Optical Microscopy and Imaging

Dr Anita Jones 3 lectures Recommended web sites:

http://microscope.fsu.edu/index.html
links to useful sites

http://www.microscopyu.com/
Nikon site

www.snom.omicron.de

Microscopy and Imaging

Optical Microscopy and Imaging

Optical Microscopy (far field) Scanning Near Field Optical Microscopy (SNOM) Fluorescence Microscopy Confocal Laser Scanning Fluorescence Microscopy Multiphoton Excitation (MPE) Microscopy Fluorescence Lifetime Imaging (FLIM)
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Optical Microscope

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Optical Microscopy
Numerical Aperture (NA)
brightness of image depends on amount of light gathered by objective depends on NA of objective

NA = n sin
where n is refractive index of medium between specimen and objective; is half angle of maximum cone of light picked up by objective

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Optical Microscopy
Numerical aperture

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Optical Microscopy
Numerical aperture - dependence on focal length of objective

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Optical Microscopy
theoretical maximum NA of lens objective with air as imaging medium (n=1.0, =90o) is 1.0; in practice <0.95 increasing n of imaging medium increases NA e.g. use of immersion oil n=1.51 gives NA up to 1.4 NA determines resolving power of objective

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Optical Microscopy
Resolution
determines smallest observable structure minimum distance between two object that can be resolved (appear as two separate objects) in the image fundamental limit (recognised by Abbe 1873) determined by wavelength of light and NA

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Optical Microscopy
resolution defined by Rayleigh criterion, minimum distance between 2 resolvable points:

d=

for high NA objective, resolution approximately /2

100s of nanometres

0.61 NA

practical limit ( ~350nm, NA ~1.4) is ~150nm

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Optical Microscopy
resolution limited by diffraction - diffraction limit when a point source is imaged by a lens a diffraction pattern (blurred spot) is produced the Airy pattern

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Optical Microscopy
central bright spot is Airy disc limit of resolution defined as d (centre-to-centre) = radius of Airy disc

in (c) d = rAiry; in (b) d > rAiry

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Optical Microscopy
Magnification
apparent enlargement of object by optical system product of magnification of objective and eyepiece up to several 100x brightness of image depends on 1/(Mag)2 and on (NA)2
for high magnification require large NA oil immersion objective

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Scanning Near Field Optical Microscopy (SNOM)

High resolution optical microscopy Overcomes the diffraction limit In the optical near field (distance << wavelength of light) diffraction effects eliminated Very small spot of light, diameter 50100nm, scanned across specimen and reflected or transmitted light detected for image formation
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SNOM
Light source is tip of metal coated-optical fibre

aperture in metal coating at tip 50-100nm diameter aperture-sample distance ~10nm resolution of image defined by size of aperture
in practice limited by intensity of light passing through aperture, typically 80-200nm apertures used
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SNOM
Scanning probe microscope
optical analogue of STM electric field of light penetrates short distance from optical fibre tip (evanescent field)
decays exponentially with distance from tip presence of sample causes disturbance of optical nearfield leading to emission of light from sample point opposite aperture

technically very similar to STM and AFM sample scanning and sample-probe distance control
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SNOM
can be combined with STM or AFM in single instrument

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SNOM
Applications
applicable to virtually any type of sample
opaque sample - reflection mode thin transparent sample - transmission mode

images surface of sample e.g.materials research, semiconductor devices, nanotechnology, biology, polymers, thin films

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SNOM
Imaging of IC test structures
next slide shows images of test structures of conducting lines with widths of 250 nm, 100 nm and 70 nm. The structures are gold lines written on a Si substrate by electron lithography. Images obtained with conventional (far field) optical microscopy, shear-force SFM and SNOM are compared.

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SNOM
250nm lines

Far field microscopy

Shear force SFM

SNOM

100nm lines

70nm lines
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SNOM
Imaging of biological tissue
SNOM image (left) of section of tissue from fish inner ear. Concentric rings which build up as fish grows, provide information about the age of the fish. Rings are not visible on topographic SFM image (right).

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Why use far-field microscopy?

Electron and scanning probe microscopy give much higher resolution than far-field light microscopy, but have drawbacks:
electron microscopy
in vacuo may require metal coating of sample to avoid charging of specimen unsuitable for imaging living cells

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Why use far-field microscopy?

scanning probe
slow image can be difficult to interpret

Most important limitation of EM and SPM restricted to investigation of specimen surface; interior of intact specimen inaccessible
particular disadvantage for biological and medical applications
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Why use far-field microscopy?

Far-field light microscopy can image inside of specimen e.g. cell
focused light penetrates translucent specimen without damage confocal microscopy can deliver 3-dimensional images of whole specimens and living biological samples

Recent important development - fluorescence microscopy

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Optical Microscopy - Contrast

To obtain image in optical microscopy need contrast In conventional optical microscopy contrast based on absorption of light
bright/dark coloured stains for biological specimens polarised light (birefringence)

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Optical Microscopy - Contrast

Photomicrographs of crystalline material using polarised light

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Fluorescence Microscopy
Contrast based on emission of light fluorescence
use of fluorescent molecules (fluorophores) as fluorescent labels/probes - molecular highlighter

Most important mode of imaging in biological and medical microscopy Development of new techniques using ultrafast (ps/fs) laser excitation sources
multiphoton excitation fluorescence lifetime imaging
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Fluorescence
Emission of light by electronically excited molecule
transition between two electronic states of same multiplicity - spin-allowed occurs on ns timescale

Large aromatic molecules with extensive delocalised -electron systems emit visible fluorescence with high efficiency
* excited state
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Fluorescence
Typical fluorophores

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Fluorescence
Excited state decay processes
Jablonskii diagram - photophysical processes
internal conversion (10-12s)
S2 S1 T1

vibrational relaxation intersystem crossing

Fluorescence (10-8 s)

Phosphorescence ( 10-6-1 s)

S0
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Fluorescence
Radiative decay
energy lost by emission of a photon

E = h =

hc

fluorescence, S1 to S0, spin-allowed phosphorescence, T1 to S0, spin-forbidden

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Fluorescence
Non-radiative decay - intramolecular
also known as radiationless decay energy lost without emission of light photophysical processes
electronic energy vibrational energy internal conversion, between states of same multiplicity - S2 to S1, S1 to S0 intersystem crossing, between states of different multiplicity - S1 to T1, T1 to S0

photochemical decomposition
photobleaching
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Fluorescence
Vibrational relaxation in condensed phase
rapid loss of vibrational energy to solvent / matrix as heat fluorescence always from lowest vibrational levels of S1 state (Boltzmann population) fluorescence spectrum independent of excitation wavelength

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Fluorescence Spectrum

Wavelength/nm

Fluorescence spectrum at lower energy/longer wavelength than absorption spectrum

Stokes shift
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Fluorescence
Intermolecular non-radiative decay
quenching electronic energy transfer
excitation energy transferred from an excited molecule to a ground state molecule without emission of a photon Forster resonance energy transfer or fluorescence resonance energy transfer (FRET)
interaction of transition dipoles of donor and acceptor molecules occurs over distance of 1-10nm can be used as spectroscopic ruler to measure intermolecular distance
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Fluorescence
Fluorescence decay kinetics
excited state decay is first order kinetic process
d [M *] = k F [M *] dt

[M *] = [M *] 0 exp( k F t )
rate constant, k, is sum of rate constants for all radiative and non-radiative decay processes

k = k R + k NR k = k R + k IC + k ISC + ...
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Fluorescence
Fluorescence intensity = rate of emission of photons
I F ( t ) = k R [M *] I F ( t ) = I F ( 0 )k R exp( k F t ) 1 =F kF

fluorescence intensity decays exponentially after excitation pulse F is fluorescence lifetime

time taken for excited state population to fall to 1/e of that initially excited
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Fluorescence
Fluorescence quantum yield, F
F =
F =
number of photons emitted number of photons absorbed
kR = k R F kF

kR essentially constant for particular fluorophore F is a measure of F

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Fluorescence
Sensitivity to microenvironment
fluorescence spectrum and lifetime sensitive to molecular environment sensitive to polarity, oxygen concentration etc increase in solvent polarity shifts spectrum
for many fluorophores S1 state has greater dipole moment than S0 S1 stabilised relative to S0 in polar solvents fluorescence spectrum red-shifted in polar environment
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Fluorescence
O2 quenches fluorescence
decrease in fluorescence intensity decrease in fluorescence lifetime (increase in rate of non-radiative decay)

ion-specific fluorescent probes

intracellular measurement of concentration of ions in vivo e.g Calcium Green 1
change in fluorescence intensity/ fluorescence lifetime on binding of calcium

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Fluorescent Probes
A few intrinsic probes
tryptophan, a fluorescent amino acid, is intrinsic fluorophore in proteins aromatic groups in synthetic polymers

Generally use extrinsic probes

specific fluorescent label introduced into specimen for imaging potential problems
perturbation of system in living cells: phototoxicity - generation of highly reactive singlet oxygen
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Fluorescent Probes
Wide variety of fluorescent probes with different emission spectra
e.g. coumarins - blue; fluorescein - green; rhodamines - orange, red

Probe molecular structure can be modified to bind to specific targets

labelling of specific molecular cell constituents
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Fluorescent Probes
Immunofluorescence
use of highly specific fluorescently labelled antibodies antibodies can be covalently labelled with fluorescent molecules without losing their immunological specificity fluorescently labelled antibody binds specifically to a particular protein (antigen) identification, location and visualistion of cell and tissue components
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Fluorescent Probes
Green fluorescent protein (GFP)
naturally fluorescent protein produced by jellyfish Aequorea victoria 27kDa monomer, 238 amino acid residues chromophore is cyclic tripeptide derived from Ser-Tyr-Gly in primary protein sequence and is only fluorescent when embedded within complete GFP protein absorption max 395nm emission max 510nm
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Fluorescent Probes
gene for GFP has been cloned and can be introduced into other organisms (genetic modification)
intracellular production of natural fluorescent probe

different coloured variants (mutants) of GFP have been engineered e.g. BFP (blue) 450nm emission applications
monitoring gene expression studying protein distribution and transport in cells studying protein-protein interactions
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Fluorescent Probes
Strategy for use of FRET between BFP and GFP to detect protein-protein interaction at molecular level

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Fluorescence Microscopy
Advantages of fluorescence microscopy
high specificity
sophisticated labelling techniques selective excitation/detection - analysis of complex mixtures of molecular species

high sensitivity
can detect fluorescence of very small number of molecules can detect objects below diffraction-limited resolution single molecule detection is possible
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Fluorescence Microscopy
Advantages (continued)
environmental sensitivity
spectral/lifetime measurements probe immediate physical/chemical environment of fluorophore viscosity, polarity, pH, ionic concentration etc. probe intermolecular interactions measure intermolecular distances on nm scale

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Fluorescence Microscopy
Triple fluorescent staining of bovine endothelial cells showing the blue nucleus, red mitochondria, and green actin cytoskeleton. (endothelial cells line internal
body cavities)

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Fluorescence Microscope
Epifluorescence microscope
episcopic illumination (epiillumination) excitation and detection via objective lens excitation beam reflected by dichroic mirror (beam splitter) into objective fluorescence collected by objective and transmitted by dichroic mirror to detector
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Fluorescence Microscope
dichroic mirror has wavelength-dependent reflectivity
e.g. reflects shorter wavelengths and transmits longer wavelengths separates excitation light from fluorescence

residual transmitted excitation light excluded by barrier filter in front of detector

absorbs excitation wavelength, transmits fluorescence

full field of view of objective is illuminated by excitation light fluorescence from full field of view imaged onto detector Microscopy and Imaging 50

Fluorescence Microscope

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Fluorescence Microscopy
Excitation sources
xenon arc lamp
continuous emission over uv and visible spectrum desired wavelength selected by filter

argon ion laser or krypton ion laser

continuous wave (CW) laser monochromatic light high intensity emission lines in uv and visible
commonly used lines: 488nm, 514 nm from Ar+ laser
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Detectors
Photomultiplier tube (PMT)
measures light intensity does not record image very sensitive
photon counting can detect single photon

used in scanning confocal fluorescence microscopy

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Detectors - PMT
operating principle
photon strikes photocathode causing emission of an electron amplification by secondary emission of electrons in dynode chain gain ~106 for each incident photon current pulse produced at anode

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Detectors
CCD - charge-coupled device
CCD camera records digital image silicon-based integrated circuit consisting of dense array of photodiodes (pixels) light energy striking pixel converted to electronic charge intensity integrated to give sufficient signal:noise accumulated charge read pixel by pixel in serial output register
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Detectors - CCD
frame transfer CCD
one half of parallel array used as storage region, protected by light proof mask rapid parallel transfer of accumulated charge pattern from image array to storage array new image acquired on image array during serial readout from storage array
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Detectors - CCD
Image-intensified CCD
CCD less sensitive than PMT sensitivity enhanced by addition of image intensifier image intensifier:
photocathode microchannel plate electron multiplier output phosophor screen 1 photon at photocathode produces pulse ~105 photons from phosphor screen
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Detectors - CCD
Image-intensified CCD

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Fluorescence Microscopy
In conventional epi-fluorescence microscope entire specimen in field of view exposed to excitation light
fluorescence excited throughout depth of sample

Fluorescence collected from throughout specimen including from above and below focal plane of objective
creates image with depth of field of 2-3m

Superposition of detail from above and below focal plane blurs the image
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Confocal Fluorescence Microscopy

In confocal fluorescence microscope, out-offocus information (blur) is removed Diffraction-limited spot of excitation light focused laser beam
with focused beam excitation intensity falls off rapidly above and below plane of focus

Fluorescence from above and below excitation focal plane is rejected

only confocal fluorescence detected
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Confocal Fluorescence Microscopy

Confocal laser scanning microscpe
tightly focused laser excitation spot scanned across sample pinhole in image focal plane prevents out-offocus fluorescence from reaching detector (PMT)
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Confocal Fluorescence Microscopy

image built up point by point as excitation spot is scanned across sample laser beam moved in regular 2-D raster pattern across sample by scanning mirror output from PMT (fluorescence intensity) read at each point and image built up on PC

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Confocal Fluorescence Microscopy

Optical sections
record optical sections of specimen by moving excitation focal plane through depth of specimen (z axis) and collecting image (xy plane) as function of z optical sections ~500nm thick can be created image processing software converts optical sections into 3-D image of sample
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Applications
Biological imaging
expression of GFP in tobacco pollen grain and pollen tube scale bar 20m

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Applications
Newt lung cell in mitosis (cell division); 240x
blue: chromosomes; green: protein fibres

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Applications
neurons in a section of brain tissue

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Applications
Materials science
3-D image of fluorescently labelled polymer - polybutadiene confocal 3-D reconstruction

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Applications
Geochemistry
3-D image of sample of Porites coral intrinsic fluorescence confocal 3-D reconstruction recorded in COSMIC, Edinburgh
100m

Pseudo 3D image comprising 15 consecutive 0.5m sections superimposed

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Multiphoton Excitation Microscopy

Use of two-photon excitation
S1 h h h S0
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MPE Microscopy
2-photon excitation
1-photon absorption
Intensity

fluorescence

2-photon absorption

200

250

300

350

400 450 Wavelength (nm)

500

550

600

650

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MPE Microscopy
Simultaneous (coherent) absorption of two photons by fluorophore
requires high local instantaneous intensity (high power density) ultrafast pulsed Ti:sapphire laser
100fs pulses at 80MHz repetition rate; low pulse energy (10nJ) but high peak power (0.1MW) wavelength tunable 700-1000nm

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MPE microscopy
Intrinsically confocal
only excite fluorescence at intense focus of excitation beam

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MPE Microscopy
Advantages
self-confocality
no need for detection pin hole

elimination of background fluorescence

no fluorescence excited as excitation beam passes through sample, only at high intensity focal point

good discrimination against scattered excitation light

very large wavelength separation between excitation and fluorescence
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MPE -advantages
reduced radiation damage - long excitation reduced photobleaching - v.small excitation volume (femtolitre)
no photobleaching outside focal spot

deeper imaging - deeper penetration of long excitation

important for medical tissue imaging

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MPE Microscopy
Disadvantages
cost of fs laser source
100,000

excitation wavelength range limited by water absorption at >1000nm

problem for biological imaging - thermal damage

lack of knowledge of 2-photon excitation spectra of fluorophores

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Applications
Geochemistry
2-photon excitation image of sample of Porites coral 15m below surface of solid (porous) sample localised centres of luminescence visible
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Fluorescence Lifetime Imaging

Very new technique Use fluorescence lifetime of fluorophore as contrast parameter
time-gated imaging record fluorescence intensity in different time windows after excitation pulse
ultrafast excitation laser gated intensified CCD camera minimum gate width ~100ps

Intensity

time/ns
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FLIM
Produce image showing distribution of fluorescence lifetimes in sample Fluorescence lifetime sensitive to molecular environment of fluorophore
image microenvironments

Measure intermolecular distances using FRET

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FLIM
Other advantages
lifetime independent of:
excitation intensity concentration fading due to photobleaching

very good discrimination against scattered laser light and background fluorescence

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FLIM
Disadvantages
cost of excitation source
ultrafast laser 200,000

cost of detector
gated intensified CCD 50,000

lack of knowledge of probe photophysics

need to develop well characterised fluorescence lifetime probes

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FLIM
Intensity images
ANS

ANS MeOH 60 M

ANS MeOH 2 M

ANS MeOH 60 M
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ANS MeOH/H2O (1:1) 60 M

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FLIM
ANS MeOH 60 M

500 ps gate width 40 gates, 8 sec. total acquisition

ANS MeOH 2 M ANS MeOH 60 M ANS MeOH/H2O (1:1) 67 M

6.2 ns
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2.4 ns
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Lifetime / ns

FLIM
GFP in cells
Same lifetime at different locations in cell Intensity differences due to concentration variation, not quantum yield variation
103

cell4a cell4b cell4c cell4d

102 Counts 101 100 0

6 8 Time/ns

10

12

14

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FLIM
Investigating signal pathways in mammalian cells FRET between GFP and YFP (COSMIC, Edinburgh and Beatson Cancer Institute,Glasgow)

X-ray structure of GFP

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FLIM

Conventional microscope image of group of cells

Fluorescence image only one cell expressing GFP

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FLIM
LHS GFP only RHS GFP- and YFP- labelled proteins (fusion proteins)
No change in F of GFP (donor) no protein-protein interaction

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Counts

102

101

100

8 10 Time/ns

12

14

16

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