Cris Lecture1

7/29/2019 Cris Lecture1

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Digital Imaging Systems

Light Microscopyconventional optical microscopes,contrast techniques, fluorescence

Cris [email protected]


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Microscopy:

Optical Microscopy (visible light)

Micro-CT (X-ray)

Electron Microscopy (electron beam)

Atomic Force Microscopy (no radiation)

Uses:

Biology

Crystallography

Material Sciences

Engineering

Introduction1st CompoundMicroscope(ca 1595)

van LeeuwenhoekMicroscope(late 1600s)

Galileo Microscope(late 1600s)


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Contents

The Bright Field Microscope

Dark Field Microscopy

Phase Contrast Microscopy

Differential Interference Contrast Microscopy

Structured Illumination

Fluorescence

The Epifluorescence Microscope

Spectral Unmixing


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Simplest, oldest form of microscopy

All other microscopy techniques we will see today (and mostwe'll see tomorrow) are implemented by adding opticalelements into the bright field microscope's optical path

Uses wide-field illumination (vs spot illumination)

Contrast obtained by absorption

Less transparent regions seen darker


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eye

ocular

a.k.a. eyepiece

objective

sample

intermediateimage plane

imageplane

lenses typically arecompound lenses,not a single lens

light


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projectionlensobjective

sample


imageplane

CCD

light


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Infinity Corrected Optics

projectionlensobjective

sample


imageplane

CCD

light

afocal

space

tubelens


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Khler IlluminationIlluminating your

samples since 1893

lampcollector

lenscondenser

lens

samplecondenserdiaphragm

oraperture

diaphragm

field

diaphragm

Note how each point of the light source illuminates the whole sample.This makes for a very uniform illumination.Improved uniformity often accomplished with frosted glass over the lamp.


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Putting It All Together

projectionlens

sample imageplane

CCD

afocal

space

collectorlens

field

diaphragm

condenserdiaphragm

objectivetubelens

condenser

intermediate

image plane

lamp


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Putting It All Together

projectionlens

objective

sample imageplane

CCD

afocal

space

tubelenscollector

lens condenser

field

diaphragm

objectiveback focal

plane

condenserdiaphragm

lamp


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Old, but Still Current!


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The Point Spread Function

Rayleigh's inequality for resolving power: r 1.22 / 2 NA

psfr =2 J1 2NA / r 2NA / r 2log (psf)

For a diffraction-limited microscope:

NA = n sin()

refractiveindex ofmedium

objective

working distance


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Spherical Aberration

When the lens curvature deviates from the ideal, light going

through different parts of the lens focuses at different distancesfrom the lens

There is no perfect focus point : loss of resolution

Correction always takes into account the cover slip

slide

cover slip

different cover slip thicknesswill cause spherical aberration!


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Chromatic Aberration

A normal lens focuses each wavelength at a different spot

Compare to a prism

Chromatic corrections: achromat, fluorite, apochromat

Using different materials: crown glass, flint glass, fluorspar

normal lens achromatic lens


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Field of View Flatness

normal lens plan lens

Field curvature corrected lenses are able to keep the whole

field of view in focus

Very important for quantitative microscopy even moreimportant for 3D microscopy!


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cover slip (n = 1.50 - 1.54)

immersion oil (n = 1.51)

Immersion Media

cover slip (n = 1.50 - 1.54)

air (n = 1.00)

also used: water (n = 1.33)


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Objectives

pixel size < M 1.22 / 2 NA

minimum CCD sampling density:

x60

0.37m

22m

1 pixel < 11m


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Objective


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Tiny modification to the bright field setup

Contrast obtained by diffraction


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projectionlens

objective

sample imageplane

CCD

tubelenscollector

lens

fielddiaphragm


condenserdiaphragm

lamp

light stop at condenser diaphragmstops all light that would godirectly into the objective

light scatters at sample

objective collectsonly scattered light

condenser


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Yet another small modification to bright field setup

Contrast obtained by change in phase


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Phase Contrast MicroscopyZernike, Nobel

Prize 1953

projectionlens

objective

sample imageplane

CCDtubelens

condenser

phase ring lets only a ring of lightthrough to illuminate the sample

direct light gets delayed by (90 degree phase change)

light scattered at sampleis not delayed

light interferes

intensity object

phase object

constructive

destructive

interference

phaseplate

phasering


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Delay or advance phase with ?


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DIC: Differential Interference Contrast Microscopy

A slightly more difficult modification

Contrast obtained by difference in phase change

That is: optical gradients converted to intensity differences

Produces fake 3D effect

Orientation-dependent


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On Polarization and Wollaston Prisms

Birefringent crystal (quartz or calcite)

linear

elliptic

circular


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DIC: Differential Interference Contrast Microscopy

projectionlens

sample imageplane

CCD

afocal

space

collectorlens

fielddiaphragm

condenserdiaphragm

objective

tubelens

polarizer analyzer

Wollastonprism Wollaston

prismlamp

unpolarizedlinearly

polarized45 deg.

0 deg. and 90 deg.polarized light

separated spatially

ellipticallypolarized

linearlypolarized

the two perpendicular components aredelayed in phase differently

condenser


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Structured Illuminationimage in

frequency

domain

uniformilluminationcorresponds

to impulsein FD

sinusoidalilluminationcorresponds

to shiftedimpulse in FD


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normal area ofFD imaged inmicroscope

multiplication

in imagedomain isconvolutionin frequency

domain

normal field ofview of frequencies

is shifted

new area of frequencydomain becomes visible

resolution can beincreased to aboutdouble the system's


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from: Stefan W. Hell, Nature Biotechnology 21:1347-1355, 2003.


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Fluorescence Microscopy


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Fluorescence Microscopy

The use of fluorescence in microscopy is the most important

innovation to quantitative biology since the invention of themicroscopy itself

Images show only location of fluorescent markers, all non-stained tissue remains perfectly transparent

Perfect when measuring the extent of stained tissues or organelles

Amount of fluorescence can be measured accurately

Direct relation between amount of fluorescence and number of proteins

l


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Fluorescence

FITC absorption & emission

Stokes shift

Fluorescence emission is alwaysa longer wavelength (less energy)than the absorption (or excitation)

The difference is the Stokes shift

Jablonski energy diagram

electronabsorbsphoton

electronrelaxesand emitsphoton

Fl h


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Fluorophoresfluoresceinderivatives

cy3

cy5

rhodaminederivatives

rhodamine greenrhodamine phalloidin

cyaninederivatives

(e.g. FITC)

Most common fluorophores:

1. Small molecules (this slide)2. Fluorescent proteins

3. Quantum dots

Ph t Bl hi d Ph t T i it


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Photo-Bleaching and Photo-Toxicity

Fluorescent molecules can loose their fluorescence ability

An excitation/emission cycle has a fixed probability of causing the loss offluorescence

Molecule reacts with something in its environment (usually oxygen), andconverts into a non-fluorescent molecule

Limit light exposure to avoid photobleaching

Fluorescent molecules can kill the cells stained with it

Fluorescence excitation can result in free oxygen radicals

Free radicals kill the cell because they are chemically highly reactive

Excitation light can also destroy the specimen through heattransfer

G Fl t P t i


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Green Fluorescent ProteinM. Chalfie, O. Shimomura, R.Y. Tsien

2008 Nobel Prize in Chemistry

man-made GFP derivatives:EGFP (enhanced GFP)

BFP (Blue)

CFP (Cyan)

YFP (Yellow)

mCherry

mRaspberrymPlum

mBanana

mStrawberry

dTomato

Q t D t


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Quantum Dots

QDot 525

QDot 565QDot 605QDot 655QDot 705

~20 atoms diameter



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projectionlens

sample imageplane

CCD

collectorlens

fielddiaphragm

condenserdiaphragm

objective

tube lens

lamp

objective works also as condenseremission filter

excitation filter

dichroic

field lens

The Filter Cube


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The Filter Cube

shorter wavelengths are reflectedselected wavelengths are transmitted

long pass: and longer are transmittedband pass: dare transmitted

filterdichroic

there also exist mirrors that reflectonly a small band of wavelengths

white light in

excitation light

fluorescenceemissionlight out

emission



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Autofluorescence


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Autofluorescence

A single optical section from a thicksection of human skingreen: anti-basal lamina proteinred: neuronal processescollagen and elastin autofluoresce in blue(source: zeiss.com)

autofluorescencein fruit fly

pollen is strongly

autofluorescent

Cross Talk


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Cross-Talk

blue green red

red

g

reen

red

g

reen

Spectral Unmixing


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Spectral Unmixing

stained with 3 different red dyes...

705 nm QDot (shown green)food autofluorescence (red)

skin autofluorescence (white)

By imaging many channels

(hyperspectral image), it ispossible to distinguishoverlapping emission spectra

source: www.cri-inc.com

Further Reading


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Further Reading

Microscopy U (Nikon)

http://www.microscopyu.com/

Microscopy Primer (Molecular Expressions)

http://microscopy.fsu.edu/

Olympus Microscopy Resource Center

http://www.olympusmicro.com/

The Handbook (on fluorescent probes)

http://probes.invitrogen.com/handbook/

Optical Microscopy

M.W. Davidson & M. Abramowitz, in: Encyclopedia of Imaging Scienceand Technology, Hornak, J. (ed.), vol. 2, pp. 1106-1141, 2002.

Images in these slides that I didn't draw myself, came mostly from

Microscopy U, some from Wikipedia, and some from other, quoted sources.

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