september 2003 chuck dimarzio, northeastern university 10379-5-1 an example charles a. dimarzio...

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

An Example

Charles A. DiMarzio

GEU110

Northeastern University

September 2003 Chuck DiMarzio, Northeastern University 10379-5-2

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

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

September 2003 Chuck DiMarzio, Northeastern University 10379-5-4

Taxonomy of 3DFM Microscopy TechniquesDIC TPLSMQTM RCM LSCM

3DFM

Staring Scanning

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:

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

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M. Beverly & I. Zhdanova, unpublished data transgenic line courtesy of D. Goldman; U. Mich.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

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

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?

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

September 2003 Chuck DiMarzio, Northeastern University 10379-5-11

Mitochondrial DistributionsAggregatedUniformly Distributed

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

September 2003 Chuck DiMarzio, Northeastern University 10379-5-13

Confocal Microscopy

PolygonalMirrorScanner

GalvoScanner

Laser

Sample

Detector

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

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)

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)

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.

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

September 2003 Chuck DiMarzio, Northeastern University 10379-5-19

3DFM Layout

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

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)

September 2003 Chuck DiMarzio, Northeastern University 10379-5-22

Status of the Keck 3DFM

qtm

dic

2h

rcmfcm

Thanks to Gustavo Herrera, Northeastern ECE

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

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