PALM MicroBeam
®Non
Non- -contact contact Laser
Laser Microdissection Microdissection for pure DNA, RNA, for pure DNA, RNA, Proteins and Living Cells Proteins and Living Cells
Agenda
1. What is LMPC?
2. Character of PALM 3. Publication
4. Pathology
5. Living cell manipulation 6. Optical tweezer
7. Upgrade device
8. Summary
Why Mi
Why Microdissection? crodissection?
Uncovering of cellular mechanisms Uncovering of cellular mechanisms by investigating DNA, RN
by investigating DNA, RNA and Proteins A and Proteins
to resolve the cellular & molecular mechanisms underlying physiological & pathological changes within Tissue or Living Cells
Samples must be … Samples must be …
… Well Defined
… Pure
… Contamination Free
… No damage
Microdissection
Microdissection & Micromanipulation & Micromanipulation
Needle transfer Needle transfer Laser Cutting
Laser Cutting
1 1
ststGeneration Needle transfer Generation Needle transfer
- - Time consuming Time consuming - - Cross contamination Cross contamination
Conclusion
Principle:
Principle: A Near Infra- A Near Infra -Red (NIR) diode Red (NIR) diode laser point
laser point- - heats (at heats (at ≈ ≈ 90° 90 °C) C) t the selected he selected specimen and melts it with an entirely specimen and melts it with an entirely contacting transfer membrane, coated by contacting transfer membrane, coated by an heat
an heat- -activated adhesive film (LCM: activated adhesive film (LCM:
“Laser Capture Melting”)
“Laser Capture Melting”)
90°C 90°C NIRNIR--diode laserdiode laser
5 mm 5 mm
2 2
ndndGeneration LCM Generation LCM
cleaning procedure (!) cleaning procedure (!)
with adhesive tape with adhesive tape
-
-may cause specimen lossmay cause specimen loss --does not clear-does not clear-off all debris!off all debris!
- -Heat damage Heat damage - -Sample lossing Sample lossing
- -Non resued adhesive tape Non resued adhesive tape - -Cross contamination Cross contamination
2 2
ndndGeneration LCM Generation LCM
3 3
rdrdGeneration LMPC Generation LMPC
Laser Microdissection and
Pressure Catapulting (LMPC)
Is really only light- driven and therefore
contact free
Laser
Laser Microdissection Microdissection & Micromanipulation & Micromanipulation
LMPC Laser Microdissection and Pressure Catapulting
Courtesy of A. Vogel, MLL, Lübeck 400 µm Speed
26 m/s
Radius 40 µm Camera delay: 15 ms
Physics of Laser Cutting
UV-A pulsed Laser Focus < 1 Focus < 1 µµm; m;
energy density energy density
> 10
> 10MW/cmMW/cm22
Objective
Photofragmentation within laser focus is due to high photon density
- - Locally restricted effects Locally restricted effects
- - No heat transfer to adjacent material No heat transfer to adjacent material
260 280 300 320 340 240
220
PALMPALM UV UV--A A 335 nm 335 nm DNA
Protein
Absorbance Spectra Absorbance Spectra
nm
Precission
Precission Cutting Cutting
Pulsed UV
Pulsed UV- -A Laser 335nm A Laser 335nm Advantages:
Advantages:
-No harm to DNA, RNA and Proteins-No harm to DNA, RNA and Proteins -
-Long LifetimeLong Lifetime
-High Precission Cutting-High Precission Cutting
Picture taken in 400x magnification 100100xx
10x
20 20xx
40x40x
Methods of LMPC
LMPC from Membrane LMPC from Membrane
Mounted Tissue
Mounted Tissue LMPC from Glass Mounted LMPC from Glass Mounted Tissue
Tissue
Object Slide
Membrane Mounted Tissue
Object Slide
Mounted Tissue
LMPC
High precision cutting
Less than 0.6 um cutting line
No contact.
LPC against gravity when in an inverted microscope system - no danger of contamination with debris.
No heat.
No impact to subsequent DNA, RNA or protein recovery.
No manual steps.
A high degree of automation is possible with the system.
Advantage of PALM
Agenda
1. What is LMPC?
2. Character of PALM
3. Publication 4. Pathology
5. Living cell manipulation 6. Optical tweezer
7. Upgrade device
8. Summary
Zeiss AxioVert
Zeiss AxioVert 200M 200M
Zeiss Objectives Zeiss Objectives
Particularly suitable because of high numerical aperture and best transmission for UV light
Fluorescence:
Fluorescence:
Highgrade fluorescence optics
Contrast Methods:
Contrast Methods:
Phase Contrast PlasDIC
ElementList
Image caperture PALM RoboSoftware
PALM
PALM RoboSoftware RoboSoftware Features Features
Multi collecting device
Fluorescence caperture
PALM Software
Laser Operations Laser Operations Drawing Functions
Drawing Functions Laser
Laser Settings
Settings Microscope Microscope
Management Management with Autofocus with Autofocus Cap Check
Cap Check
Selective
Selective Caperture Caperture
Fluorescence attachment Fluorescence attachment
- consisting of: light source for fluorescence illumination Filter wheel for 6 excitation filters
HEP-G2 cells fixed on supporting LMPC-membrane - F-actin labelled with TexasRed-Phalloidin - interphase nuclei counterstained with DAPI
Fluorescence mode Fluorescence mode
Controller Controller
Single tube 8 caps strip Living cell
CapMover
CapMover
CapMover
Agenda
1. What is LMPC?
2. Character of PALM 3. Publication
4. Pathology
5. Living cell manipulation 6. Optical tweezer
7. Upgrade device 8. Summary
PALM MicroBeam Enabling technology
MicroBeam
Functional Downstream Analysis
Visualization – Imaging
Isolation - LMPC
Specimen Preparation and Selection
Application of PALM
Reference list >300
> 700 publication
Publication of PALM
神經醫學 ：Biol Reprod 63, 643 (2000) 分子醫學 ：Nature Medicine 4, 1329 (1998) 癌症醫學 ：Int. J. Cancer 85, 82 (2000) 細胞生物學：Nature 409, 1, 630 (2001)
生殖醫學 ：Photomed. in Gynecol. and Reprod., 340 (2000) 發育學 ：Nature 376, 6, 57 (1995)
遺傳學 ：PNAS, 102, 25, 8905 (2005)
新生兒診斷：CMLS Cell Mol Life Sci, 57: 96 (2000) 病理學 ：Am J Pathol, 156, 1, 57 (2000)
Publication of PALM
Protocol of PALM
Application note 1. DNA & RNA
2. Tissue section and staining 3. Living cell
4. Chromosome
Agenda
1. What is LMPC?
2. Character of PALM 3. Publication
4. Pathology
5. Living cell manipulation 6. Optical tweezer
7. Upgrade device
8. Summary
Tissue Section and Cell Culture Tissue Section and Cell Culture
Before cut After cut Tube cap
Selective LMPC Selective LMPC
Cryo section, human prostate duct, cresylviolet stain, 40x
• selective elimination of unwanted material
• clear cut gap
between selected
and unwanted
specimen
native tissue cut
motoneuron catapult
motoneuron cut glial cell catapult glial cell
Rat spinal cord - cells grown on LPC membrane
courtesy of B. Meurers, The R.W. Johnson Pharmaceutical Research Inst. San Diego, USA
Benefits:
Selectively prepared single cells, cell clusters or other clustering material without any contamination with unwanted surrounding material.
Application in
Application in NeuroScience NeuroScience
RNA in Toxicology
RNA in Toxicology (cDNA (cDNA- -arrays) arrays)
Identification of region-specific gene expression changes and signalling pathways affected by dibutyl phthalate in foetal rat testes. (GD19)
INT (Leydig cell region) TUB (Sertoli cell region)
60mer oligo array hybridised with RNA from foetal testes (Cy3 or 5)
Microarray analysis Microarray analysis
By courtesy of S. Plummer, CXR Biosciences, UK; Society of Toxicology Annual meeting 2006
RNA/Protein analysis Prostate Cancer
40 different patients:
Each with tumor and non-tumor material
Sample collection for
RNA / Protein analysis with P.A.L.M. MicroBeam
Schlomm T et al. Int J Oncol, 27, 3: 713-720 (2005)
P.A.L.M. MicroBeam Sample collection
Schlomm T et al. Int J Oncol, 27, 3: 713-720 (2005)
RNA analysis Agilent Bioanalyzer results
RNA / Protein analysis Prostate Cancer
40 different patients:
Each with tumor and non-tumor material
Sample collection for
RNA / Protein analysis with P.A.L.M. MicroBeam
Schlomm T et al. Int J Oncol, 27, 3: 713-720 (2005)
cDNA Microarray analysis RT-PCR of PSA
~ 5,000 cells One amplification Chip with ~ 37.000 sequences
RNA analysis Microarray results
Array result of 5 patients (tumor against non
Array result of 5 patients (tumor against non--tumor)tumor)
●
● 200 ng total RNA200 ng total RNAamplified with Ambion MessageAmp Kitamplified with Ambion MessageAmp Kit
●● label: Cy3 and Cy5 with label: Cy3 and Cy5 with „„dyedye--swapswap““
●
● Chip with 37,531 cDNA ClonesChip with 37,531 cDNA Clones Result:
Result: in tumor in tumor 191 191 genes genes upregulatedupregulated, , 25 downregulated25 downregulated among these some
among these some prostate cancer relevant genesprostate cancer relevant genes, like, like AMACR (upreg.), CAV1, CLU, THBS1 (downreg.)
AMACR (upreg.), CAV1, CLU, THBS1 (downreg.) Schlomm T et al. Int J Oncol, 27, 3: 713-720 (2005)
RNA/Protein analysis Prostate Cancer
40 different patients:
Each with tumor and non-tumor material
Sample collection for
RNA / Protein analysis with P.A.L.M. MicroBeam
cDNA Microarray analysis RT-PCR of PSA
~ 5,000 cells One amplification Chip with ~ 37.000 sequences
SELDI Protein analysis
~1,500 cells
Schlomm T et al. Int J Oncol, 27, 3: 713-720 (2005)
11000 11500 12000
11000 11500 12000
45 N 32 N 15 N 12 N 47 N 9 N 6 N 1 N 19 N 86 N 32 T 47 T 9 T 86 T 12 T 45 T 15 T 1 T 6 T 19 T
Protein analysis SELDI results
normal
tumor
Laser
Laser Mi Microdissection for Proteomics crodissection for Proteomics
Histological cancer section (HNC); the white line shows the area to be excised by LMPC
normal pharyngeal epithelium
carcinoma
A
B
Christian M et al. Molecular & Cellular Proteomics 27, 443-452 (2005)
Signal identification: 2
Signal identification: 2- -DE; in DE; in- -gel gel- -digestion digestion
7 20.1 28.5 35.4 51.1 94 Mr (kDa)119
3.0 pI 10.0
tumor normal
7 20.1 28.5 35.4 51.1 94 Mr (kDa)119
3.0 pI 10.0
1 0 0 0 2 0 0 0 3 0 0 0 4 0 0 0 5 0
1 0 0 0 2 0 0 0 3 0 0 0 4 0 0 0 5 0
0 25 50 75
659.0+H 954.4+H
1020.4+H1106.7+H 1234.9+H1274.8+H
1340.9+H
1434.0+H1614.7+H 1705.5+H
1734.7+H1750.6+H 1800.8+H
1851.9+H2097.8+H 2164.2+H
2185.9+H 2274.6+H
2290.9+H
2311.6+H2421.3+H 2437.5+H2552.1+H
2889.7+H 3155.3+H
3213.7+H3229.8+H
3399.6+H3616.0+H 3797.4+H 3814.1+H
4045.1+H 49
Trypsin digestion
Christian M et al. Molecular & Cellular Proteomics 27, 443-452 (2005)
Single Cell Analysis Single Cell Analysis
LightCycler PCR: single cells from glass LightCycler PCR: single cells from glass
DNA isolation with:
(2) (1)
(5)
(10) (neg)
Single Chromosome Preparation
Metaphase spread on LMPC-membrane Laser ablation of unwanted chromatin
Laser-isolated mouse chromosomes;
FISH after MSE-adaptor PCR
Chromosome specific paint probes
1
4 3
2
LMPC of hair follicle
LMPC of hair follicle (CresylViolet stain) (CresylViolet stain)
Arabidopsis stalk, phloem, LMPC
Agenda
1. What is LMPC?
2. Character of PALM 3. Publication
4. Pathology
5. Living cell manipulation
6. Optical tweezer
7. Upgrade device
8. Summary
Working with Living Cells Working with Living Cells
LD Plan
-N
EO FL UAR
40x / 0.6
421361-9970
ZEIS S
Cloning from “picked” cells
Capture of living cells for “ex vivo”
analyses or ongoing cultivation
LMPC
LMPC of Living Cells of Living Cells
PALM
®MicroBeam Genetic Proof
Not catapulted
Not catapulted 5x catapulted5x catapulted
Both have the same karyotype :
45,X,der(10)dup(10)t(10;16),der(16)t(8;16),der(18)t(17,18)
CGHCGH
M M--FISHFISH
Langer S. et al. Cancer Genetics and Cytogenetics 161: 174-177(2005)
Generation of Pure Cell Populations
Cell cultures of
two different murine cell lines, immunostained against CD34
Balb C RM26
Mixed culture grown on DuplexDish
Pure clones after LMPC
Embryonic Stem Cells Embryonic Stem Cells
Do they survive the LMPC - procedure ?
Do they keep their stem cell character ?
i.e. can they maintain themselves indefinitely in a stem cell state by
“self-renewal”, while also producing (through division) more specialized cells
day1 day5 day10 day13
LMPC of
LMPC of murine murine stem cell line RM26 and stem cell line RM26 and subsequent
subsequent recultivationrecultivation
9
MIKROSO
By courtesy of Dr. A. Buchstaller, LMU Munich
The expression of the
The expression of the pluripotencypluripotency--marker marker „„OctOct--44““
9
Oct-4Actin Cytoskeleton
before LMPC after LMPC
After re-cultivation of this single cell on a feeder layer the cell divided in two daughter cells
Murine stem cell (ES) clones with transgenic EGFP (enhanced green fluorescent protein) expression
Courtesy of Level Biotechnology INC.
Research & Development Division Associate Scientist Wen-Jen Yu Ph. D.
mailto: jacksonyu@mail.level.com.tw EGFP mouse chimera production
500 EGFP bp
PCR genotyping of transgenic EGFP
in ES clone
Mouse Embryonic Stem Cell LMPC
LMPC of Whole Living Organisms LMPC of Whole Living Organisms
Caenorhabditis elegans
Courtesy of Ralf Baumeister
And it stays alive !
A. Vogel MLL-Lübeck
Microinjection and cell fusion
Living Cell Manipulation
Microinjection (2 μg/ml EGFP plasmid in medium)
WJ. Yu, Dept.R&D, Level Biotechnology, 2003
Precise Single Cell Transfection !!
Microinjection
Agenda
1. What is LMPC?
2. Character of PALM 3. Publication
4. Pathology
5. Living cell manipulation 6. Optical tweezer
7. Upgrade device
8. Summary
Ashkin, A. et al. PNAS 94: 4853 (1997)
MIE REGIME:
MIE REGIME:
trapping due to refraction of rays a and b;
i.e. radiation pressure force due to change in momentum
Objective Objective
a b
F b F a particle
center
NIR laser NIR laser
(1064nm) (1064nm)
laser focus
Optical tweezer
The Dual
The Dual- -system concept system concept
LD Pl an -N EOFLUA R
40x / 0.6
421361-9 970
ZEIsS
LD Pl an -N EOFLUA R
40x / 0.6
421361-9 970
ZEIS S
Laser Microdissection Laser Tweezers
Optical Tweezer
Sperm Catching
(I)
Optical Tweezers Optical Tweezers
NIR laser NIR laser
Robo
Robo- -MicroTweezers MicroTweezers
Positioning, catching P ositioning, catching - - catch and move - catch and move -
Sperm Catching
(II)
(micro-surgery)
Optical Tweezer
---
Huber R. et al. Nature, 1995, 376: 57-58
P.Beck and R.Huber FEMS Microbiol.Lett. 147:11-14 (1997)
Specimen Segregation Specimen Segregation
syringe needle
bacterial-mixture trap
Laser
erythrocytes
Prefertilization Diagnostics
a. Opening of the zona pellucida with laser zona drilling
b. Polar body trapping and dragging with optical tweezers
c. Isolated polar body d. Empty oocyte
e. Isolated polar body (40x) f. Empty oocyte (40x)
Opening of zona pellucida by LMM
Trapping of sperm using positioning laser
Bovine Oocyte Laser-medited oocyte penetration
Laser-medited fusion=fertilization
In vitro fertilization
Agenda
1. What is LMPC?
2. Character of PALM 3. Publication
4. Pathology
5. Living cell manipulation 6. Optical tweezer
7. Upgrade device 8. Summary
PALM
PALM RoboMover RoboMover
Single Tube
8 Tubes
8-Strip Collector
Excellent for high-quality bright field color images
For brightfield AxioCam MRc
Excellent for high-quality fluorescence imaging with variable exposure time
Obtain low-noise images in extraordinary quality
For fluorescence AxioCam MRm
Highly sensitive 2/3" CCD image sensor with 1.4 megapixels
AxioCam MR family
AxioCam
Apotome
Axons of a dorsal root ganglia
Drosophila melanogaster embryo
Image Scan and Navigator
Set markers at Set markers at regions of interest regions of interest Scan 3 slides
Scan 3 slides to get overview to get overview
ReScan in higher ReScan in higher magnification to get a magnification to get a
closer look closer look
Axiovision
Agenda
1. What is LMPC?
2. Character of PALM 3. Publication
4. Pathology 5. Proteomic
6. Living cell manipulation 7. Optical tweezer
8. Summary
One System
One System – – Many Advantages Many Advantages
PALM MicroBeam
• Flexible applications from archival material to living cells – DNA, RNA, Protein
• Patented LMPC system for non-contact and, thus, contamination-free capture
• Upgradeable technology with additional solutions from Carl Zeiss
• PALM Application Laboratory: years of experience and specialized know-how
Chromosomes Sperms Cytogenetics
Cancer Research
Cells on Forensic Tape Plas DIC Fluorescence Immunohistochemistry
Plant Research Cell Biology
Phase Contrast
Stem Cells
One System
One System – – Many Application Many Application
N.A.
Yes Local R&D support
No Yes
Routine Slide
N.A.
Combisystem Optical Tweezer
High Low
Consumable cost
N.A.
Yes SALDI Compatible
No Yes
Living cells
Automated multi-disk positioning
Quick access, automated multi-group sample collection, automated multi-caps positioning
Automation
Microdissection with 40X objective only
Ranged from 0.6 μm to 1mm in any shape Precision
Low, Direct contact High, LPC
Purity
Inverted microscope, Heat direct contact, restricted application Inverted microscope,
Cold ablation, Broadest application
General features
LCM LMPC
