1. Concerted Project at CRAW For Microdissection & Micromanipulation applications Project developed by following partners : D1 : Dr Geerts Pascal, Ing Terzi-Jean Michel D7 : Dr Veys Pascal, Dr Olivier Fumière Supported and interested persons : D1 : Mauro M; D7 : Berben G., Baeten V., P. Dardenne; D3 : Chandelier A. Objective : sensibilisation and call for interest to all scientific staff of CRAW Note from Geerts P. : simplified PPT presentation developed in agreement with Zeiss company Contact persons : p.geerts@cra.wallonie.be or p.veys@cra.wallonie.be p.geerts@cra.wallonie.bep.veys@cra.wallonie.be

2. DNARNAProteins Main question in modern biomedical research Understanding the cellular and molecular mechanisms underlying function or dysfunction (pathological changes) on a molecular, cellular or tissue level Challenge in microscopy (limited molecular biology tools) Identify structures or molecules and study their function by labeling with marker molecules e.g. GFP (living cells), immuno-histochemical stain (histological samples) Challenges downstream to microscopy Apply the full toolbox of molecular biology for DNA, RNA and protein analysis to defined cellular structures or defined tissue areas seen in the microscope to investigate molecular function (e.g. PCR, RT-PCR, DNA-Fingerprinting, MALDI) I. Motivation Why Microdissection & Micromanipulation?

3. Prerequisits and instrumental challenges … Separate the areas or interest from any unwanted surrounding material (i.e. contamination by surrounding tissue) with a high spatial resolution on a microscopic level … Transport the material into reaction tubes for further downstream analysis (DNA, RNA, Protein) and avoid any potential contamination in this process Decrease the background noise level in the downstream analysis II. Prerequisites & Challenges What is Microdissection & Micromanipulation? Visualization Isolation

4. Laser Microdissection and Pressure Catapulting (LMPC)  Well defined  Pure  Contamination free The New Generation: PALM MicroBeam III. An existing tool : The PALM Microbeam from Zeiss The Laser Microdissection & Pressure Catapulting combined with The LMPC Method

5. Laser Microdissection and Pressure Catapulting (LMPC)  Contamination Free  Non-contact (laser pulse)  Against gravity LD Plan-NEOFLUAR 40x / 0.6 421361-9970 ZEISS Slide and PCR tube arrangement 1 2 3 IV. The basic function of the microdissector

6. … a complete workflow PALM MicroBeam Enabling technology MicroBeam Specimen Preparation and Selection Functional Downstream Analysis  Visualization – Imaging  Isolation - LMPC

7. Solid State Laser 355nm (FTSS) Advantages:  No harm to DNA, RNA and Proteins  Long Lifetime  High Precission Cutting 100x 10x 20x 40x 0.6  m Cut precision: AxioObserver microscopy platform  Developed for observation, manipulation and analysis of biological material  Ideal microscope arrangement for live cell work 10x 0.6  m 20x 40x 100x V. Observations through Axio technology “See more. Discover more. Know more”

8.  Multichannel Fluorescence Image acquisition in several individual fluorescence channels DAPI Overlay FITC VI. Fuorescence possibilities - Highend Imaging Platform Overlay  Extended Focus Acquisition of image series from different focus positions TexasRed

9. Microdissection at your finger tips: New user interface Simple Convenient Well structured Laser management on the left Microscope management on the right Drawing tools at the bottom Navigator, Information Center and Element List included Optional Modules: Extended Focus (EF) Multichannelfluorescence TimeLapse Database AxioVision Commander (Scripting) VII. Adapted and simplified Software PALM RoboSoftware 4.0

10. Laser Microdissection from Glass Mounted Tissue Object Slide Mounted Tissue Glas Mounted Tissue Can be used on archival material Homogenizes the tissue Unique PALM feature Mounted Tissue PALM MembraneSlide Laser Microdissection from Membrane Mounted Tissue Object Slide Membrane Preserves tissue morphology Enables dissection of any shape & size Facilitates ablation & tissue separation Allows fixation & staining VIII. Applications 8.1. Tissue section Unique flexibility for tissue section - AutoLPC

11. ChromosomesSperms Cytogenetics Cancer Research Cells on Forensic Tape Plas DICFluorescence Immunohistochemistry Plant ResearchCell Biology Phase Contrast Stem Cells 8.2. Various type of source material & applications

12.  DNA extraction  Detection of loss of heterozygosity in different cell types  Translocation analysis of chromosomes  Single cell analysis of tumor cells  DNA fingerprinting even using forensic tapes DNA RNA  RNA extraction from different tissues (e.g. human, animal, plant) for expression analysis  RNA integrity analysis (Agilent Bioanalyzer)  RT-PCR from immunostained tissue  Microarray analysis Proteins  HPLC from laser microdissected samples  „High resolution protein analysis“ ( nano-LC/MS/MS ) of human brain tissue  Mass spectrometry of protein composition ( MALDI-TOF, SELDI- TOF )  2D SDS-PAGE analysis of kidney cancer tissue 8.3. DNA, RNA, Protein Applications

13. 8.3.1. DNA applications DNA Application and Laser Microdissection Gene-specific analysis of a low amount of cells Specific analysis of even single chromosomes DNA „Finger-Print“ analysis Frozen or paraffin embedded tissue Chromosomes Sperms and Epithelial Cells from forensic tapes upstream source downstream PALM MicroBeam (PALM Appilcation Note Forensics) (e.g. Thalhammer04, Langer05) (e.g. Cardoso04, Gallardo06)

14. 8.3.2. RNA applications RT-PCR even from single cells Roche LightCycler specific melting curves for murine PBGD Membrane (neg. control) 50 cells 10 cells One single cell -Extraction of RNA done with QuickPick mRNA Kit from BioNobile (utilzing magnetic particles) -Analysis with gene-specific RT-PCR with murine PBGD (Porphobilinogen deaminase, 154 bp) -Negative control: Membrane without cell 1 2 3 Test with: -frozen tissue (liver, kidney) -plant tissue -live cells negative controls control water control RNA number of cells 50 10 5 1

15. 8.3.2. RNA applications Toxic effects on specific cell types in foetal rat testes -Amplification of RNA and Microarray Technology by Agilent 1 2 Courtesy of S. Plummer, CXR Biosciences, UK Oligo MicroArray hybridised with RNA from foetal testes (Type1 RNA: Cy3) (Type2 RNA: Cy5) Identification of region-specific gene expression changes in foetal rat testes. 1 2 Type 1 (Sertoli cell region) Type 2 (Leydig cell region) Scatter Plot interpretation of microarray result Reliable results for cDNA Microarrays guaranteed. Sertoli cell regions Leyding cell regions

16. 8.3.3. Protein applications Protein Application and Laser Microdissection Pooling: With the PALM MicroBeam you can collect big amounts of cells from different slides into the same cap SELDI: Test on influence of different stains 2D SDS-PAGE: Comparison of protein expression in tumor versus non-tumor 2D SDS-PAGE: Comparison of protein expression in tumor versus non-tumor Frozen tissue e.g., mouse liver Renal Cell Carcinoma: tumor – non-tumor Pharyngeal Epithelium: tumor – non-tumor downstream upstream source PALM MicroBeam (Pub.: von Eggeling06) (Pub.: Ernst06) (Pub.: Poznanovic05)

17. 8.4. Live Cell applications Cultivation of laser microdissected live cells Isolation of specific cell types from primary cultures and subsequent cultivation (clonal picking) Positive and negative selection of cells Live cells

18. The MicroBeam offers a convenient solution in live cell work. Recultivation: After laser microdissection Gene expression of functional cardiac markers Pure Clones: Recultivated after selection with laser microdissection Adherent live cells: Picking live cells for recultivation ES-derived cardiomyocytes isolated with LMPC Mixed cell culture (Fluorescences) 8.4.1. Laser Microdissection of cultured cells downstream upstream source PALM MicroBeam (Pub.: Chaudhary 06) (Pub.: Burgemeister06, Langer05 )

19. LMPC has no effect on phenotype After LMPC and clonal expansion the cells keep their stell cell character (expression of pluripotency markers like Oct-4) day 1day 5day 10day 13 Clonal expansion LMPC of mouse stem cell line RM26 and subsequent recultivation (clonal expansion out of one single cell) Courtesy of Dr. A. Buchstaller, LMU Munich 8.4.2. Working with embryonic stem cells Oct-4Actin before LMPC after LMPC Oct-4Actin

20. Sterile Work / CapMover SingleCap, EightCap Collector and Microtiter Plate Formate Single experiment Automated sample collection and object recognition Degree of Automation  Cutting: Manual, interactive and automated image object recognition (AxioVision, Definiens)  Collection: Sterile for life cell, single, 8 up to 96 capture plate 8.4. Extended possibilities… Single experiment to full automation

21. Genetic Analysis of laser microdissected live cells: Langer S et al. Cancer Genetics and Cytogenetics 161:174-177(2005) 8.5. Important advantage No harm to DNA, RNA, Proteins and Live Cells Setup: Laser microdissection of living cells (Pool 0) Reculture of dissected cells (PoolA) Again laser microdissection out of Pool A DNA extraction (PoolA) Reculture of second time dissected cells (PoolB) … finished after 5 times of microdissection and recultivation DNA extraction (PoolB) Idea: Comparison of genetic pattern : native against laser microdissected cells Genetic analysis done with CGH (Comparitive Genomic Hybridization) ->allows you to easily detect genetical differences Used cell line: HCT116 (Colon Cancer Cell line) -> is described in different publications as absolutely genetically stable with the same genetic pattern -> genetic changes in chromosome 8, 10, 17 & Y Expected result: After several laser microdissection steps and followed genetic analysis, no change in the genetic pattern should occur

22. IX. Consumables A full spectrum for any application Consumables for Live Cell Work: look like regular cell culture dishes immunostaining using fluorescence marker For isolation and recultivation of adherent cells with no need for a TRYPSINIZATION step Consumables for Histology: Look like regular consumables Easy handling Not impairing standard protocols (1-2-3 step protocol)

23. Expert consultation service RentalLab Application training Validation service R&D Protocol development palm-labs@zeiss.de X. PALM ServiceLab and facilities in Munich “You don’t have to reinvent the wheel again …”

24. CONCLUSION ON PALM Beam Technology by Zeiss “ Building Bridges between Microscopy and Molecular Analysis ” The New Generation PALM MicroBeam Very flexible in applications from archival material to living cells – DNA, RNA, Protein LMPC method as a non-contact against gravity and, thus, contamination-free collection method Workflow extendibility: from individual experiments to automation digital cameras for brightfield and fluorescence with advanced imaging capabilities automated image analysis Standardized and tested consumables from P.A.L.M. and accessories Strong workflow competency and specialized know-how at PALM Application Laboratory to support you

25. CALL for interest All scientific staff of CRAW is invited to send by email their potentiel interest within a short one page document description to the following person : Fumiere@cra.wallonie.be