Microscopy II - UFCH JH
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Transcript Microscopy II - UFCH JH
CZECH TECHNICAL UNIVERSITY IN PRAGUE
FACULTY OF BIOMEDICAL ENGINEERING
Fluorescence microscopy II
Advanced approaches
Martin Hof, Radek Macháň
Microscope resolution:
The lateral resolution of an optical microscope d:
0,5
d
NA
2
The axial resolution (in the direction of optical axis) dz:
1,4 n
dz
NA2
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
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
I(z) I(0) exp(z / d)
~ 3 – 300 nm
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
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
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
CCD
PMT
MPD
…
dichroic
whole image at once
confocal
image is scanned point by point
Confocal microscopy – Scanning systems:
spinning disk
M. Petráň 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
o low excitation efficiency – only a
small fraction of light passes pinhole
nowadays enhanced by microlens
arrays on another Nipkow disk
more points in parallel possible –
faster imaging
laser scanning microscope (LSM)
Collimated laser beam focus is
scanned through the sample:
sample scanning by a piezo crystal
o slow
possible combination with scanning
probe microscopy (AFM, STM, …)
beam scanning by a mirrors mounted
on galvanometers
X
Y
optical path for excitation and
emission formed by the same mirrors
Axial scanning (Z) usually by a piezo or stepper motor actuator
Confocal vs. Wide field microscopy:
Wide-field:
Confocal:
Elimination of out-of-focus light improves contrast and, thus,
resolution
Confocal vs. Wide field microscopy:
Focusing only in one plane axial sectioning of the
sample to ~ mm slices
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
Two-photon microscopy – Basic idea:
E = h
c =
E~1/
h*
h*
E* = 1/2 E
Emission
h
h
two-photon excitation
Absorption
Emission
Absorption
single-photon excitation
h
E* ~ 1 / 2
Two photons at the same time and at the same place with doubled wavelength
photons from the infra red spectrum (> 750 nm) – typically Ti:Sa 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 )
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
photon
non-excited
dye molecule
2p-excited
dye molecule
1p-excitation
2p-excitation
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
o more costly and complicated
instrumental setup
o higher bleaching in the focus
o broader excitation spectra –
decreased selectivity of excitation
o scanning technique like confocal
microscopy
General features of scanning microscopy:
Advantages
Limitations
improved contrast
o more complicated and costly setup
optical sectioning ability
o limited speed of image acquisition
possibility to perform fluorescence
o longer imaging more photobleaching
measurements in individual points
(lifetime, spectra, FCS, …)
Fluorescence lifetime
imaging (FLIM)
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)
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
o limited only to surfaces
Effective increasing of numerical aperture:
structured illumination
Sample is illuminated by a periodically
modulated light. Interference of
structures in the sample and
illumination results in Moiré fringes
4Pi microscopy
2 opposing objectives – PSF closer
to spherical symmetry – 3-7 times
improved axial resolution (depends
on type)
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
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)
MSD(t ) r(t ) r(0)
2
Schmidt et al. (1996) PNAS 93:2926-2629
4 Dt
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 3:793-795
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
Optical saturation and resolution enhancement:
Optical saturation results in nonlinear relation between excitation and
fluorescence intensities
broadening of the PSF
1.0
PSF(x)
Fluorescence [a.u.]
1.0
0.5
0.5
0.0
0.0
0
0
200
400
Excitation rate [MHz]
400
x (nm)
800
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
0 4
0
0 2
0 3
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
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
Imax>> Isaturation
Fluorescence
x
x
STED pulse
• Saturation of the stimulated
emission in the STED pulse is
essential for breaking the
diffraction limit
saturation parameter:
x = I max/ Isaturation
Excitation
spot
x
~
kIC >kSE >> kfluor
STED:
Theoretically unlimited resolution, usually ~ 3 times in lateral and ~ 6 times in axial
direction is achieved
Selective plane illumination microscopy:
Based on q microscopy (uses excitation and detection optics at 90˚ instead of epifluorescence to generate isotropic PSF) – combination with light sheet illumination
faster imaging of 3D objects
http://www.lmg.embl.de/home.html
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