BME 101 Biomedical Optics and Lasers

Instructor: Irene Georgakoudi

TA: Cherry GreinerClass meeting time: T/Th: 10:30 -11:45 AM

Office hours: Tuesdays, 3:00-4:30 PM

Blackboard site: http://blackboard.tufts.eduReading material on reserve (Tisch):

Handbook of Biomedical Photonics, Tuan Vo Dinh

Introduction to Biophotonics, Prasad

Biological spectroscopy, Campbell

Electo-optics library: Optics, Hecht

Evaluation• 10% Class participation• 30% Homeworks/Lab reports (due every Tuesday)

• 15% Midterm exam• 25% Final exam• 20% Final paper and presentation

– 10-15 page paper on specific molecular imaging method and its impact on a clinical problem

» Introduction» Theoretical background of method» Background on clinical problem» Instrumentation» Methods/Results» Advantages/Disadvantages» Suggested improvements

What is biomedical optics?

• Biomedical optics is typically defined as the area of study of methods/technologies based on the use of visible light (applications cover UV-NIR) for:– Improving basic understanding of biological

processes (from gene to tissue level)– Enhancing the detection and treatment of

human diseases (from acne to atherosclerosis and cancer)

Syllabus

• Basic principles

• Spectroscopic methods

• Microscopy and Imaging

• Photodynamic Therapy/Flow Cytometry

Basic Principles

• Light matter interactions– Basic wave definitions– Schrodinger’s equation

-Bonds and orbitals

-Biological chromophores

kzttz o cos,

Basic Principles

• Laser basics– Principles of operation

• Stimulated emission• Critical inversion• Pumping schemes

– Major laser components– Laser beam properties– Diode lasers

Cell and Tissue basics• Cell basics

– Major cellular components– Origins of intrinsic cellular

optical signals

• Tissue basics– Major epithelial types– Connections to disease and optical

sources of contrast

Spectroscopic methods

• Absorption• Scattering• Fluorescence

Epithelium

Connective Tissue

Spectroscopic methods• Basic Theoretical principles

• Instrumentation

• Applications

),(3),(),(),(),(1

12 trSDtrStrtrDtr

tc oa

Source

Polarizer

Filters

BeamSplitter

CCD

Tissue

Polarizer

Mirror

0 0.5 1 1.5

0

0.5

1

1.5

EnlargedNuclei, %

mm

mm40-50

30-40

20-30

10-20

Non-dysplasticmucosaAdenoma

AdenomaAdenoma

NormalNormal

0 2 4 6 8 10

20

18

16

14

12

10

8

6

4

2

0y (cm)

x (c

m)

Colon cancer

Breast cancer

800 1000 1200 1400 1600 1800

-0.5

0

0.5

1

Raman Shift (cm-1)

Inte

nsi

ty (

a.u

.)

DataFitResidual

atherosclerosis

Microscopy and Imaging• Basic Principles

4-Pi microscopyMitochondrial network of live bacterial cell

80 nm res

From organelles to cells

Triple stained endothelialCell of pulmonary artery

From

cel

ls

to ti

ssue

s

Engineered tissue:Fibroblast (red) in collagen matrix (green)Endogenous signal

From

tissues to

animals

Tumors and blood vessels imaged in vivo

Confocal in vivo H&E “En face” SECTION of human skin

From

anim

als to

hum

ans

Optical imaging of cell-matrix interactions

Excitation wavelength: 488 nmGreen channel: 485-490 nm (scattering)Red channel: 500-620 nm (GFP fluorescence)Stack size: 238x238x125 mImages acquired using 63X, 1.2 NA, water immersion objective

Collagen gel embedded with GFP-expressing fibroblasts

Leica TCS SP2 confocal microscope

Photodynamic Therapy

• Basic Principles

• Applications

1O2

3O2

C ollisionalQuenching

Ground StateTriplet Oxygen

Excited stateSinglet oxygen

Type II: Oxygen radicals

Type I: Free radicals

C YTOTOXIC ITY

Ground State So

Exited SingletState S1

Excited Triplet State T1A

bsorption

Fluorescence

Phosphorescence

1O2

3O2

C ollisionalQuenching

Ground StateTriplet Oxygen

Excited stateSinglet oxygen

Type II: Oxygen radicals

Type I: Free radicals

C YTOTOXIC ITY

Ground State So

Exited SingletState S1

Excited Triplet State T1A

bsorption

Fluorescence

Phosphorescence

Maculardegeneration

Flow Cytometry

• Basic Instrumentations

• Advanced Methods

• Reading assignment:

• Introduction to biophotonics, ch. 1 and 2.1

• Lecture notes

• Also posted on the blackboard site:– Calculus review– Electromagnetic waves review

Biomedical opticsExploiting interactions of light with matter

Wavelengths used typically: 300-900 nm

Why biomedical optics?

• Major advantages: non-invasive; high resolution• Continuous or repetitive monitoring• Study/characterize process/disease in natural

environment (no artifacts)• More sensitive/accurate monitoring• Real-time information

– Triage with therapy– Accurate dosimetry– Psychological impact

A bit of optics history

It all started with the Greeks…

Plato (427-347 BC)

Believer of extramission theory: Eye emits a “fire” providing man the capability of vision by seizing objects

It all started with the Greeks…

Aristotle (384-322 BC)

Light emitted by a source is captured by the eyes when reflected by an object

• Euclid (circa 325-265 BC)• Treatise entitled

“Catoptrics”• Foundations of geometric

optics• First law of reflection

It all started with the Greeks…

Galen of Pergamum (Claudius Gelenus: 130-201 AD)

• Described anatomical details of the eye

• Identified lens as principle eye instrument

• Believed in extramission theory

It all started with the Greeks…

Philosophers from the Middle-East followed…

Mohammad ibn Zakariya al-Razi (864-930AD)

• Also known as Rhazes • Observed that pupil

contracts in response to light

Philosophers from the Middle-East followed…

Abu Ali al-Hasan ibn al-Haytham (965-1040 AD)

• Also known as Alhazen• Considered by some as the father of

Optics• Wrote comprehensive treatise on

optics (Katib-al-Manazir/Book on Optics), translated in Latin in 1270– Proved that extramission theory is not

correct– Detailed description of human eye – Theory of vision which prevailed until

17th century– Discussed primary and secondary light

sources, light propagation and colors– Studied spherical and parabolic mirrors– Laws of reflection and refraction

Western philosophers/scientists

Leonardo da Vinci (1452-1519 AD)

• Initially believed in extramission, but later changed his view in support of external light sources based on experiments he performed with ox eyes

Western philosophers/scientists

Johannes Kepler (1571-1630)

• Established retinal image formation theory based on experiments with ox eyes

• Law of refraction for small angles of incidence

Theories on nature of light:Light as a particle vs. Light as a wave

• Only corpuscular theory of light prevalent until 1660

• Francesco Maria Grimaldi (Bologna) described diffraction in 1660

Light as a particle

Sir Isaac Newton (1642-1727)• Embraces corpuscular theory of

light because of inability to explain rectilinear propagation in terms of waves

• Demonstrates that white light is mixture of a range of independent colors

• Different colors excite ether into characteristic vibrations---sensation of red corresponds to longer ether vibration

Light as a wave

Christiaan Huygens (1629-1695)Huygens’ principle (Traite de la

Lumière, 1678):Every point on a primary

wavefront serves as the source of secondary spherical wavelets, such that the primary wavefront at some later time is the envelope of these wavelets. Wavelets advance with speed and frequency of primary wave at each point in space

http://id.mind.net/~zona/mstm/physics/waves/propagation/huygens1.html

Light as a wave

Thomas Young (1773-1829)

1801-1803: double slit experiment, showing interference by light from a single source passing though two thin closely spaced slits projected on a screen far away from the slits

http://vsg.quasihome.com/interfer.htm

Light as a waveAugustine Fresnel (1788-

1827)

1818: Developed mathematical wave theory combining concepts from Huygens’ wave propagation and wave interference to describe diffraction effects from slits and small apertures

Electromagnetic wave nature of light

• Michael Faraday (1791-1865)

• 1845: demonstrated electromagnetic nature of light by showing that you can change the polarization direction of light using a strong magnetic field

Electromagnetic theory

• James Clerk Maxwell (1831-1879)

• 1873: Theory for electromagnetic wave propagation

• Light is an electromagnetic disturbance in the form of waves propagated through the ether

Quantum mechanics• 1900: Max Planck postulates that

oscillating electric system imparts its energy to the EM field in quanta

• 1905: Einstein-photoelectric effect– Light consists of individual energy quanta, photons,

that interact with electrons like particle• 1900-1930 it becomes obvious that concepts

of wave and particle must merge in submicroscopic domain

• Photons, protons, electrons, neutrons have both particle and wave manifestations– Particle with momentum p has associated

wavelength given by p=h/• QM treats the manner in which light is

absorbed and emitted by atoms

Max Planck

Niels Bohr

Louis de Broglie

Schrödinger

Heisenberg

Wave definitions

Classical Description of Light

Wave Equation (derived from Maxwell’s equations)

Any function that satisfies this eqn is a wave

It describes light propagation in free space and in time

operatorLaplacian

fieldinductionmagnetic

fieldelectricE

lightofspeedc

wheretc

t

E

cE

2

2

2

22

2

2

22

,

1

1

B

BB

(see calculus review handout)

Classical Description of Light Plane Wave Solution

One useful solution is for plane wave

frequencyangular

vectornpropagatioornumberwavek

where

eBB

eEeeEEtrki

o

trkio

tirkio

,

E

B

r

Classical description of lightConsidering only the real part of the previous solution to make things simpler, we have for the electric field propagating along one dimension, z

kzttz o cos,

(or distance)

0

Period

time

angle) to timeconverts(2

2

)angle todistance converts(2

)/(1

)(sec

)(

frequencyangular

wavenumberk

Hzorscyclesfrequencyc

ondsperiod

meterswavelength

Light as a wave: Basic concepts• Phase of a wave is the offset of

the wave from a reference point o

• We typically talk about a phase shift

• When light interacts with matter (e.g. as it travels through a biological specimen), its speed of propagation slows down. The wave emanating from the specimen exhibits a phase shift when compared with the initial wave

• The refractive index , n, and the thickness of a specimen determine by how much the wave is retarded

Green-incident waveBlue-wave after passing through specimen shifted by /4

in vacuumlight of speed where, cc

n

Phase==t-kz

Monochromatic (only onewavelength/frequency)waves traveling in phase

Monochromatic (only onewavelength/frequency)waves traveling out of phase

Phase==t-kz

Coherent Light

Incoherent Light

Incoherent Light

Constructiveinterference

Destructiveinterference