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TechnologyTransition Workshop
Carl Zeiss® PALM®
Technology Transition Workshop| Jack Garner
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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®
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TechnologyTransition Workshop
The New Carl Zeiss®PALM® MicroBeam
Physical Separation of Forensic Mixtures Using Laser Microdissection Techniques 3Zeiss® PALM®
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TechnologyTransition Workshop
The Next Generation of Laser Microdissection
Physical Separation of Forensic Mixtures Using Laser Microdissection Techniques 4Zeiss® PALM®
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TechnologyTransition Workshop
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
Physical Separation of Forensic Mixtures Using Laser Microdissection Techniques 5Zeiss® PALM®
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TechnologyTransition Workshop
Optical Density of DNA and Protein
FTSS Laser
355 nm
Physical Separation of Forensic Mixtures Using Laser Microdissection Techniques 6Zeiss® PALM®
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TechnologyTransition Workshop
• Incise with light, transport with light, work with light
• Non-contact manipulation by the force of focused light!
Physical Separation of Forensic Mixtures Using Laser Microdissection Techniques 7Zeiss® PALM®
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TechnologyTransition Workshop
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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TechnologyTransition Workshop
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®
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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
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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
Physical Separation of Forensic Mixtures Using Laser Microdissection Techniques 11Zeiss® PALM®
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TechnologyTransition Workshop
The Next Generation: PALM® MicroBeam IV
Physical Separation of Forensic Mixtures Using Laser Microdissection Techniques 12Zeiss® PALM®
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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
Physical Separation of Forensic Mixtures Using Laser Microdissection Techniques 13Zeiss® PALM®
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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
Physical Separation of Forensic Mixtures Using Laser Microdissection Techniques 14Zeiss® PALM®
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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
Physical Separation of Forensic Mixtures Using Laser Microdissection Techniques 15Zeiss® PALM®
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TechnologyTransition Workshop
Functional Downstream
Analysis
Specimen Preparation
and Selection
PALM® MicroBeamEnabling Technology
MicroBeamForensic Specimen DNA Fingerprint
Analysis
Laser Microdissection: Understanding the Complete Forensic Workflow
Physical Separation of Forensic Mixtures Using Laser Microdissection Techniques 16Zeiss® PALM®
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TechnologyTransition Workshop
Contact-free Collection of Forensic Evidence with PALM® MicroBeam
• Adhesive tape
• Sperm cells
• Epithelial cells
• Hair
• DNA fingerprinting
Physical Separation of Forensic Mixtures Using Laser Microdissection Techniques 17Zeiss® PALM®
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TechnologyTransition Workshop
Forensic Evidence – Hair
Physical Separation of Forensic Mixtures Using Laser Microdissection Techniques 18Zeiss® PALM®
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TechnologyTransition Workshop
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
Physical Separation of Forensic Mixtures Using Laser Microdissection Techniques 19Zeiss® PALM®
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TechnologyTransition Workshop
Fluorescence
Physical Separation of Forensic Mixtures Using Laser Microdissection Techniques 20Zeiss® PALM®
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TechnologyTransition Workshop
PALM® Sample Selection and Identification
Physical Separation of Forensic Mixtures Using Laser Microdissection Techniques Zeiss® PALM® 21
AxioVision® Example Script
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TechnologyTransition Workshop
Goal Example: Selection of Nuclei in Liver Tissue
High Throughput: Image Recognition Software Interfaces
Carl Zeiss® AxioVision®
Physical Separation of Forensic Mixtures Using Laser Microdissection Techniques 22Zeiss® PALM®
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TechnologyTransition Workshop
• 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®
Physical Separation of Forensic Mixtures Using Laser Microdissection Techniques 23Zeiss® PALM®
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TechnologyTransition Workshop
Carl Zeiss® AxioVision®
High Throughput: Image Recognition Software Interfaces
Physical Separation of Forensic Mixtures Using Laser Microdissection Techniques 24Zeiss® PALM®
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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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TechnologyTransition Workshop
Carl Zeiss® AxioVision®
High Throughput: Image Recognition Software Interfaces
Physical Separation of Forensic Mixtures Using Laser Microdissection Techniques 26Zeiss® PALM®
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TechnologyTransition Workshop
Imaged Sample in AxioVision®
Physical Separation of Forensic Mixtures Using Laser Microdissection Techniques 27Zeiss® PALM®
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TechnologyTransition Workshop
Example Script
Physical Separation of Forensic Mixtures Using Laser Microdissection Techniques 28Zeiss® PALM®
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TechnologyTransition Workshop
Binary Results of Auto ID
Physical Separation of Forensic Mixtures Using Laser Microdissection Techniques 29Zeiss® PALM®
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TechnologyTransition Workshop
Discriminatory Selection
Physical Separation of Forensic Mixtures Using Laser Microdissection Techniques 30Zeiss® PALM®
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TechnologyTransition Workshop
Selected Samples Filled
Physical Separation of Forensic Mixtures Using Laser Microdissection Techniques 31Zeiss® PALM®
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TechnologyTransition Workshop
Samples Selected for Collection
Physical Separation of Forensic Mixtures Using Laser Microdissection Techniques 32Zeiss® PALM®
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TechnologyTransition Workshop
Questions?
Physical Separation of Forensic Mixtures Using Laser Microdissection Techniques 33Zeiss® PALM®
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TechnologyTransition Workshop
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.