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TechnologyTransition Workshop
Carl Zeiss® PALM®
Technology Transition Workshop| Jack Garner
TechnologyTransition Workshop
Laser Microdissection & Pressure Catapulting (LMPC)
• New horizons for microgenomics, proteomics and live cell micromanipulation
− Laser capture microscopy
− The technology
− Forensic science application
Physical Separation of Forensic Mixtures Using Laser Microdissection Techniques 2Zeiss® PALM®
TechnologyTransition Workshop
The New Carl Zeiss®PALM® MicroBeam
Physical Separation of Forensic Mixtures Using Laser Microdissection Techniques 3Zeiss® PALM®
TechnologyTransition Workshop
The Next Generation of Laser Microdissection
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Safety of LMPC for Biological Applications
• Focally restricted adiabatic process
• No UV-A absorbance of DNA, RNA, proteins
• No impact on DNA, RNA, protein recovery
300 320 340240220
Absorbance Spectra
nm
PALM
UV-A
355
nm
260
DNA
280
Protein
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Optical Density of DNA and Protein
FTSS Laser
355 nm
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• Incise with light, transport with light, work with light
• Non-contact manipulation by the force of focused light!
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Srinivasan R. “Ablation of Polymers and Biological Tissue by Ultraviolet Lasers.”
Science 234 (4776) (October 1986): 559-565.Photodecomposition:UV laser energy in focal volume causes breakdown of molecular bonds without lateral heat transfer
Nd-Yag UV-A Laser(355 nm; >90 µJ; 1-100 Hz)
Focal spot <1 µm
PALM® – Laser Microdissection
Physical Separation of Forensic Mixtures Using Laser Microdissection Techniques 8Zeiss® PALM®
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How Does LMPC Work?
LD Plan-
NEOFLUAR
40x / 0.6
421361-9970
ZEISS
LD Plan-
NEOFLUAR
40x / 0.6
421361-9970
ZEISS
Laser Microdissection Laser Pressure Catapulting
Physical Separation of Forensic Mixtures Using Laser Microdissection Techniques 9Zeiss® PALM®
TechnologyTransition Workshop
Laser Plasma Formation:An adiabatic process (no lateral energy transfer during formation & collapse of laser plasma)
PALM® – Laser Microdissection
Srinivasan R. “Ablation of Polymers and Biological Tissue by Ultraviolet Lasers.”
Science 234 (4776) (October 1986): 559-565.
Physical Separation of Forensic Mixtures Using Laser Microdissection Techniques 10Zeiss® PALM®
To see a video depiction of this principle in action, go to: http://www.zeiss.com/micro
TechnologyTransition Workshop
The PALM® MicroBeam LMPC Method
LD Plan-NEOFLUAR
40x / 0.6
421361-9970
ZEISS
• Laser microdissection pressure catapulting
– Contamination-free
– Non-contact
– Against gravity
1
2
3
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The Next Generation: PALM® MicroBeam IV
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TechnologyTransition Workshop
Laser Microdissection from Glass-mounted Tissue
Glass-mounted Tissue
• Can be used on archival material
• Unique PALM® feature
Laser Microdissection from Membrane-mounted Tissue
Object Slide
PEN (Polyethylene-Naphtalene) Membrane
• Serves as a backbone – Preserves tissue morphology
• Holds selected specimen together – Facilitates ablation and tissue
separation• Enables dissection of any shape and
size– Allows fixation and staining
Mounted TissueMembrane
Object Slide
Mounted Tissue
Tissue Microdissection from Membrane and Glass Slides
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TechnologyTransition Workshop
Laser Microdissection from Glass-mounted Tissue
Laser Microdissection from Membrane-mounted Tissue
Tissue Microdissection from Membrane and Glass Slides
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TechnologyTransition Workshop
The Zeiss® Advantage• Collection accuracy and efficiency
− Stage movement to sub-micron accuracy
− Laser cutting remains perpendicular to focal plane
− Laser pressure catapulting provides increased efficiency under normal laboratory conditions, and better collection efficiency under challenging laboratory conditions
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Functional Downstream
Analysis
Specimen Preparation
and Selection
PALM® MicroBeamEnabling Technology
MicroBeamForensic Specimen DNA Fingerprint
Analysis
Laser Microdissection: Understanding the Complete Forensic Workflow
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Contact-free Collection of Forensic Evidence with PALM® MicroBeam
• Adhesive tape
• Sperm cells
• Epithelial cells
• Hair
• DNA fingerprinting
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Forensic Evidence – Hair
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Sperm Identification Methods
• Manual
− Visual identification and selection by laboratory personnel
• Automatic
− Identification with pattern recognition feature of AxioVision® software
− This is a significant time saver where larger numbers of samples are required
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Fluorescence
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PALM® Sample Selection and Identification
Physical Separation of Forensic Mixtures Using Laser Microdissection Techniques Zeiss® PALM® 21
AxioVision® Example Script
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Goal Example: Selection of Nuclei in Liver Tissue
High Throughput: Image Recognition Software Interfaces
Carl Zeiss® AxioVision®
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• Find sperm/forensic evidence according to staining/morphology • Reject particles of wrong morphologies• Prepare correct objects for LMPC• Export to RoboSoftware®
Segmentation
by Color
Removal of
ScrapDilation
High Throughput: Image Recognition Software InterfacesCarl Zeiss® AxioVision®
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Carl Zeiss® AxioVision®
High Throughput: Image Recognition Software Interfaces
Physical Separation of Forensic Mixtures Using Laser Microdissection Techniques 24Zeiss® PALM®
TechnologyTransition Workshop
Carl Zeiss® AxioVision®
High Throughput: Image Recognition Software Interfaces
Physical Separation of Forensic Mixtures Using Laser Microdissection Techniques 25Zeiss® PALM®
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Carl Zeiss® AxioVision®
High Throughput: Image Recognition Software Interfaces
Physical Separation of Forensic Mixtures Using Laser Microdissection Techniques 26Zeiss® PALM®
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Imaged Sample in AxioVision®
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Example Script
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Binary Results of Auto ID
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Discriminatory Selection
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Selected Samples Filled
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Samples Selected for Collection
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Questions?
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Contact Information
Jack GarnerPALM® Microlaser TechnologiesCarl Zeiss® MicroImaging Group
Physical Separation of Forensic Mixtures Using Laser Microdissection Techniques 34Zeiss® PALM®
All images courtesy of Carl Zeiss® unless otherwise noted.
