edited, labelled, analyzed and evaluated, image series are easily managed and linked. Specimen after microdissection Inspection mode Image of the dissected material in the lid of the PCR tube IM1000 screenshot 10

11 Technical Data Microscope Leica DM LA Motorized objective nosepiece Motorized specimen movement (xy) Motorized focusing (z) Laser Maintenance-free nitrogen laser Wavelength nm Pulse repeat rate up to 30 Hz (up to 60 Hz in burst mode) Pulse duration < 4 ns Peak performance 75 kw Beam divergence 0.3 mrad Laser Microdissection Module Motorized optical beam scanning Motorized setting of laser illumination aperture, focus offset and laser attenuation Collection tray for dissected material, with quick-change mount, Motorized positioning of collection tray Software Control of all motorized microscope functions and laser movements Display of microscope image on the PC monitor using 3CCD camera Incision path drawing with correction facility; Starting and execution of the cutting process, Storing of images for documentation of different processing steps of the experiment Simple calibration of the whole system. 11

12 Leica Microsystems the brand for outstanding products Leica Microsystems mission is to be the world s first-choice provider of innovative solutions to our customers needs for vision, measurement, lithography and analysis of microstructures. Leica, the leading brand for microscopes and scientific instruments, developed from five brand names, all with a long tradition: Wild, Leitz, Reichert, Jung and Cambridge Instruments. Yet Leica symbolizes innovation as well as tradition. Leica Microsystems an international company with a strong network of customer services Australia: Gladesville Tel Fax Austria: Vienna Tel Fax Canada: Richmond Hill/Ontario Tel Fax Denmark: Herlev Tel Fax France: Rueil-Malmaison Tel Fax Cedex Germany: Bensheim Tel Fax Italy: Milan Tel Fax Japan: Tokyo Tel Fax Korea: Seoul Tel Fax Netherlands: Rijswijk Tel Fax Portugal: Lisbon Tel Fax Republic of China: Hong Kong Tel Fax Singapore: Tel Fax Spain: Barcelona Tel Fax Sweden: Sollentuna Tel Fax Switzerland: Glattbrugg Tel Fax United Kingdom: Milton Keynes Tel Fax USA: Bannockburn/llinois Tel Fax and representatives of Leica Microsystems in more than 100 countries. The companies of the Leica Microsystems Group operate internationally in five business segments, where we rank with the market leaders. Microscopy Our expertise in microscopy is the basis for all our solutions for visualization, measurement and analysis of microstructures in life sciences and industry. Specimen Preparation We specialize in supplying complete solutions for histology and cytopathology. Imaging Systems With confocal laser technology and image analysis systems, we provide three-dimensional viewing facilities and offer new solutions for cytogenetics, pathology and materials sciences. Medical Equipment Innovative technologies in our surgical microscopes offer new therapeutic approaches in microsurgery. With automated instruments for ophthalmology, we enable new diagnostic methods to be applied. Semiconductor Equipment Our automated, leading-edge measurement and inspection systems and our E-beam lithography systems make us the first choice supplier for semiconductor manufacturers all over the world. Copyright Leica Microsystems Wetzlar GmbH Ernst-Leitz-Straße Wetzlar Germany 2000 Tel. ( ) 29-0 Fax ( ) LEICA and the Leica Logo are registered trademarks of Leica IR GmbH. Order nos. of the editions in: English German Part-No Printed on chlorine-free bleached paper. X/01/AX/K.H. Leica Microsystems Wetzlar GmbH Ernst-Leitz-Straße D Wetzlar (Germany) Tel. +49 (0) Fax +49 (0)