1 Agenda1.What is LMPC?2.Character of PALM 3.Publication4.Pathology 5.Living cell manipulation6.Optical tweezer7.Upgrade device 8.Summary PALM MicroBeam

(1)

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

(2)

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

(3)

Needle transfer Needle transfer Laser Cutting

Laser Cutting

1 1

^st^st

Generation 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”)

90°C 90°C NIRNIR--diode laserdiode laser

5 mm 5 mm

2 2

^nd^nd

Generation LCM Generation LCM

(4)

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

^nd^nd

Generation LCM Generation LCM

3 3

^rd^rd

Generation LMPC Generation LMPC

Laser Microdissection and

Pressure Catapulting (LMPC)

Is really only light- driven and therefore

contact free

(5)

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

(6)

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

(7)

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

(8)

Zeiss AxioVert

Zeiss AxioVert 200M 200M

Zeiss Objectives Zeiss Objectives

Particularly suitable because of high numerical aperture and best transmission for UV light

Fluorescence:

Highgrade fluorescence optics

Contrast Methods:

Phase Contrast PlasDIC

ElementList

Image caperture PALM RoboSoftware

PALM

PALM RoboSoftware RoboSoftware Features Features

Multi collecting device

Fluorescence caperture

(9)

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

(10)

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

(11)

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

(12)

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

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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

(14)

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

(15)

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)

(16)

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)

(17)

P.A.L.M. MicroBeam Sample collection

RNA analysis Agilent Bioanalyzer results

(18)

RNA / Protein analysis Prostate Cancer

40 different patients:

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

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)

(19)

RNA/Protein analysis Prostate Cancer

40 different patients:

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

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

(20)

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

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)

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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)

(22)

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)

(23)

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

(24)

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

(25)

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

(26)

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-4

Actin 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

(27)

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

(28)

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

(29)

Ashkin, A. et al. PNAS 94: 4853 (1997)

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

(30)

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

(31)

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

(32)

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

(33)

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

(34)

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

(35)

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

(36)

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

Comparison of LCM

(37)