carl zeiss® palm® - projects at nfstc.org · physical separation of forensic mixtures using laser...

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

Physical Separation of Forensic Mixtures Using Laser Microdissection Techniques 4Zeiss® PALM®

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®

TechnologyTransition Workshop

Optical Density of DNA and Protein

FTSS Laser

355 nm

Physical Separation of Forensic Mixtures Using Laser Microdissection Techniques 6Zeiss® PALM®

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®

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®

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®

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

Physical Separation of Forensic Mixtures Using Laser Microdissection Techniques 11Zeiss® PALM®

TechnologyTransition Workshop

The Next Generation: PALM® MicroBeam IV

Physical Separation of Forensic Mixtures Using Laser Microdissection Techniques 12Zeiss® PALM®

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®

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®

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®

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®

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®

TechnologyTransition Workshop

Forensic Evidence – Hair

Physical Separation of Forensic Mixtures Using Laser Microdissection Techniques 18Zeiss® PALM®

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®

TechnologyTransition Workshop

Fluorescence

Physical Separation of Forensic Mixtures Using Laser Microdissection Techniques 20Zeiss® PALM®

TechnologyTransition Workshop

PALM® Sample Selection and Identification

Physical Separation of Forensic Mixtures Using Laser Microdissection Techniques Zeiss® PALM® 21

AxioVision® Example Script

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®

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®

TechnologyTransition Workshop

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®

TechnologyTransition Workshop

Carl Zeiss® AxioVision®

High Throughput: Image Recognition Software Interfaces

Physical Separation of Forensic Mixtures Using Laser Microdissection Techniques 26Zeiss® PALM®

TechnologyTransition Workshop

Imaged Sample in AxioVision®

Physical Separation of Forensic Mixtures Using Laser Microdissection Techniques 27Zeiss® PALM®

TechnologyTransition Workshop

Example Script

Physical Separation of Forensic Mixtures Using Laser Microdissection Techniques 28Zeiss® PALM®

TechnologyTransition Workshop

Binary Results of Auto ID

Physical Separation of Forensic Mixtures Using Laser Microdissection Techniques 29Zeiss® PALM®

TechnologyTransition Workshop

Discriminatory Selection

Physical Separation of Forensic Mixtures Using Laser Microdissection Techniques 30Zeiss® PALM®

TechnologyTransition Workshop

Selected Samples Filled

Physical Separation of Forensic Mixtures Using Laser Microdissection Techniques 31Zeiss® PALM®

TechnologyTransition Workshop

Samples Selected for Collection

Physical Separation of Forensic Mixtures Using Laser Microdissection Techniques 32Zeiss® PALM®

TechnologyTransition Workshop

Questions?

Physical Separation of Forensic Mixtures Using Laser Microdissection Techniques 33Zeiss® PALM®

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.