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Biomedical Imaging Optical Imaging Charles A. DiMarzio EECE–4649 Northeastern University June 2019

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  • Biomedical Imaging

    Optical Imaging

    Charles A. DiMarzio

    EECE–4649

    Northeastern University

    June 2019

  • Optical Imaging

    • Basics; µs, µa, n

    • Optical Instruments: Lens Equation, Magnification

    • Fourier Transform: NA, and more

    • Sources and Detectors

    • Microscopy

    – Brightfield Microscopy

    – Fluorescence

    – Phase Contrast

    – Confocal Microscopy

    – Multi–Photon and Harmonic Microscopy

    • Optical Coherence Tomography

    • Diffusive Optical Tomography

    June 2019 Chuck DiMarzio, Northeastern University 12286..slides5–1

  • Waves Interactions

    (and of course, emission)

    June 2019 Chuck DiMarzio, Northeastern University 12286..slides5–2

  • Skin OpticalProperties

    400 450 500 550 600 650 700 750 80010

    0

    101

    102

    103

    a(e)

    s(e)

    a(d)

    s(d)

    400 450 500 550 600 650 700 750 800

    , Wavelength, nm

    0.7

    0.75

    0.8

    0.85

    0.9

    g(e)

    g(d)

    June 2019 Chuck DiMarzio, Northeastern University 12286..slides5–3

  • Blood and Water

    200 300 400 500 600 700 800 900 1000

    ,Wavelength, nm

    10-4

    10-3

    10-2

    10-1

    100

    101

    102

    103

    104

    a,

    Ab

    sorp

    tio

    n C

    oef

    f, /

    cm

    Oxy in Skin

    Deoxy

    Oxy in Blood

    Deoxy

    Water

    June 2019 Chuck DiMarzio, Northeastern University 12286..slides5–4

  • Light Penetration

    • Best in Near–IR Window

    • Ballistic to 100s of micrometers

    • Diffuse to centimeters

    • Except in the Eye

    June 2019 Chuck DiMarzio, Northeastern University 12286..slides5–5

  • Lenses

    1s +

    1s′= 1f m =

    x′

    x = −s′

    s

    June 2019 Chuck DiMarzio, Northeastern University 12286..slides5–6

  • Optical FourierTransform

    .

    June 2019 Chuck DiMarzio, Northeastern University 12286..slides5–7

  • 2–D FourierTransform Pairs

    A. Aperture B. Airy Function PSF

    C. Gaussian Apodization D. Gaussian PSF

    June 2019 Chuck DiMarzio, Northeastern University 12286..slides5–8

  • Pupil as Low–PassFilter

    A. Knife Edge Object B. Image Slices

    C. Image with Aperture D. Image with Gaussian

    June 2019 Chuck DiMarzio, Northeastern University 12286..slides5–9

  • Resolution

    • Transverse

    fx =u

    λ=

    sin θ cos ζ

    λMAX =

    NA

    λ

    δ =λ

    NA

    • Axial

    δz =λ

    NA2

    • Examples

    NA = 0.95 λ = 500 nm → 526 nm fmax = 1900/mm

    NA = 0.25 λ = 800 nm → 3.2 µm fmax = 312/mm

    June 2019 Chuck DiMarzio, Northeastern University 12286..slides5–10

  • Light Sources

    • Tungsten Lamp (3200K)

    • Quartz–Halogen–Tungsten Lamp (3500K - Melts at 3683K)

    • Mercury Lamp (Some Useful Narrow Lines)

    • Light–Emitting Diode (≈ 20 nm Linewidth)

    • Laser (Pulsed, CW, Narrow, Strong Lines)

    June 2019 Chuck DiMarzio, Northeastern University 12286..slides5–11

  • Detectors

    • Photon Detectors vs. Thermal Detectors

    • Some Vacuum Photomultipliers

    • Mostly Silicon Photon Detectors

    • Arrays

    – Slower

    – Massively Parallel

    – Pixel Size Choices (Resolution, Full Well, etc.)

    June 2019 Chuck DiMarzio, Northeastern University 12286..slides5–12

  • Early Microscopes

    • Compound Microscope (Jansen, 1590)

    • Simple Microscope (m=300) (Leeuwenhoek, early 1600s)

    • Physiological Observation (Hooke 1665)

    • Diffraction Theory (Abbe, 1860)

    • Diffraction–Limited Imaging (Spencer, mid 1880s)

    June 2019 Chuck DiMarzio, Northeastern University 12286..slides5–13

  • Modern Microscopy

    • What’s so Modern?

    Microscopy has been around since 1590. . .

    • . . . But a Lot Has Happened in the Last Few Decades

    • Three Reasons why the Time is Right

    – Illumination Sources (From Tungsten to Lasers, LEDs)

    – Fast, Low–Cost Computers (and Cameras, etc.)

    – Chemistry (Molecular Tags)

    June 2019 Chuck DiMarzio, Northeastern University 12286..slides5–14

  • Microscope Layout

    Fourier Transform Between Field Planes and Pupil Planes

    June 2019 Chuck DiMarzio, Northeastern University 12286..slides5–15

  • Example

    • 10X 0.25 Objective with Green Light

    NA = 0.25 λ = 500 nm → 2 µm

    • Resolution on Camera

    2 µm× 10 = 20 µm

    • Camera Pixel 5 micrometers

    • Point–Spread Function Covers 4 Pixels

    June 2019 Chuck DiMarzio, Northeastern University 12286..slides5–16

  • Sampling with anArray

    • Pixel Pitch vs. Pixel Size

    • Pixel Pitch vs. Object Size

    • Blurring

    • Aliasing

    • Nyquist

    • Anti–Aliasing Filter

    June 2019 Chuck DiMarzio, Northeastern University 12286..slides5–17

  • Sampling Example

    Keeping Nyquist Happy . . .

    0 0.2 0.4 0.6 0.8 1

    t, Time, sec

    -1

    -0.8

    -0.6

    -0.4

    -0.2

    0

    0.2

    0.4

    0.6

    0.8

    1

    V,

    Vo

    lta

    ge,

    Vo

    lts

    f = 10 Hz., fs = 333.3333Hz., = 0 Radians

    0 0.2 0.4 0.6 0.8 1

    t, Time, sec

    -1

    -0.8

    -0.6

    -0.4

    -0.2

    0

    0.2

    0.4

    0.6

    0.8

    1

    V,

    Vo

    lta

    ge,

    Vo

    lts

    f = 10 Hz., fs = 33.3333Hz., = 0 Radians

    . . . or Not

    0 0.2 0.4 0.6 0.8 1

    t, Time, sec

    -1

    -0.8

    -0.6

    -0.4

    -0.2

    0

    0.2

    0.4

    0.6

    0.8

    1

    V,

    Vo

    lta

    ge,

    Vo

    lts

    f = 10 Hz., fs = 16.6667Hz., = 0 Radians

    0 0.2 0.4 0.6 0.8 1

    t, Time, sec

    -1

    -0.8

    -0.6

    -0.4

    -0.2

    0

    0.2

    0.4

    0.6

    0.8

    1

    V,

    Vo

    lta

    ge,

    Vo

    lts

    f = 10 Hz., fs = 16.6667Hz., = 1 Radians

    June 2019 Chuck DiMarzio, Northeastern University 12286..slides5–18

  • Pathology Slide

    Hematoxilyn (Blue) and Eosin (Red)

    Milind Rajadhyaksha

    June 2019 Chuck DiMarzio, Northeastern University 12286..slides5–19

  • Wavelength–ChangingProcesses

    Fluorescence 2–Photon Fluorescence Second Harmonic

    June 2019 Chuck DiMarzio, Northeastern University 12286..slides5–20

  • Fluorescence Imaging

    Gal, OCT4, Dapihttp://www.mediacy.com/index.aspx?page=UManchester stemcellanalysis

    June 2019 Chuck DiMarzio, Northeastern University 12286..slides5–21

  • DIC and Phase

    Epi–Fluorescence with Hoechst Dye, vs. DIC and OQM

    Newmark Microscopy and Microanalysis, 2007

    June 2019 Chuck DiMarzio, Northeastern University 12286..slides5–22

  • Confocal Microscopy

    Trans–Illumination Epi–Illumination (Usual)

    Reflectance or Fluorescence

    Adapted from Milind Rajadhyaksha

    June 2019 Chuck DiMarzio, Northeastern University 12286..slides5–23

  • 2–Galvo System

    June 2019 Chuck DiMarzio, Northeastern University 12286..slides5–24

  • Polygon/GalvoSystem

    June 2019 Chuck DiMarzio, Northeastern University 12286..slides5–25

  • Brightfield Focusing

    In–Focus Image Out–Of–Focus Image

    June 2019 Chuck DiMarzio, Northeastern University 12286..slides5–26

  • Confocal Focusing

    Very High 145µm 150µm 155µm

    160µm 165µm 170µm Very low

    Judy Newmark (Warner Group), Bill Warger

    June 2019 Chuck DiMarzio, Northeastern University 12286..slides5–27

  • Normal Skin

    CRM, Spinous Layer Basal Layer

    H&E, Spinous Layer Basal Layer

    Milind Rajadhyaksha

    June 2019 Chuck DiMarzio, Northeastern University 12286..slides5–28

  • Skin Cancers

    CRM, Nodular BCC Infiltrative BCC

    H&E, Nodular BCC Infiltrative BCC

    Milind Rajadhyaksha

    June 2019 Chuck DiMarzio, Northeastern University 12286..slides5–29

  • Large 3–D Mosaics

    .

    Mouse Embryo at Day 9

    Z–Stack from Confocal Reflectance Microscopy

    −3µm 27µm 84µm 114µmSelected Sample Z Locations from Mosaic

    3200 wide by 4800 high by 160 deep, Decimated for Display

    Irina Larina (Baylor), Kirill Larin (Houston), Joe Kerimo

    June 2019 Chuck DiMarzio, Northeastern University 12286..slides5–30

  • Multi–Modal Slices

    Inverted

    Microscope

    Red: DIC

    Blue:

    Hoechst CFM

    Green: CRM

    Hoechst

    Confocal shows

    nuclei

    Weak CRM deep

    suggests lack of

    ballistic light.

    1. Top (Deep) 2.

    3. 4. Bottom

    June 2019 Chuck DiMarzio, Northeastern University 12286..slides5–31

  • 2–Photon Microscopy

    Huang, UCF

    June 2019 Chuck DiMarzio, Northeastern University 12286..slides5–32

  • 2–P Advantages

    • IR Light to Reduce Photodamage

    • Nonlinearity to Reduce Photodamage

    • IR Light to Increase Penetration

    • No Pinhole (Better Alignment, Better Sectioning)

    • Wide Detector (Collects All Light, including Scattered)

    • Easier Filtering

    June 2019 Chuck DiMarzio, Northeastern University 12286..slides5–33

  • Melanin 3–P

    Before Activation After Activation

    Kerimo Photochemistry and Photobiology, 2011

    June 2019 Chuck DiMarzio, Northeastern University 12286..slides5–34

  • Collagen Fibrils inSHG

    • Long–Range Goal: Understand Organization Under Load

    • Current Goal: Measure Organization in Cornea

    Thanks to Yair Mega, Mike Robitaille, Ramin Zareian

    Collaboration with Kai–Tak Wan and Jeff Ruberti

    June 2019 Chuck DiMarzio, Northeastern University 12286..slides5–35

  • Collagen FibrilOrganization

    June 2019 Chuck DiMarzio, Northeastern University 12286..slides5–36

  • 2–Photon vs. SHG

    2–Photon SHG

    λem > λex/2 λem = λex/2Depends on λem Less Dependent on λemExponential Time Decay Instantaneous

    Random Direction Forward Direction

    Unpolarized (Maybe) Polarized

    June 2019 Chuck DiMarzio, Northeastern University 12286..slides5–37

  • Optical CoherenceTomography

    • Short Coherence Source

    – Super–Luminescent

    Diode

    – Ti:Sap Laser

    – Other

    • M1 is Reference

    • Moving Reference Mirror

    • M2 is Target

    • Interference? Compare. . .

    – Path Difference

    – Coherence Length

    • Michaelson Interfereometer

    g

    June 2019 Chuck DiMarzio, Northeastern University 12286..slides5–38

  • OCT Signals

    • Examples with Partial Reflectors

    • Air–Glass Interfaces (Simulated Signals)

    • Idea Extends to Thick “Distributed” Targets

    A. Target at Zero B. Added target at 8 µm

    June 2019 Chuck DiMarzio, Northeastern University 12286..slides5–39

  • Lung Images (OCT)

    Initial Lung Image with Transparent Probe (Detail)

    Partially Indented Lung Image (Detail)

    Scale Bar 300 µm

    Andrew Gouldstone, Maricris Silva, MIE Ph.D. 2011

    June 2019 Chuck DiMarzio, Northeastern University 12286..slides5–40

  • Bubble Phantom

    Golabchi, Biomedical Optics Express, 2012

    June 2019 Chuck DiMarzio, Northeastern University 12286..slides5–41

  • Diffusive Imaging

    June 2019 Chuck DiMarzio, Northeastern University 12286..slides5–42

  • DOT and Ultrasound

    June 2019 Chuck DiMarzio, Northeastern University 12286..slides5–43

  • Some Safety Issues

    • Chemical Toxicity

    • Light Toxicity

    – Photochemical

    – Thermal

    • Issues for Patient and Operator

    June 2019 Chuck DiMarzio, Northeastern University 12286..slides5–44

  • Summary

    • Imaging with Light Offers

    – Imaging Deep in the Body

    – Imaging with Sub–Micrometer Resolution

    – Non–Invasive Imaging

    June 2019 Chuck DiMarzio, Northeastern University 12286..slides5–45

  • Summary

    • Imaging with Light Offers

    – Imaging Deep in the Body

    – Imaging with Sub–Micrometer Resolution

    – Non–Invasive Imaging

    • Pick Any Two

    June 2019 Chuck DiMarzio, Northeastern University 12286..slides5–46