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

September 2003 Chuck DiMarzio, Northeastern University 10379-5-1

An Example

Charles A. DiMarzio

GEU110

Northeastern University


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


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


Taxonomy of 3DFM Microscopy TechniquesDIC TPLSMQTM RCM LSCM

3DFM

Staring Scanning


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:


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


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M. Beverly & I. Zhdanova, unpublished data transgenic line courtesy of D. Goldman; U. Mich.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


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


Some Questions About Embryos• Where are the

mitochondria?• Multi-Cell: How

many cells in the Inner Cell Mass?


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


Mitochondrial DistributionsAggregatedUniformly Distributed


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


Confocal Microscopy

PolygonalMirrorScanner

GalvoScanner

Laser

Sample

Detector


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


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)


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)


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.


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


3DFM Layout


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


3DFM Fabrication Timeline

Table, 22 Sept

Ti:Sapphire Laser, 25 Oct Scanner 8 NovMicroscope 15 Dec(Demo Shown Below)


Status of the Keck 3DFM

qtm

dic

2h

rcmfcm

Thanks to Gustavo Herrera, Northeastern ECE


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


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

Documents

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