Technology Transition Workshop Jack Garner. Carl Zeiss PALM. Technology Transition Workshop

Size: px

Start display at page:

Download "Technology Transition Workshop Jack Garner. Carl Zeiss PALM. Technology Transition Workshop"

Bonnie McDowell
6 years ago
Views:

1 Jack Garner Carl Zeiss PALM

2 Laser Microdissection & Pressure Catapulting (LMPC) New horizons for microgenomics, proteomics and live cell micromanipulation Laser capture microscopy The technology Forensic science application 2

3 The New Carl Zeiss PALM MicroBeam 3

4 The Next Generation of Laser Microdissection 4

5 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 Absorbance Spectra DNA PALM UV-A 355 nm Protein nm 5

6 Optical Density of DNA and Protein FTSS Laser 355 nm 6

7 Incise with light, transport with light, work with light Non-contact manipulation by the force of focused light! 7

8 PALM Laser Microdissection Focal spot <1 µm Nd-Yag UV-A Laser (355 nm; >90 µj; Hz) Photodecomposition: UV laser energy in focal volume causes breakdown of molecular bonds without lateral heat transfer Srinivasan R. Ablation of Polymers and Biological Tissue by Ultraviolet Lasers. Science 234 (4776) (October 1986):

9 How Does LMPC Work? ZEISS LD Plan- NEOFLUAR 40x / ZEISS LD Plan- NEOFLUAR 40x / Laser Microdissection Laser Pressure Catapulting 9

Ablation of Polymers and Biological Tissue by Ultraviolet Lasers.

10 PALM Laser Microdissection Laser Plasma Formation: An adiabatic process (no lateral energy transfer during formation & collapse of laser plasma) Srinivasan R. Ablation of Polymers and Biological Tissue by Ultraviolet Lasers. Science 234 (4776) (October 1986): To see a video depiction of this principle in action, go to: 10

11 The PALM MicroBeam LMPC Method Laser microdissection pressure catapulting Contamination-free Non-contact Against gravity 3 2 LD Plan-NEOFLUAR 40x / ZEISS 11

12 The Next Generation: PALM MicroBeam IV 12

13 Tissue Microdissection from Membrane and Glass Slides Laser Microdissection from Membrane-mounted Tissue Membrane Mounted Tissue Laser Microdissection from Glass-mounted Tissue 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 Object Slide Glass-mounted Tissue Can be used on archival material Unique PALM feature 13

14 Tissue Microdissection from Membrane and Glass Slides Laser Microdissection from Membrane-mounted Tissue Laser Microdissection from Glass-mounted Tissue 14

pressure catapulting provides increased efficiency under normal laboratory

15 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 15

16 Laser Microdissection: Understanding the Complete Forensic Workflow Specimen Preparation and Selection PALM MicroBeam Enabling Functional Downstream Analysis Forensic Specimen MicroBeam DNA Fingerprint Analysis 16

17 Contact-free Collection of Forensic Evidence with PALM MicroBeam Adhesive tape Sperm cells Epithelial cells Hair DNA fingerprinting 17

18 Forensic Evidence Hair 18

19 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 19

20 Fluorescence 20

21 PALM Sample Selection and Identification AxioVision Example Script 21

22 High Throughput: Image Recognition Software Interfaces Carl Zeiss AxioVision Goal Example: Selection of Nuclei in Liver Tissue 22

staining/morphology Reject particles of wrong morphologies Prepare

23 High Throughput: Image Recognition Software Interfaces Carl Zeiss AxioVision 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 Scrap Dilation 23

24 High Throughput: Image Recognition Software Interfaces Carl Zeiss AxioVision 24

25 High Throughput: Image Recognition Software Interfaces Carl Zeiss AxioVision 25

26 High Throughput: Image Recognition Software Interfaces Carl Zeiss AxioVision 26

27 Imaged Sample in AxioVision 27

28 Example Script 28

29 Binary Results of Auto ID 29

30 Discriminatory Selection 30

31 Selected Samples Filled 31

32 Samples Selected for Collection 32

33 Questions? 33

34 Contact Information Jack Garner PALM Microlaser Technologies Carl Zeiss MicroImaging Group All images courtesy of Carl Zeiss unless otherwise noted. 34

Bosch Institute Advanced Microscopy Facility Workshops Summer/Autumn 2016

Bosch Institute Advanced Microscopy Facility Workshops Summer/Autumn 2016 Presented by Dr Louise Cole and Dr Cathy Payne, Advanced Microscopy Facility, Bosch Institute, School of Medical Sciences, The