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7/29/2019 Cris Lecture1
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Digital Imaging Systems
Light Microscopyconventional optical microscopes,contrast techniques, fluorescence
Cris Luengocris@cb.uu.se
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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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The Bright Field Microscope
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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The Bright Field Microscope
eye
ocular
a.k.a. eyepiece
objective
sample
intermediateimage plane
imageplane
lenses typically arecompound lenses,not a single lens
light
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The Bright Field Microscope
projectionlensobjective
sample
intermediateimage plane
imageplane
CCD
light
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Infinity Corrected Optics
projectionlensobjective
sample
intermediateimage plane
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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Dark Field Microscopy
Tiny modification to the bright field setup
Contrast obtained by diffraction
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Dark Field Microscopy
projectionlens
objective
sample imageplane
CCD
tubelenscollector
lens
fielddiaphragm
intermediateimage plane
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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Phase Contrast Microscopy
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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Phase Contrast Microscopy
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 Illumination
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Structured Illuminationimage in
frequency
domain
uniformilluminationcorresponds
to impulsein FD
sinusoidalilluminationcorresponds
to shiftedimpulse in FD
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Structured Illumination
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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Structured Illumination
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
The Epifluorescence Microscope
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The Epifluorescence Microscope
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
The Epifluorescence Microscope
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The Epifluorescence Microscope
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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