1. Fluorescence microscopy II Advanced approaches
CZECH TECHNICAL UNIVERSITY IN PRAGUE
FACULTY OF BIOMEDICAL ENGINEERING
Fluorescence microscopy II Advanced approaches
Martin Hof, Radek Macháň

2. 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

3. 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

4. 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

5. 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

6. image is scanned point by point
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

7. laser scanning microscope (LSM)
Confocal microscopy – Scanning systems:
spinning disk
laser scanning microscope (LSM)
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
Collimated laser beam focus is scanned through the sample:
sample scanning by a piezo crystal
slow
possible combination with scanning probe microscopy (AFM, STM, …)
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

8. Confocal vs. Wide field microscopy:
Elimination of out-of-focus light improves contrast and, thus, resolution

9. Confocal vs. Wide field microscopy:
Focusing only in one plane  axial sectioning of the sample to ~ mm slices

10. 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

11. single-photon excitation two-photon excitation
Two-photon microscopy – Basic idea:
single-photon excitation h
Absorption
Emission
two-photon excitation h
h*
Absorption
Emission
E ~ 1 / l
E = hn
c = ln
E* ~ 1 / 2l
E* = 1/2 E
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 l)

12. the required photon density for two-photon excitation
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

13. 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

14. Fluorescence lifetime imaging (FLIM)
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)

15. 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)

16. 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

17. Effective increasing of numerical aperture:
structured illumination
4Pi microscopy
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

18. 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 93:

19. 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:
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 ~ nm
PALM – Photoactivated localization microscopy the same principle with switching of dyes between on and off states

20. 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

21. Stimulated emission excited state depletion (STED):
Developed by Stefan Hell (
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
Fluorescence
STED pulse
Imax>> Isaturation
Saturation of the stimulated emission in the STED pulse is essential for breaking the diffraction limit ~
Excitation spot x
saturation parameter:
x = I max/ Isaturation
kIC >kSE >> kfluor

22. STED:
Theoretically unlimited resolution, usually ~ 3 times in lateral and ~ 6 times in axial direction is achieved

23. 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

24. 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