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雷射顯微擷取儀雷射顯微擷取儀(Laser Capture Microdissection )原理與應用

劉恬均

產品技術專員

鎂陞科技股份有限公司

E-mail: Ivy.liu@mass-solutions.com.tw

OutlineOutline

Laser Capture Microdissection Technology

Arcturus LCM Systems

Applications of LCM

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Sample preparation

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Right Samples, Valued ResultsRight Samples, Valued Results

Sequence Microarray

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PCR, QPCR

Mass Spec

2D Gel

The LCM ProcessThe LCM Process

2. Identify Target Cells 3. Isolate Target Cells1. Visualize Specimen

ADH

Normal

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LCM of premalignant breast cancer progressionMa, Xiao-Jun et al. (2003) Proc. Natl. Acad. Sci. USA 100, 5974-5979

DCIS

IDC

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See What You've Been Missing

Why Microdissect?Why Microdissect?

Gene 1Proteoglycan 1

Gene 2CD53 antigen

Gene 3Proteosome subunit

Cell-specificMolecular Analysis

Meaningful expression data

Mixed Pure

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Meaningful expression data based on specific cell types.

Manual MicrodissectionManual Microdissection

Time-consuming process

In 1970’sIn 1970’s

Time-consuming process.

Requires patience and dexterity.

Difficult to microscopically verify the purity of the cells and the accuracy of the dissection.

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Back to 1976…..Back to 1976…..

UV Laser Cutting Technology

Histochem J., Vol 8, Issue 4, 387 - 401, 1976

The laser in the Lowry techniquefor microdissection of freeze-dried tissue slides.

UV Laser Cutting Technologyfor Microdissection

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Near IR laserNear IR laser

Lowest Absorption by biomoleculesLowest Absorption by biomolecules

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Since 1996Since 1996

Science, Vol 274, Issue 5289, 998-1001, 1996Science, Vol 278, Issue 5342, 1481-1483, 1997

National Institutes of Health & National Cancer Institute

LCM Caps

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Near IR Laser

Single Cell Capture via LCMSingle Cell Capture via LCM

Before LCM

After LCM

CapSure

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Frozen Mouse Brain – HistoGene Staining

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Over 1,700 systems installed world-wide

Leading the Laser Microdissection RevolutionLeading the Laser Microdissection Revolution

PixCell® AutoPix™

Veritas™ ArcturusXT™

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Veritas Arcturus

LCM Publication ListLCM Publication List

1. Ccr2 deficiency impairs microglial accumulation and accelerates progression of Alzheimer-like diseaseNature Medicine, 13(4):432 –438, 2007

2. Gene expression profiling of tumor microenvironment during breast cancer progressionBreast Cancer Res. 2009 Feb 2;11(1):R7

3. TelomereMaintenance in Laser Capture Microdissection PurifiedBarrett’s Adenocarcinoma Cells and Effect of TelomeraseInhibition In vivoClin Cancer Res 2008;4971 14(15) August 1, 2008)

4. DNA fingerprinting tags novel altered chromosomal regions and identifies the involvement of SOX5 in the progression of prostate cancer

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identifies the involvement of SOX5 in the progression of prostate cancer.Int J Cancer. 2008 Dec 23.

5. HOXB13-to-IL17BR Expression Ratio Is Related With Tumor Aggressiveness and Response to Tamoxifen of Recurrent Breast Cancer: A Retrospective StudyJ Clin Oncol 25:662-668, 2007

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1. Immunohistochemical and Biogenetic Features of Diffuse-TypeTenosynovial Giant CellTumors:The Potential Roles of Cyclin A,P53,a nd Deletion of 15q in SarcomatousTransformation

Taiwan LCM Publication ListTaiwan LCM Publication List

Clin Cancer Res 2008;6023 14(19) October 1,2008

2. Heat Shock Protein 90 Overexpression Independently PredictsInferior Disease-Free Survivalwith Differential Expression ofthe A and B Isoforms in Gastrointestinal StromalTumorsClin Cancer Res 2008;14(23) December 1, 2008

3. Increased expression of Dyrk1a in HPV16 immortalized keratinocytes enable evasion of apoptosis

Int. J. Cancer: 120, 2377–2385 (2007)

4 Increase of disintergin metalloprotease 10 (ADAM10) expression

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4. Increase of disintergin metalloprotease 10 (ADAM10) expressionin oral squamous cell carcinomaCancer Letters 245 (2007) 33–43

5. Expression and localization of Tmie in adult rat cochleaHistochem Cell Biol (2008) 130:119–126

Dual solidDual solid--state lasersstate lasersAutomatic Laser Microdissection SystemAutomatic Laser Microdissection System

CapSure

Tissue

Glass Slide

IR Laser LCM

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a i e

UV Laser Cutting and Ablation

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Laser Cutting Laser Cutting vs.vs. LCM: LCM: Choice based on experimental objectiveChoice based on experimental objective

Small Area Captures~30um areas

UV - Laser CuttingIR - Laser Capture

Same size dissections

30 um diameter

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IR Laser Capture preserves RNA quality for small areas and single cell dissections

Large difference in RNA quality

Laser Cutting Laser Cutting vsvs LCM: LCM: Choice based on Experimental objectiveChoice based on Experimental objective

UV - Laser CuttingIR - Laser Capture

Large Area Captures

Same size dissections

650 um diameter

Co a able

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UV laser cutting enables quick dissection of large areas and hard tissues

Comparable RNA quality

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VeritasVeritas®

Working StageWorking Stage

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Load up to 8 caps

Modular insert configured for 3 

slides

Veritas®

Modular Sample TrayModular Sample Tray• Modular format for 3

slides or possible lt d i titculture and microtiter

plate options

• Enclosed in Veritas design to prevent contamination of precious samples

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Load up to 8 caps

Modular insert configured for 3 

slides

Positions for 3 slides

Removable unload station

QC Station

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Veritas®

NearNear IR Laser Capture IR Laser Capture MicrodissectionMicrodissection

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Veritas®

UV Laser CuttingUV Laser Cutting

H d Ti Pl t Ti E b Sub-Cellular L AHard Tissue Plant Tissue Embryo Sub Ce u aOrganelle Large Area

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Fast, Easy, and Efficient 3 Step OperationFast, Easy, and Efficient 3 Step Operation

VeritasVeritas®®

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Veritas®

Materials HandlingMaterials Handling• Automatically detects caps and • Compatibility

with ALL slideslides to ensure accuracy

• Integrated QC station for easy

with ALL slide types – glass and membrane

• Easy slide naming with

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CapSure inspection or ablation of unwanted material

naming with built-in bar-code integration

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Veritas®

Automated Microscope ControlsAutomated Microscope Controls

• Rapid light• Automated objective selection

• AutoFocus -removes the guess work!

• Stored positions

• Rapid light intensity and focus adjustment in real time

• Live or Static image modes

• Dynamic camera t l ith l

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Stored positions allow for easy tracking of dissected areas

controls with real time color balancing for the best image quality

Veritas®

MicrodissectionMicrodissection ToolsTools

Operate in multiple modes:• Ruler option to

measure in• Operate in multiple modes:- LCM single cells

- Cut & Capture for large areas and hard tissues

- Ablation to remove unwanted material

measure in microns

• Quick “go capture”

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• Draw regions, freehand or circles with ease

• Autoscan integrated automatic target cell identification and capture

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Veritas®

Capture GroupsCapture GroupsSelect up to 8 Capture Groups

User selected name, number and total area of regions in each group

Capture Groups

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Step thrucapture regions

Capture Groupsproperties

Area of last drawn or selected region

Cell RecognitionCell Recognition

Multiple Modes of Microdissection

P i t d h t l ti i di ti• Point-and-shoot real-time microdissection• Pick single cells or tissue structures of any shape using a

variety of drawing tools • Auto Scan: Train the system to automatically target high-

contrast or fluorescently-labeled cells

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RGB levels

Gray scale intensity level

Color ratiosColor

Cell RecognitionCell Recognition

Mean intensity

Max-Min pixel intensity variation

Spatial frequency variation

Horizontal difference variation

Vertical difference variationTexture

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Major/minor axis length

Orientation of axes

Eccentricity

Area

Circle equivalent diameter

Texture

Object

ArcturusArcturusXTXT™™ Microdissection InstrumentMicrodissection Instrument

Simplest user interfaces

LCM near IRLCM – near IR

UV laser cutting

Objective:

2x,4x,10x,20x,40x,60x,100x

Fluorescence system

DIC

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DIC

Phase contrast

Live cell culture system

Image analysis (MetaMorph)

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Veritas & ArcturusXTVeritas & ArcturusXT

Leading the Laser Microdissection Revolution

•Latest technology•Latest technologyIR Laser Capture MicrodissectionUV Laser Cutting

•Unmatched flexibility, precision, and speedIR laser capture is a gentle technique, maximizing bio-molecule integrity Ideal for single cell or small number of cell gene expression studies

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Ideal for single cell or small number of cell gene expression studies

UV laser cutting provides unprecedented speed and flexibility. Ideal for hard tissues and large # of cell harvesting for proteomics

applications

LCMApplications

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LCM LCM –– FFPE tissue sectionFFPE tissue section

Rat FFPE kidney section. Paradise stain.

Before LCM

After LCM

CapSure Cap

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LCM of Prostate Epithelium, LCM of Prostate Epithelium, ImmunostainedImmunostained for bclfor bcl--22

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LCM of adjacent cellsLCM of adjacent cells

Adjacent cells can be collected because they are not ablated or damaged during collection

Large Dorsal Root Ganglion Rat Neurons, Nissl stained

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Courtesy of Dr. Lin Luo and Dr. Mark Erlander, R. W. Johnson Pharmaceuticals

Nissl stained

Undamaged cells remain

Before LCM After LCM Captured LCM Cell

LCM of Human Blood SmearLCM of Human Blood Smear

Before LCM After LCM Captured LCM Cell

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Drosophila Embryo X-gal-Stained

Before Laser Cutting

FFluorescence microdissectionluorescence microdissection

FluorescenceAfter Laser Cutting

CapSure Cap

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laser cutting laser cutting –– live plant microdissectionlive plant microdissection

Whole mount preparation. Blade of grass on frame membrane slide.

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Human Chromosome Giemsa-stained Peripheral Lymphocyte

Before Laser Cutting

SubSub--cellular organellescellular organelles

After Laser Cutting

CapSure Cap

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FFPE Human Breast Tissue

Before Laser Cutting

Large area dissectionLarge area dissection

After Laser Cutting

CapSure Cap

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ArcturusXTArcturusXT Live Cell Live Cell MicrodissectionMicrodissection

Arcturus Application Note #11 Isolate living cells for re-culture or molecular analysis

After LCMBefore LCM

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39 CapSure® Cap

3T3 cells visualized with DIC optics

ArcturusXTArcturusXT Live Cell Live Cell MicrodissectionMicrodissection(Petri (Petri DishDish))

0 Hours Post-Microdissection

24 Hours Post-Microdissection

48 Hours Post-Microdissection

NEW!

RNA Yield: 2.7 ng/μlRIN 9 5

RNA Yield: 2.6 ng/μl RNA Yield: 3.1 ng/μlRIN 9 7

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Cultured CHO and TE671 cells

RIN: 9.5 RIN: 9.6 RIN: 9.7

Microdissection does not affect cell or nucleic acid viability

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ArcturusXTArcturusXT Petri Dish Stage InsertPetri Dish Stage Insert

Modular stage insert accommodates 50mm x 7mm Petri dishes ArcturusXT operating software accommodates stage insertEasy swap out for live cell or tissue based applications

Stage insert installation guide provided

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ApplicationsLive cell imagingLive cell microdissection

Identifying Cell-Specific Expression Signaturesin Various Stages of Breast Cancer

Molecular Signatures of Cancer ProgressionMolecular Signatures of Cancer Progression

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Ma, Xiao-Jun et al., PNAS 100(10): 5974-5979, 2003.

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The basal layer in human squamous tumors harbors more The basal layer in human squamous tumors harbors more UVA than UVB fingerprint mutationsUVA than UVB fingerprint mutations

PNAS 101(14): 4954-4959, 2004Regions sampled by LCM

for mutational analysis

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1. uninflamed upper 2. uninflamed lower 3. inflamed upper 4. inflamed lower 5. Stromal 6. normal upper 7. normal lower

Developmental Biology 312 (2007) 596–612

Gene expression analysis of zebrafish Gene expression analysis of zebrafish retinal ganglion cellsretinal ganglion cells

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Gene ontological analysis of changes in gene expression in RGCs during optic nerve regeneration

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Genesis, 2004, 39: 58-64

Laser Capture Microdissection of Fluorescently Labeled Laser Capture Microdissection of Fluorescently Labeled Embryonic Cranial Neural Crest CellsEmbryonic Cranial Neural Crest Cells

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2D DIGE Analysis2D DIGE Analysis

Molecular & Cellular Proteomics 1:117–124, (2002)

Cy3 image of Proteins fromnormal cells

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Cy5 image of Proteins fromTumor cells

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MALDIMALDI--Image AnalysisImage Analysis

Clinical Chemistry 46(9): 1513-1515 (2000)

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LCM + MALDI MSLCM + MALDI MS

Summary of application

Frozen tissue

QPCR

!! Only !!

Near IR (form NIH)+UV

QC function

Fluorescence tissue

Pl

Array

2D Gel

FFPE

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Plant

Chromosome

Live cell

Mass spec

Sequence

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ApplicationApplication

Some Topics of InterestDNA/RNA ResearchMiMicroarrayProteomicsDevelopmental biologyNeurologyChromosome / CGHLive cellPlantVirology

Sequence

PCR, QPCR 2D Gel

Microarray

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gyBoneForensics (single cell, crime scene)

• Approximately 2000 peer-reviewed articles

PCR, QPCR

Mass Spec

Cell Identification

Cell Selection Labeling and AnalysisPurification

Integrated System for MicrogenomicsIntegrated System for Microgenomics®®

Amplification

PicoPure®

& Paradise®

l i i

RiboAmp®

& Paradise®

AmplificationKits

TURBOLabeling™ Kits

ArcturusXTTM VeritasTM

CONFIDENTIAL

HistoGene® & Paradise® TissuePreparation Kits

Isolation Kits

• Frozen Samples(HistoGene, PicoPure,

RiboAmp)

• Formalin-Fixed, Paraffin-embedded Samples

(Paradise)

ArcturusXTTM, VeritasTM

Microdissection Systems

CapSure® Caps

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Getting Startedwith LCM

ProcessFrozen tissue FFPE tissueStaining & dehydration Deparaffin & Staining & dehydration

Capture cells

Isolation、extraction

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DNA RNA protein

Amplification & labeling

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Frozen Tissue PreparationFrozen Tissue Preparation

Specimen FreezingAdd a layer of OCT to cryomold.Add tissue specimen to OCTAdd OCT to cover specimen Freeze the cryomoldOnce specimen is completely frozen transfer to –80℃ freezer for storage.

Clean all surfaces.Use new section of microtome blade for each specimen.

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specimen.Optimal section thickness

7-10 μm for LCM5-100 μm for LC/LCM

Do all sectioning at -20 ℃Store sections at -80 ℃ to preserve RNA

Fix biopsy in 10% Neutral Buffered Formalin as soon as possible after dissection of tissue.

Paraffin Tissue ProcessingParaffin Tissue Processing

Clean all surfaces.

Use nuclease free or DEPC treated water for tissue floatation water bath.

Optimal section thickness 7 10 m

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Optimal section thickness 7-10 μm.

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Frozen Section Staining and DehydrationFrozen Section Staining and Dehydration

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FFPE SectionsFFPE SectionsDeDe--paraffinizationparaffinization, Staining, Dehydration, Staining, Dehydration

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Staining and Dehydration Staining and Dehydration ––IHCIHCFluorescent StainingFluorescent Staining

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Once staining is complete sample cannot be stored.Use immediately for LCM!!!

DehydrationDehydration

***Use fresh 100% Ethanol for all samples.

75%EtOH

95%EtOH

100%EtOH Xylene

Air Dry

p

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30 sec 30 sec 30 sec 5 min 5 min

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HistoGene™ HistoGene™ LCM Frozen Tissue Staining KitLCM Frozen Tissue Staining Kit

Less than 10 minutes !!Less than 10 minutes !!

Mouse Small Intestine

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RNA QualityRNA Quality

1. Histogene stained

2. Unstained

3. Unstained & dehydrated

Mouse Kidney

Important Points!!Important Points!!

For RNA isolation

Wear disposable gloves and change them frequentlyUse RNase-free solutions, glassware, plasticware and instrumentsWipe working area down with RNASE-ZAP.Use sample for microdissection with 2 hours of stainingDO NOT refreeze a stained tissue section

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Use it or Lose it !

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CapSureCapSure® LCM Caps® LCM Caps

CapSure Macro LCM CapsMacro Cap ideal for large area capture, many cellsContacts specimen surface for optimal adhesionContacts specimen surface for optimal adhesion50 microliters of extraction volume neededCouples directly with 0.5 microfuge tubes

CapSure HS LCM Caps“HS” = High Specificity12μm stand-off railsOptimal for smaller #’s of cells, especially rare single cells

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gOnly 10 microliters of extraction volume neededCaptures cells with speed and high specificityUse of ExtracSure Device, Alignment Tray, Incubation Block

CapSure® HS LCM CapsWork with ExtracSure™ Sample Extraction

CapsCaps

Sample Extraction Devices that minimize the dilution of biological molecules extracted from captured cells.

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2 4

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The Extraction of The Extraction of BioMoleculesBioMolecules Using Using CapSureCapSure HS LCM CapsHS LCM Caps

1. Pipette Lysis Buffer 2. Incubate Sample

0 5 l Mi t if t b

Pipette tip

ExtracSure® Device

CapSure® HS LCM Cap

0.5 ml Microcentrifuge tube

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0.5 ml microcentrifuge tubes for extraction

(Applied BioSystems Catalog # N8010611)

ArcturusArcturus LCM InstrumentsLCM Instruments

• Ultimate sample preparation flexibility

Frame Membrane

Glass Membrane

Standard Glass

Standard Glass Slides Glass Membrane Slides Frame Membrane Slides

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64Arcturus LCM only commercial instruments that

enable use of ALL slide formats

• IR capture only • Archived specimens • Economical

• IR capture only or IR capture + UV Cutting

• Thicker sections

• IR capture + UV cutting• Non-contact, no contamination

microdissection• Hydrated or dehydrated

specimens

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Standard Glass Slides Glass Membrane Slides Frame Membrane Slides

LCM l LCM & UV Abl ti LCM & UV Ablation

ArcturusArcturus LCM InstrumentsLCM Instruments

• Multiple Slide Formats Allow LCM and LC/LCM

CapSure

IR Laser LCM IR Laser LCMIR Laser LCM

CapSureCapSure

Tissue M bFrame

LCM or UV Ablation LCM only or LCM & UV Ablation LCM & UV Ablation

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TissueGlass Slide

UV Laser Ablation

TissueTissue

UV Laser Cutting and Ablation

UV Laser Cutting and Ablation

Membrane Slide

Membrane

Glass Slide

RNA IsolationRNA Isolation

PicoPureTM RNA Isolation Kit• Highest recovery ( > 90% )Highest recovery ( > 90% )• Low-volume elution ( 11 uL )• Optimally shaped column for high-yield

recovery• Column lid to prevent contamination

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RNA AmplificationRNA Amplification

RiboAmpTM RNA Amplification Kit

CONFIDENTIAL

DNA ExtractionDNA Extraction

PicoPureTM DNA Extraction Kit• Go Direct to PCR• Go Direct to PCR

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at 65°C

FFPE >16 hrs

Frozen ~ 3 hrs

Centrifuge

1 min.

at 1,000 x g

Macro Caps: 50uL

HS Caps: 10uL

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Protein Isolation Protein Isolation -- 11

1. The methodology and buffers utilized for processing microdissected samples for protein-based studies is variable depending on the downstream molecular analysis methoddownstream molecular analysis method.

2. For two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) studies, from NCI:

100 µl of IEF lysing solution containing 7M Urea2M Thiourea4% CHAPS1% MEGA-101% Octyl-b-Glucopyranoside,40 mM Tris

CONFIDENTIAL

50 mM DTT2 mM tri-butyl phosphine (TBP)0.5% (v/v) Pharmalytes (pH ranges 3-10, 4-7, or 6-11)

3. Place the buffer in an Eppendorf tube, place the LCM cap on the tube, invert the tube, and vortex vigorously for one minute until all cells are completely lysed.

Protein Isolation Protein Isolation -- 22

1. The extraction buffer formulation is

- 45 mM Tris-HCl- 0.0188 M EDTA- 4.61 M GITC- 1% Triton X-100- 1% Mercaptoethanol

2. Extraction Procedure:

Add 50 l of this b ffer to the

CONFIDENTIAL

- Add 50ul of this buffer to the - 0.5ml microcentrifuge tube, place each cap in a tube, invert to

cover the cap surface with buffer.- Incubate the sample for 3 hours at 65 degrees.- After incubation spin down the sample and collect the lysate.

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Protein Isolation Protein Isolation -- 33

Scheme for Extraction of ProteinScheme for Extraction of Proteinfrom Multiple LCM Capsfrom Multiple LCM Caps

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Average Biomolecules Recovery from LCM Average Biomolecules Recovery from LCM Captured CellsCaptured Cells

h ll• Average human cell is 20 um in size• Total DNA (0.25% of cell weight): ~ 5pg per cell• Total RNA (1.1% of cell weight): ~30 - 50pg/cell• Total Protein (18% of cell weight): ~0.5ng/cell

• Average tissue section cut to 10um

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• an average single cell LCM capture ~ 2.5pg per cell• an 1500 single cell LCM capture ~ 2.5pg per cell x 1500• a 300 multiple cell (5 cells per laser spot) LCM capture ~ 12.5pg per cell

x 300

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Average Biomolecules Recovery from LCM Average Biomolecules Recovery from LCM Captured CellsCaptured Cells

CONFIDENTIAL

More Information of LCMMore Information of LCM

CONFIDENTIAL

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http://www.moleculardevices.com/http://www.moleculardevices.com/

CONFIDENTIAL

Application NoteApplication Note

CONFIDENTIAL

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Literature CitationLiterature Citation

MDS AT http://www.moleculardevices.com/citation/

More Information of LCMMore Information of LCM

CONFIDENTIAL

LCM Web Seminar Series

CONFIDENTIAL

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Cell Identification

Cell Selection Labeling and AnalysisPurification

Integrated System for Integrated System for MicrogenomicsMicrogenomics®®

Amplification

PicoPure®

& Paradise®

l i i

RiboAmp®

& Paradise®

AmplificationKits

TURBOLabeling™ Kits

ArcturusXTTM VeritasTM

CONFIDENTIAL

HistoGene® & Paradise® TissuePreparation Kits

Isolation Kits

• Frozen Samples(HistoGene, PicoPure,

RiboAmp)

• Formalin-Fixed, Paraffin-embedded Samples

(Paradise)

ArcturusXTTM, VeritasTM

Microdissection Systems

CapSure® Caps

Thanks for your attention!!!

鎂陞科技股份有限公司

y

For Technical Support feel free to contact us

CONFIDENTIAL

劉恬均

TEL: 02-2698-9511 #616E-mail: ivy.liu@mass-solutions.com.tw