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 : [email protected]or [email protected]
Transcript
Concerted Project at CRAWFor 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
Main question in modern biomedical researchUnderstanding 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?
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
Laser Microdissection and Pressure Catapulting (LMPC)
Well defined Pure Contamination free
The New Generation:
PALM MicroBeam
III. An existing tool : The PALM Microbeam from ZeissThe Laser Microdissection & Pressure Catapultingcombined with The LMPC Method
Laser Microdissection and Pressure Catapulting (LMPC)
Contamination Free Non-contact (laser pulse) Against gravity
LD Plan-NEOFLUAR40x / 0.6
421361-9970
ZEISS
Slide and PCR tube arrangement
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IV. The basic function of the microdissector
… a complete workflow
PALM MicroBeamEnabling technology
MicroBeam
Specimen Preparation and Selection
Functional DownstreamAnalysis
Visualization – Imaging Isolation - LMPC
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: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”
Multichannel FluorescenceImage acquisition in several individual fluorescence channels
DAPI
OverlayFITC
VI. Fuorescence possibilities - Highend Imaging Platform
Overlay Extended FocusAcquisition of image series from different focus positions
TexasRed
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
VII. Adapted and simplified Software PALM RoboSoftware 4.0
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. Applications8.1. Tissue section
Unique flexibility for tissue section - AutoLPC
Chromosomes Sperms
CytogeneticsCancer Research
Cells on Forensic Tape
Plas DICFluorescence Immunohistochemistry
Plant Research Cell Biology
Phase Contrast
Stem Cells
8.2. Various type of source material & applications
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
8.3.1. DNA applications DNA Application and Laser Microdissection
Gene-specific analysis of a low amountof cells
Specific analysisof even single chromosomes
DNA „Finger-Print“ analysis
Frozen or paraffin embedded tissue
Chromosomes
Spermsand Epithelial Cellsfrom forensic tapes
upstream source downstream
PALM MicroBeam
PALM MicroBeam
PALM MicroBeam
(PALM Appilcation Note Forensics)
(e.g. Thalhammer04, Langer05)
(e.g. Cardoso04, Gallardo06)
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
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Test with:- frozen tissue (liver, kidney)- plant tissue- live cells
negative controls
control water
control RNA
number of cells501051
8.3.2. RNA applications Toxic effects on specific cell types in foetal rat testes
- Amplification of RNA and Microarray Technology by Agilent
Identification of region-specific gene expression changes in foetal rat testes.
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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
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 ofprotein expressionin tumor versus non-tumor
2D SDS-PAGE:Comparison ofprotein expressionin tumor versus non-tumor
Frozen tissuee.g., mouse liver
Renal Cell Carcinoma:tumor – non-tumor
PharyngealEpithelium:tumor – non-tumor
downstreamupstream source
PALM MicroBeam
PALM MicroBeam
PALM MicroBeam
(Pub.: von Eggeling06)
(Pub.: Ernst06)
(Pub.: Poznanovic05)
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
The MicroBeam offers a convenient solution in live cell work.
Recultivation:After laser microdissection
Gene expressionof functional cardiac markers
Pure Clones: Recultivated after selection with laser microdissection
Adherent live cells:Picking live cellsfor recultivation
ES-derived cardiomyocytesisolated with LMPC
Mixed cell culture(Fluorescences)
8.4.1. Laser Microdissection of cultured cells
downstreamupstream source
PALM MicroBeam
PALM MicroBeam
PALM MicroBeam
(Pub.: Chaudhary 06)
(Pub.: Burgemeister06, Langer05 )
LMPC has no effect on phenotypeAfter LMPC and clonal expansion the cells keep their stell cell character (expression of pluripotency markers like Oct-4)
day 1 day 5 day 10 day 13Clonal expansionLMPC 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-4 Actin
before LMPC
after LMPC
Oct-4 Actin
Sterile Work / CapMover SingleCap, EightCap Collector and Microtiter Plate Formate
Single experimentAutomated sample collection and object recognitionDegree 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
Genetic Analysis of laser microdissected live cells: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, After several laser microdissection steps and followed genetic analysis, no change in the genetic pattern should occurno change in the genetic pattern should occur
IX. ConsumablesA 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)
• Expert consultation
service• RentalLab• Application training• Validation service• R&D• Protocol development
“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-contactagainst 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
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 :
