Arcturus® XT Microdissection System
王森右Field Applications Scientist
• Theory of Laser Capture Microdissection
• Introduction to Arcturus XT™ Laser Microdissection System
• Tissue Preparation Seminar
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Why Use Laser Capture Microdissection (LCM) in Gene Expression Studies?
To reveal accurate, cell-specific expression profiles otherwise
obscured in mixed cell samples
―Pure‖ populations‖ vs. ―Mixed‖ populations
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2. Identify Target Cells 3. Isolate Target Cells1. Visualize Specimen
LCM of premalignant breast cancer progressionMa, Xiao-Jun et al. (2003) Proc. Natl. Acad. Sci. USA 100, 5974-5979
ADH
Normal
DCIS
IDC
LCM Process
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The Power of Microgenomics in Cell-Specific Molecular Analysis
Homogeneous cell populations reveal hidden molecular signatures
Gene 1:
Proteoglycan 1
Gene 2:
CD53 antigen
Gene 3:
Proteosome subunit
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Introduction to LCM
Microgenomics
Quantitative genomic and proteomic molecular
analysis of single cells or small groups of cells
Systems for Microgenomics
Integrated and complete sets of instruments,
reagents and protocols for the study of
microgenomics
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Microgenomics Process
Biopsy, FACS sorting, cell culture
ArcturusXT™, Veritas™,
PixCell® , AutoPix®
RiboAmp family, Paradise
PCR, QPCR, Microarray, Tissue
Array, 2-DGE, LC/MS, etc.
EtOH/Frozen, FFPE
HistoGene, Standard and IHC
PicoPure RNA, DNA, Paradise
Sample Collection
Molecular Analysis
Cell Selection
Cell Identification
Extraction and Purification
Amplification
Specimen Preservation
Labeling Turbo Biotin, Cy™3, Cy™5
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Ciphergen
Arcturus® System LCM Applications Gene Expression –
Microarray Mutation Analysis
Gene Expression –
Real-Time PCR
Proteomics – Protein
Chip/Mass Spec
Proteomics – 2D Gels
Forensics – STR
analysis
Proteomics – Reverse
Phase Protein Arrays
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Two fundamental approaches to Microdissection
− IR Laser Capture (LCM)
>Developed at the NIH- exclusive license to Life Tech
>Low energy laser preserves biomolecule integrity (RNA)
>Best choice for single cell or small number of cells
>Allows reliable use of plain glass slide preparations
>Maintains Morphology, nondestructive to adjacent tissue
>Well Published- Over 1,000 citations
− UV Laser Cutting
>Provides additional speed and flexibility
>Ideal for difficult tissues and large samples
>Unwanted material can be ablated
>Supports Contact or Non-Contact Microdissection
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How LCM Works
• Near-Infrared (IR) laser activates thermoplastic polymer transfer film
− Thermoplastic film:
> Absorbs IR laser energy
− Prevents laser energy from reaching sample
− Laser energy never directly absorbed by sample
> Becomes adhesive
− Polymer film activates and melts near 70° C
− Sticks to cells of interest
> Increasing IR energy increases activated film area
> Distends predictably, evenly, reproducibly to enable selective
targeting
> Adhesion overcomes opposing forces to enable selective capture
• Cell(s) removed with polymer
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Laser Capture Microdissection Forces
For successful LCM, Tissue-Activated Film force must be greatest.
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SEM Image of LCM pulses
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The LCM Spot
• Ensure film is able to contact the glass.
− Take a practice shot where there is no tissue.
− Adjust IR laser power and duration settings to insure polymer film touches the glass.
− If film touches glass –IR laser and LCM cap are ready for for capture.
Proper Film contact No Film contact
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LCM Caps
• CapSure™ Macro LCM Caps
− MacroCap ideal for large area capture
− Contacts tissue surface for optimal adhesion
− 50 microliters of extraction volume needed
− Couples directly with 0.5 microfuge tubes
• CapSure™ HS LCM Caps
− ―HS‖ stands for High Specificity
− Optimal for smaller#’s of cells, especially rare single cells
− Only 10 microliters of extraction volume needed
− Captures cells with speed and high specificity.
− Use of ExtracSure Device, Alignment Tray, Incubation Block
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1. Prepare tissue 2. Locate cells 3. Place cap
4. Pulse laser 5. Remove cap 6. Extract molecules
The Laser Capture Microdissection Process
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Microdissect a Variety of Samples
Microdissect any morphological shape
•Microdissect single cells or multi-cellular structures—regardless of the
• shape (linear, circular, doughnut, etc)
Preserve cell and tissue integrity
•Microdissected material and surrounding cells can never be
damaged
Utilize a variety of slide preparations techniques•Use standard slide preps or previously archived samples.
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ArcturusXT™ Microdissection Instrument
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ArcturusXT Software Graphical User Interface
1. Load Materials
2. Inspect Samples
3. Select Cells
4. Microdissect
5. QC
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Large Area Captures
Laser Cutting vs. LCM: Choice based on Experimental Objective
UV laser cutting enables quick dissection of large areas and hard tissues
UV Laser CuttingIR Laser Capture Microdissection
Large Captures
~650u areas
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Laser Cutting vs. LCM: Choice based on Experimental Objective
IR Laser Capture preserves RNA quality for small areas and single cell
dissections
Small Area Captures
UV Laser CuttingIR Laser Capture Microdissection
Small Captures
~30u areas
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Phase Contrast DIC
Cultured TM3
Cells
Chinese hamster ovary (CHO)
cells
Cultured TM3 cells
Cultured 3T3 cells
ArcturusXT – Phase Contrast and DIC
Ideal for unstained samples
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ArcturusXT – Fluorescence
Triple labeled bovine pulmonary artery endothelial (BPAE) cells.
Mitochondria = red, F-actin=green and Nuclei= blue. Visualized
simultaneously using an Omega triple band dichroic filter.
Human Breast Carcinoma, anti-
cytokeratin/Cy3
Cultured HELA cells exposed to
BCECF, a cytoplasmic pH indicator.
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Single Cell Capture - ArcturusXT™ IR Laser Capture Microdissection
Frozen Mouse Brain –
Histogene Stain
Before LCM
After LCM
CapSure
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FFPE Cresyl Violet - Stained
Human Bone Marrow
ArcturusXT™ UV Laser Cutting
Before Laser Cutting
After Laser Cutting
CapSure
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ArcturusXT LCM – FFPE tissue section
Rat FFPE kidney section.
Paradise stain.
Before LCM
After LCM
CapSure Cap
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Drosophila Embryo X-gal-
Stained Fluorescence
Before Laser Cutting
After Laser Cutting
CapSure
Fluorescence Dissection - ArcturusXT™
UV Laser Cutting
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ArcturusXT™ - Microdissected GFP Neurons
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ArcturusXT laser cutting – live plant microdissection
Whole mount preparation.
Blade of grass on frame
membrane slide.
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Multiple Stage Formats
• Large Slide Stage Insert
> 25mm, 38mm and 50mm slides
> Great for neuroscience applications
• Petri Dish Stage Insert> accommodates 50mm x 7mm Petri dishes
> Easy swap out for live cell or tissue based
applications
− Live cell imaging
− Live cell microdissection
ArcturusXT
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ArcturusXT Live Cell Microdissection
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Cell Identification
HistoGene®
Kits
Cell Isolation and
Extraction
PicoPure® Kit (frozen)
Paradise®
PLUS Kits (FFPE)
Amplification
RiboAmp® PLUS
Kit (frozen)
Paradise® PLUS
Kit (FFPE)
Labeling
Turbo
Labeling™
Kit
Cell Selection
ArcturusXT™
LCM
System
Downstream
Analysis
Integrated Systems for Microgenomics®
Tissues preparation
王森右Field Applications Scientist
• Tissue Preparation
• Tissue Sectioning
• Staining
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Preparing Samples for Laser Capture Microdissection
Many common histological preparations
are compatible with laser cutting and laser
capture microdissection
Downstream analysis of microdissected
samples include RNA, DNA and protein
For microgenomic analysis, the sample
preparation process must facilitate cell
identification and preserve the integrity of
biomolecules of interest
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General Guidelines for Specimen Preparation
Tissue should be processed as soon as possible upon removal
All cells contain proteases including nucleases that are still active
after removal or upon death
Autodegradation of biomolecules
RNase-Free conditions should be observed at all times during
handling of tissues and sections.
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RNase-Free Technique
• Wear disposable gloves and change frequently
• Use new or clean instruments between each animal or patient
specimen
• Use RNase-free or Nuclease free solutions, glassware and
plasticware
• Use RNase Zap or similar product to clean equipment
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Laser Capture Microdissection Forces
For successful LCM, Tissue-Activated Film force must be greatest.
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Frozen Tissue Preparation
• Tissues should be frozen in OCT or a similar product
• Preferred Freezing Methods
Isopentane cooled over liquid Nitrogen
Isopentane cooled with dry ice
• Other Freezing Methods
Dry Ice Alone (not optimal, slow)
Liquid Nitrogen (not optimal, sectioning difficulties)
Cryostat (NO!)
• Tissues can be sectioned immediately or stored in a
–70C freezer
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Protocol for Freezing Tissue
Specimen Freezing with Isopentane
1. Add a thin layer of OCT to bottom of cryomold
2. Place tissue specimen on top OCT layer
3. Add additional OCT to cover specimen
4. Place cryomold into cooled isopentane to freeze
5. Once tissue block is completely frozen transfer to dry ice or to
a –70C freezer for storage
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Frozen Tissue Sectioning
Gloves must be worn at all times
Cryostat must be cleaned prior to use. All surfaces must be wiped
down with 95-100% ethanol, especially knife holder and anti-roll plate Recommended Section thickness
LCM alone = 8-10umLC+LCM = up to 100um
Mount sections onto room temperature slides. After mounting the
sections place in slide box stored on dry ice. SLIDES MUST REMAIN
COLD!
For mounting of sections onto frame membrane slides refer to
Arcturus Protocol #9
Use separate areas of the microtome blade for each specimen
Sections can be stored in a –70C freezer until further processing
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Formalin-Fixed Paraffin Embedded (FFPE) Tissues
• Formalin-fixation
Cross-linking by aldehyde
groups affects structural integrity
of nucleic acids and proteins.
• Best for working with DNA
Presents additional challenges when working with RNA
• Paraffin processing and embedding
Extraction of nucleic acids from paraffin embedding causes
degradation of RNA and DNA
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FFPE Tissue Processing
• Fixation in 10% Neutral Buffered Formalin as soon as possible after
harvesting
• Fixation should not exceed 24 hours at room temperature with tissue
thickness not exceeding 5mm during fixation process
• Tissues should be processed into paraffin immediately after fixation.
Storage in ethanol or PBS is not recommended
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Preparation of Tissue Sections
Formalin Fixed Paraffin Embedded Tissue
Use Nuclease Free or DEPC treated water for tissue floatation bath
Float sections for minimal amount of time, no more than 1-2 mins
Once sections mounted on slides, prop up vertically to allow water to drain away from sections
Air dry for about 2 hrs at room temperature. Do not use oven to dry sections.
Slides can be store for up to 2wks at room temperature with dessicant, for longer terms store at –70C.
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Optimal Staining vs. RNA Quality
RNA QualityVisualization
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Staining and Dehydration
• Staining
− Light staining of tissue sections improves visualization
− Staining agents and protocols can affect quality and yield of
recovered molecules
− Some samples do not require staining – ex. GFP label
• Dehydration Removal of water inactivates nucleases
> Graded ethanol series – 75%, 95%, 100%
> Ethanol concentrations must be freshly prepared
> Xylene steps performed in fume hood
> Air Drying no more than 5 minutes in a fume hood
− Provides conditions that maximizes the LCM process
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General Staining Guidelines (Frozen and FFPE)
• Total staining time should be as short as possible
• Staining dishes should be nuclease free
• Staining solutions should be dedicated for use with LCM samples
• Solutions are prepared with nuclease free water
• Stained slides can be held in xylene until initiation of laser capture
microdissection
• Once removed from xylene, microdissection should be completed within 2
hours
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Histochemical Staining
HistoGene Stain for Frozen Tissue
• 75% ETOH - 30secs
• NF dH20 - 30 secs
• Histogene stain -10-30 secs
• NF dH20 - 30 secs
• 75% ETOH - 30 secs
• 95% ETOH - 30 secs
• 100% ETOH - 30 secs
• Xylene - 5 mins
Total Time = 8.5 mins
Aqueous Time = 3.5 mins
Paradise Stainfor FFPE Tissue Xylene – 2-3 mins
Xylene _ 2-3 mins
100% ETOH – 1 min
95% ETOH – 1min
75% ETOH – 1 min
NF dH20 – 30 secs
Paradise Stain – 30-45 secs
NF dH20 – 30 secs
75% ETOH – 30 secs
95% ETOH – 30 secs
100% ETOH – 1 min
Xylene – 5 mins
Total Time = 18 mins
Aqueous Time = 7 mins
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HistoGene® Immunofluorescence Kit
biotin
tissue antigen
1o Antibody
streptavidin
Cy3
Total staining time = 17 mins
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Stains Compatible with Downstream RNA Analysis
• HistoGene Frozen Section Staining Kit
• Paradise Kit for FFPE
• Hematoxylin (Mayer’s) and Eosin (frozen, FFPE)
• Cresyl Violet (frozen sections)
• Toluidine Blue (frozen section)
• HistoGene Immunofluorescence Staining Kit
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RNA Quality Assessment of Tissues
Take a look at the RNA in your tissue sample prior to LCM and
downstream analysis
Analysis can be completed from as little as one tissue section
RNA
−NanoDrop: Quantity
−Agilent Bioanalyzer: Quality
−qRT-PCR: 3’/5’ ß-Actin Ratios, transcribe ability of the RNA
(important with FFPE samples)
Frozen Tissues: Arcturus Protocol #1-Tissue Scrape Protocol for
Verifying RNA Quality
FFPE Tissues: Paradise® Plus or Paradise® Plus WT-RT Kits
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Slide Formats for LCM and LC/LCM
Standard Glass
Glass Membrane
Frame Membrane
•Multiple modes for microdissection.
•Enables flexibility w/ applications.
•Removes dependence on tissue dehydration.
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Compatible with ALL standard glassand membrane slides
Standard Glass Slides Glass Membrane Slides Frame Membrane Slides
CapSure
TissueGlass Slide
IR Laser LCM
UV Laser Ablation
IR Laser LCMIR Laser LCM
CapSureCapSure
TissueTissue
UV Laser Cutting and Ablation
UV Laser Cutting and Ablation
MembraneSlide
Membrane
Frame
Glass Slide
LCM & UV Ablation LCM, LCM & UV Ablation LCM & UV Ablation
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Cell Quantities and Downstream Applications
Source: Espina V etal, ―Laser-Capture Microdissection‖, Nature Protocols (1:2), 2006
http://www.nature.com/nprot/journal/v1/n2/pdf/nprot.2006.85.pdf
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Arcturus Protocols & App Notes
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Q&A For Research Use Only.
Not intended for any animal or
human therapeutic or diagnostic
use.