September 2003 Chuck DiMarzio, Northeastern University 10379-5-1 An Example Charles A. DiMarzio...
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![Page 1: September 2003 Chuck DiMarzio, Northeastern University 10379-5-1 An Example Charles A. DiMarzio GEU110 Northeastern University.](https://reader036.fdocuments.us/reader036/viewer/2022081504/56649e4e5503460f94b45375/html5/thumbnails/1.jpg)
September 2003 Chuck DiMarzio, Northeastern University 10379-5-1
An Example
Charles A. DiMarzio
GEU110
Northeastern University
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September 2003 Chuck DiMarzio, Northeastern University 10379-5-2
The Design Process
NeedsAssessment
ProblemFormulation
Abstractionand 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
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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
Nn = 1.39
Cell Body Nc = 1.37
• ~105 Mitocondria per cell
• (0.5µm2X1.5 / 100 µm3 ) x 105 = 4% by volume
•Volume of each cell ~100 µm3
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September 2003 Chuck DiMarzio, Northeastern University 10379-5-4
Taxonomy of 3DFM Microscopy TechniquesDIC TPLSMQTM RCM LSCM
3DFM
Staring Scanning
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September 2003 Chuck DiMarzio, Northeastern University 10379-5-5
Three Biological ModelsMouse Oocytes and Embryos
Zebrafish Neural Stem Cells
Melanoma and Non-Melanoma Skin Cancers
100m Objects 1cm Objects10-100m Cells 5-50m Cells
QTM, DIC, Some Fluorescence Confocal, A Little 2-Photon
Reflectance Confocal, Some Hyperspectral
Fluorescence Confocal
Existing Work:
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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 muscleNeurons
Other
The Embryo-Stem Cell CircleConcepts and Graphics by Carol Warner and Judy Newmark, Northeastern.Biology
Zygote
Oocyte
DIC
DIC
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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
lefteye
right eye
nose
forebrain
Thanks to Don O’Malley Northeastern.Biology
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September 2003 Chuck DiMarzio, Northeastern University 10379-5-8
Skin Cancer Geometries
keratinocytes(RCM, 2h)
melanocytes(RCM)
collagen (2h, SHG, RCM) andelastin (SHG, RCM)
StratumCorneum,5-10m
Epidermis,50-100m
Dermis,few mm
Basal cell cancer (RCM)
Thanks to Milind Rajadhyaksha Northeastern
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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?
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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 egg old unhealthy egg
Thanks to Judy Newmark, Northeastern Biology
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September 2003 Chuck DiMarzio, Northeastern University 10379-5-11
Mitochondrial DistributionsAggregatedUniformly Distributed
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September 2003 Chuck DiMarzio, Northeastern University 10379-5-12
Multi-Cell Embryo
-8
8
100 200 300 400 500 600
50
100
150
200
250
300
350
400
450
Differential InterferenceContrast
QTM UnwrappedPhase, Radians
0
50
100
150
200
250
8
-8
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September 2003 Chuck DiMarzio, Northeastern University 10379-5-13
Confocal Microscopy
PolygonalMirrorScanner
GalvoScanner
Laser
Sample
Detector
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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 10m Voxel), or
– Spatial distribution of mitochondria in an image to derive the above
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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)
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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)
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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.
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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
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September 2003 Chuck DiMarzio, Northeastern University 10379-5-19
3DFM Layout
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September 2003 Chuck DiMarzio, Northeastern University 10379-5-20
Components and ConnectionsTungsten 633
630
636
Hg
QTM
Rcvr
4xGrabobj
ill
tube
Safety Sw
x-yScanner
APD
532 TiSap
488/etc
780
PMT
PMT SetCooled Cam
z scan
Optical CxComputerInterface
Eyepiece
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September 2003 Chuck DiMarzio, Northeastern University 10379-5-21
3DFM Fabrication Timeline
Table, 22 Sept
Ti:Sapphire Laser, 25 Oct Scanner 8 NovMicroscope 15 Dec(Demo Shown Below)
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September 2003 Chuck DiMarzio, Northeastern University 10379-5-22
Status of the Keck 3DFM
qtm
dic
2h
rcmfcm
Thanks to Gustavo Herrera, Northeastern ECE
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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
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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
Biology
Imaging