Fluorescence microscopy II Advanced approaches

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Fluorescence microscopy IIAdvanced approaches
Martin Hof, Radek Machán
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Microscope resolution
The lateral resolution of an optical microscope d
The axial resolution (in the direction of
optical axis) dz
Sufficient contrast is necessary for full
utilization of the available resolution
However fluorescence from planes below and above
focus also contributes to signal ? blurred image,
decreased contrast
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Total internal reflection fluorescence - TIRF
When total reflection appears, only an
exponentially decaying evanescent wave crosses
the interface ? only fluorophores close to the
interface are excited
3 300 nm
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Total internal reflection fluorescence - TIRF
When total reflection appears, only an
exponentially decaying evanescent wave crosses
the interface ? only fluorophores close to the
interface are excited
prism-based
objective-based
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Confocal microscopy Basic principle
A pinhole in the back focal plane rejects the
light coming from outside the focal plane. The
pinhole size is a trade-off between good
rejecting ability and sufficient light throughput
(typically 30 150 mm)
wide field
confocal
detection
pinhole
tube lens
objective
focal plane
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Confocal microscopy Basic principle
The pinhole restricts the observed volume of the
sample to a single point (the size of which is
restricted by the pinhole size). Excitation by a
collimated beam (point source optically
conjugated to the pinhole) focused to a
diffraction limited spot
wide field
PMT MPD
confocal
CCD
image is scanned point by point
dichroic
whole image at once
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Confocal microscopy Scanning systems
spinning disk
laser scanning microscope (LSM)

• Collimated laser beam focus is scanned through
  the sample
• sample scanning by a piezo crystal
• slow
• possible combination with scanning probe
  microscopy (AFM, STM, )

• M. Petrán and M. Hadravský (1967)
• Wide-filed illumination passes through pinholes
  in Nipkow disk (arranged in Archimedean spiral)
• either a single pinhole for excitation and
  emission or 2 tandem disks

• low excitation efficiency only a small fraction
  of light passes pinhole

beam scanning by a mirrors mounted on
galvanometers

• nowadays enhanced by microlens arrays on another
  Nipkow disk
• more points in parallel possible faster imaging

X
Y
optical path for excitation and emission formed
by the same mirrors
Axial scanning (Z) usually by a piezo or stepper
motor actuator
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Confocal vs. Wide field microscopy
Wide-field
Confocal
Elimination of out-of-focus light improves
contrast and, thus, resolution
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Confocal vs. Wide field microscopy
Focusing only in one plane ? axial sectioning of
the sample to mm slices
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Resolution in confocal microscopy
collimated laser beam is focused by the objective
into a diffraction limited spot
PSF (point spread function) focus profile
collection efficiency of the objective. Those two
are approximately the same diffraction limited
spot.
Slightly higher resolution than in wide field
microscopy (improvement 1.4)
x 200 nm z 1 mm
3D Gaussian profile
The image is a convolution of the object and the
PSF
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Two-photon microscopy Basic idea
Two photons at the same time and at the same
place with doubled wavelength

• photons from the infra red spectrum (gt 750 nm)
  typically TiSa laser

• high photon density (6 7 orders of magnitude
  higher than in single photon confocal microscopy)

• excitation probability proportional to I2 ?
  reduced detection volume, higher resolution
  (improvement mainly in axial direction, in
  lateral it can be negligible due to larger l)

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Two-photon microscopy Focus profile
laser pulse
the required photon density for two-photon
excitation can be established only in the focal
plane ? no out-of focus fluorescence ? no pinhole
needed
focal plane
2p-excitation
1p-excitation
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Two-photon microscopy

• Advantages
• improved axial resolution
• reduced bleaching out of focus
• higher light collection efficiency (no pinhole)
• higher depth of light penetration
• broader excitation spectra simultaneous
  excitation of more dyes

• Limitations
• more costly and complicated instrumental setup
• higher bleaching in the focus
• broader excitation spectra decreased
  selectivity of excitation
• scanning technique like confocal microscopy

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General features of scanning microscopy

• Advantages
• improved contrast
• optical sectioning ability
• possibility to perform fluorescence measurements
  in individual points (lifetime, spectra, FCS, )

• Limitations
• more complicated and costly setup
• limited speed of image acquisition
• longer imaging ? more photobleaching

Fluorescence lifetime imaging (FLIM)
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Below the diffraction limit

• Going to near-field, where the diffraction limit
  does not hold Near-field Scanning Optical
  Microscope (NSOM)

• Effectively increasing the numerical aperture
  (does not really break the limit, but increases
  resolution) Structured (Patterned) Illumination
  Microscopy (SIM),

• Localization of individual fluorophores and
  fitting their PSFs, typically combined with
  switching between dark and fluorescent state
  (PALM, STORM, ) or utilizing intensity
  fluctuations of individual fluorophores
  (Superresolution Optical Fluctuation Imaging
  SOFI)

• Employing nonlinear optical effects

• Multi-photon excitation

• Optical saturation nonlinear dependence of
  fluorescence on excitation intensity, happens at
  high excitation intensities when large fraction
  of fluorophores resides in excited state and
  cannot be excited

• Other saturation phenomena Dynamic
  saturation optical microscopy (DSOM) kinetics
  of transition to triplet state, Stimulated
  emission excited state depletion (STED)

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Near-field scanning optical microscopy (NSOM)
Diffraction limit is valid in the far-filed,
where spherical wave-fronts exiting from an
aperture can be regarded locally as plane waves
coming close to the sample changes the situation
scanning probe approach
The probe usually a metal coated tapered
optical fibre moved by a piezo scanner
various operation modes purely near-field or
combining near-/far-field excitation/emission or
vice versa

• resolution 20 nm in lateral (determined by tip
  size) and 2-5 nm in axial direction

• limited only to surfaces

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Effective increasing of numerical aperture
4Pi microscopy
structured illumination

• 2 opposing objectives PSF closer to spherical
  symmetry 3-7 times improved axial resolution
  (depends on type)

• Sample is illuminated by a periodically
  modulated light. Interference of structures in
  the sample and illumination results in Moiré
  fringes

• combination with nonlinear image restoration
  improvement in 3D

• a confocal approach - scanning

• Additional spatial frequency increases the
  resolution power by factor 2

• A wide-field approach faster then scanning

• Several images with shifted illumination
  patterns are recorded and the final image is
  reconstructed by Fourier transform analysis ?
  optical sectioning

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Localization of individual molecules
Single fluorophores have dimensions much smaller
than the PSF. A single fluorophore is seen in the
image as the PSF
By fitting the PSF in the image with a Gaussian
profile, fluorophore location can be determined
with a few nm accuracy
? precise determination of distances, single
particle tracking (SPT)
Schmidt et al. (1996) PNAS 932926-2629
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Localization of individual molecules
At higher densities of fluorophores, the PSFs
overlap impossible to distinguish the centers
of peaks. Nevertheless, fluorophores need to be
densely located in the sample to be cover to all
structural details
STORM Stochastic optical reconstruction
microscopy
Rust et al. (2006) Nature Meth 3793-795

• Uses photoswitchable dyes (special organic dyes,
  GFP mutants)
• a strong red laser pulse switches off all
  fluorophores (to a nonfluorescent state)
• a green laser pulse switches on a small fraction
  of fluorophores, which emit fluorescence when
  excited with red laser until switched off, cycle
  repeated

A wide field technique, but imaging slow because
many imaging cycles needed
Resolution 20-30 nm
PALM Photoactivated localization microscopy the
same principle with switching of dyes between on
and off states
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Optical saturation and resolution enhancement
Optical saturation results in nonlinear relation
between excitation and fluorescence intensities
? broadening of the PSF
We apply a ramp of excitation intensity and the
dependence of fluorescence intensity in each
pixel on excitation intensity can be fitted with
a polynomial expansion
Ifl(x,y)
Iex -
Iex2
Iex3 -
Iex4...
Theoretically unlimited resolution, but
practically limited by noise and poor stability
of polynomial fits ( 30)
Saturated excitation microscopy (SAX)
harmonically modulated excitation, Saturated
structured illumination (SSIM) SIM combined
with nonlinearity
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Stimulated emission excited state depletion
(STED)
Developed by Stefan Hell (http//www.mpibpc.mpg.de
/abteilungen/200/STED.htm)

• A confocal approach

• Fluorophores in the detection volume are excited
  by an excitation pulse.

• A doughnut-shaped STED pulse is applied, which
  suppresses the fluorescence completely (by
  inducing stimulated emission) everywhere except
  the center of the detection volume

• Photons in STED pulse have lower energy to avoid
  excitation

• STED pulse duration should be much shorter then
  S1 lifetime 1/kfluor

• Saturation of the stimulated emission in the STED
  pulse is essential for breaking the diffraction
  limit

saturation parameter x I max/ Isaturation
kIC gtkSE gtgt kfluor
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STED
Theoretically unlimited resolution, usually 3
times in lateral and 6 times in axial direction
is achieved
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Selective plane illumination microscopy
Based on q microscopy (uses excitation and
detection optics at 90 instead of
epi-fluorescence to generate isotropic PSF)
combination with light sheet illumination ?
faster imaging of 3D objects
http//www.lmg.embl.de/home.html
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Acknowledgement
The course was inspired by courses of Prof.
David M. Jameson, Ph.D. Prof. RNDr. Jaromír
Plášek, Csc. Prof. William Reusch
Financial support from the grant FRVŠ 33/119970