Fluorescence microscopy II Advanced approaches

1. CZECH TECHNICAL UNIVERSITY IN PRAGUE FACULTY OF BIOMEDICAL ENGINEERING Fluorescence microscopy IIAdvanced 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. 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. 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. 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 • 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: Wide-field: Confocal: 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 h* h h Absorption Emission Emission Absorption h h* E = hn E ~ 1 / l E* = 1/2 E E* ~ 1 / 2l c = ln 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 (> 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. photon non-excited dye molecule 2p-excited dye molecule 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

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. 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 fluorophoresand 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: 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

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 93:2926-2629

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 3:793-795 • 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

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. Imax>> Isaturation Fluorescence STED pulse Excitation spot x ~ 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 >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 http://www.lmg.embl.de/home.html

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