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