1. September 2003 Chuck DiMarzio, Northeastern University 10379-5-1 An Example Charles A. DiMarzio GEU110 Northeastern University

2. September 2003 Chuck DiMarzio, Northeastern University 10379-5-2 The Design Process Needs Assessment Problem Formulation Abstraction and Synthesis Analysis Implementation Ch. 2 3, 4, 5 6,7 8,9,10 11 Remember these phases are not absolute The edges are rough We often use multiple loops Usually we don’t think about the process at all It’s best taught by examples

3. September 2003 Chuck DiMarzio, Northeastern University 10379-5-3 Optical Components of Pre- Implantation Embryos Mitochondria ~ 1.5 µm x 0.5 µm x 0.5 µm Nuclear Membrane Cell Membrane ≤ 100 nm Nucleus ~10 µm dimension N n = 1.39 Cell Body N c = 1.37 ~10 5 Mitocondria per cell (0.5µm 2 X1.5 / 100 µm 3 ) x 10 5 = 4% by volume Volume of each cell ~100 µm 3

4. September 2003 Chuck DiMarzio, Northeastern University 10379-5-4 Taxonomy of 3DFM Microscopy Techniques DICTPLSMQTMRCMLSCM 3DFM StaringScanning

5. September 2003 Chuck DiMarzio, Northeastern University 10379-5-5 Three Biological Models Mouse Oocytes and Embryos Zebrafish Neural Stem Cells Melanoma and Non-Melanoma Skin Cancers 100  m Objects 1cm Objects 10-100  m Cells 5-50  m Cells QTM, DIC, Some Fluorescence Confocal, A Little 2-Photon Reflectance Confocal, Some Hyperspectral Fluorescence Confocal Existing Work:

6. September 2003 Chuck DiMarzio, Northeastern University 10379-5-6 E:\images\02.10.17\blastocyst1 Embryonic Stem (ES) Cells 2-cell 8-cell Morula (16-cell) Blastocyst Skin Blood Bone Cardiac muscle Neurons Other The Embryo-Stem Cell Circle Concepts and Graphics by Carol Warner and Judy Newmark, Northeastern.Biology Zygote  Oocyte DIC

7. September 2003 Chuck DiMarzio, Northeastern University 10379-5-7 α1-tubulin/GFP expressing transgenic zebrafish larva M. Beverly & I. Zhdanova, unpublished data transgenic line courtesy of D. Goldman; U. Mich. in Transgenic Research 10:21-33, 2001. Olfactory Placodes left eye right eye nose forebrain Thanks to Don O’Malley Northeastern.Biology

8. September 2003 Chuck DiMarzio, Northeastern University 10379-5-8 Skin Cancer Geometries keratinocytes (RCM, 2h ) melanocytes (RCM) collagen (2h, SHG, RCM) and elastin (SHG, RCM) Stratum Corneum, 5-10  m Epidermis, 50-100  m Dermis, few mm Basal cell cancer (RCM) Thanks to Milind Rajadhyaksha Northeastern

9. September 2003 Chuck DiMarzio, Northeastern University 10379-5-9 Some Questions About Embryos Where are the mitochondria? Multi-Cell: How many cells in the Inner Cell Mass?

10. September 2003 Chuck DiMarzio, Northeastern University 10379-5-10 Fluorescence Confocal Images Plan to Do Full Z, Other Scanning Modes, and Fuse with Staring Modes young healthy eggold unhealthy egg Thanks to Judy Newmark, Northeastern Biology

11. September 2003 Chuck DiMarzio, Northeastern University 10379-5-11 Mitochondrial Distributions AggregatedUniformly Distributed

12. September 2003 Chuck DiMarzio, Northeastern University 10379-5-12 Multi-Cell Embryo -8 8 100200300400500600 50 100 150 200 250 300 350 400 450 Differential Interference Contrast QTM Unwrapped Phase, Radians 8 -8

13. September 2003 Chuck DiMarzio, Northeastern University 10379-5-13 Confocal Microscopy Polygonal Mirror Scanner Galvo Scanner Laser Sample Detector

14. September 2003 Chuck DiMarzio, Northeastern University 10379-5-14 Mitochondrial Distribution Data Requirements Biology goal is to determine whether mitochondria are perinuclear, uniformly distributed, or aggregated. Therefore we want to determine either; –Statistical Properties; Size distribution of clumps vs. individual mitochondria, (Per 10  m Voxel), or –Spatial distribution of mitochondria in an image to derive the above

15. September 2003 Chuck DiMarzio, Northeastern University 10379-5-15 Mitochondrial Distribution Measurement (1) Fluorescence Confocal with Mitotracker Green FM –Proven Technique –Have 2-D data, may be able to get z stacks Reflectance Confocal –Have two 3-D data sets at 1  m lateral by 3  m axial resolution with images spaced 3  m apart in the axial direction –Problems are speckle (average speckle size and mitochondria are both equal to lateral resolution) and clutter from other organelles QTM –Probably best detected by examining diffraction –Need to figure out how to scan (need a model)

16. September 2003 Chuck DiMarzio, Northeastern University 10379-5-16 Mitochondrial Distribution Measurement (2) 2h –Coming when 3DFM is assembled –Use Mitotracker CMXRos at 1156 Excitation –or NADH at 730 –Processing same as Fluorescence Confocal –Probably biggest problem will be low SNR (quantum noise)

17. September 2003 Chuck DiMarzio, Northeastern University 10379-5-17 Cell Counting Data Requirements Biology rationale is that the growth rate of cells in the inner cell mass (ICM) is an indication of health of the embryo Therefore we want to count the cells in the inner cell mass, from 1 through 64. Note: counting Nuclei is easier –Boundaries between cells are not well defined in the inner cell mass and thus harder to detect.

18. September 2003 Chuck DiMarzio, Northeastern University 10379-5-18 Cell Counting Approaches Fluorescence Confocal with Hoechst Dye and UV Excitation to count the nuclei –Limited data avalable Reflectance Confocal to count nucleii –May validate Fluorescence, but edges of nucleii are not sharp QTM to actually count the cell bodies –Data available and we can collect more Can do z stacks, but need to know how to scan –Later can do tomographic imaging

19. September 2003 Chuck DiMarzio, Northeastern University 10379-5-19 3DFM Layout

20. September 2003 Chuck DiMarzio, Northeastern University 10379-5-20 Components and Connections Tungsten 633 630 636 Hg QTM Rcvr 4x Grab obj ill tube Safety Sw x-y Scanner APD 532TiSap 488/etc 780 PMT PMT Set Cooled Cam z scan Optical Cx Computer Interface Eyepiece

21. September 2003 Chuck DiMarzio, Northeastern University 10379-5-21 3DFM Fabrication Timeline Table, 22 Sept Ti:Sapphire Laser, 25 Oct Scanner 8 Nov Microscope 15 Dec (Demo Shown Below)

22. September 2003 Chuck DiMarzio, Northeastern University 10379-5-22 Status of the Keck 3DFM qtm dic 2h rcm fcm Thanks to Gustavo Herrera, Northeastern ECE

23. September 2003 Chuck DiMarzio, Northeastern University 10379-5-23 The Team Biology –Warner, Newmark, O’Malley, Rajadhyaksha Hardware Engineering –DiMarzio, Rajadhayksha, Townsend, Katkar, Herrera Phantoms –Rockward, Quarles, Thomas Models –Rappaport, Morgenthaler, Dunn, DiMarzio, Hollman Computation –Kaeli, Meleis Signal Processing –Brooks, Miller, Karl, McKnight, Smith

24. September 2003 Chuck DiMarzio, Northeastern University 10379-5-24 Who Do We Need? Good Engineers –Electrical E/M and Optics Controls Computers –Mechanical Good Biologists Good Bio-Engineers? Bio-Imaging of Embryos BiologyBiology ImagingImaging