EPFL SV PTBIOP BIOP COURSE 2015 OPTICAL SLICING METHODS

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1 BIOP COURSE 2015 OPTICAL SLICING METHODS

2 OPTICAL SLICING METHODS Scanning Methods Wide field Methods Point Scanning Deconvolution Line Scanning Multiple Beam Scanning Single Photon Multiple Photon Total Internal Reflection Structured Illumination Selective Plane Ilmmumination Emission Slicing Excitation slicing Image processing

3 Image IMAGE FORMATION LOW PASS FILTERING Object

4 DECONVOLUTION Image

5 SIMPLE DECONVOLUTION APPROACHES object CONVOLUTION DECONVOLUTION image Schematic representation of the image formation model (without considering noise!) h(x) f (x) g(x) optical system Recall the definition of convolution: x f x hx g * u, v, whx u, y v z w f, The convolution operation corresponds to a simple multiplication in the frequency domain! The majority of the deconvolution algorithms are implemented in the frequency domain. g( x) f ( x) h( x) G( ) F( ) H( ) dudvdw

6 DECONVOLUTION TECHNIQUES COMPARISON A Confocal images of fixed epithelial cell labeled with concanavalin A and FITC

7 THEORETICAL VS MEASURED PSF Practically, the effective PSF can significantly differs from the theoretical one Theoretical PSF Measured PSF (λ=488 nm) Confocal microscope, Zeiss LSM700 Measured PSF (λ=561 nm) Confocal microscope, Zeiss LSM700

8 PSF THEORETICAL VS MEASURED How to obtain an experimental PSF? 1. record 3D stacks of sub-resolution fluorescent beads, with the same microscopy parameters and in the same conditions you acquired the images you want to deconvolve noise-free Theoretical PSF does not require additional microscopy acquisitions does not take into account specific optical system peculiarities 2. register and average the beads images to improve their SNR 3. derive the experimental PSF using the deconvolution principle Experimental PSF takes into account specific optical system peculiarities partially corrects for spherical aberration needs to be extracted from beads images

9 DECONVOLUTION IN PRACTICE Deconvolution effects deconvolution improves image resolution, particularly in the axial direction improves image contrast improves image SNR For these reasons it is a good practice to deconvolve your images, particularly when you want for example to segment objects or do colocalization analysis, besides getting nicer and cleaner images. Deconvolution applicability widefield microscopy confocal microscopy spinning disk microscopy 2P microscopy

10 OPTICAL SLICING METHODS Scanning Methods Wide field Methods Point Scanning Deconvolution Line Scanning Multiple Beam Scanning Single Photon Multiple Photon Total Internal Reflection Structured Illumination Selective Plane Ilmmumination Emission Slicing Excitation slicing Image processing

11 TOTAL INTERNAL REFLECTION CRITICAL ANGLE 2 n sin n sin n 2 n 1 n 1 C sin 1 n n n C

12 EVANESCENT FIELD 1,0 I I Z 0 exp z / d p I z / I 0 0,8 0,6 0,4 488 nm n 1 =1.51 n 2 =1.36 = 65 n 1 =1.45 = 80 d p n1 sin 1 n2 0,2 0, depth / nm

13 TIRFM ILLUMINATION

14 KINESIN MOVEMENT

15 TIRFM EB3-GFP Evanescent field Wide-field

16 TIRFM EB3-GFP

17 TIRFM SUMMARY Advantages Illumination of small optical slices ( nm) Increased S/B ratio Observation of Single Molecules Fast compared to Confocal systems Disadvantage Restricted to Surface Phenomina Photobleaching, Blinking

18 LIGHT SHEET MICROSCOPY Siedentopf, H. and Zsigmondy, R. (1902). Über Sichtbarmachung und Größenbestimmung ultramikoskopischer Teilchen, mit besonderer Anwendung auf Goldrubingläser. Ann. Phys. 10, Development 136, (2009) Huisken, J., Swoger, J., Del Bene, F., Wittbrodt, J. and Stelzer, E. H. K. (2004). Optical sectioning deep inside live embryos by selective plane illumination microscopy. Science 305, Stelzer, E. H. K. Dodt, H. U., Leischner, U., Schierloh, A., Jährling, N., Mauch, C., Deininger, K., Deussing, J. M., Eder, M., Zieglgänsberger, W. and Becker, K. (2007). Ultramicroscopy: three-dimensional visualization of neuronal networks in the whole mouse brain. Nat. Methods 4,

19 LIGHT SHEET MICROSCOPY SETUP Development 136, (2009)

20 LIGHT SHEET MICROSCOPY SETUP COMPARISON Development 136, (2009)

21 LIGHT SHEET MICROSCOPY Development 136, (2009)

22 OPTICAL SLICING METHODS Scanning Methods Wide field Methods Point Scanning Deconvolution Line Scanning Multiple Beam Scanning Single Photon Multiple Photon Total Internal Reflection Structured Illumination Selective Plane Ilmmumination Emission Slicing Excitation slicing Image processing

23 MULTIPHOTON MICROSCOPY Zipfel W.R., Nature Biotechnology (2003)

24 2 PHOTON CROSS SECTION Maria Goeppert Meyer ( ) Physicist, Nobel price 1963

25 OPTICAL SETUP Helmchen F. & Denk W., Nature Methods (2005) Zipfel W.R., Nature Biotechnology (2003)

26 BEAM PATH COMPARISON Ballistic photons: non scattered photons

27 APPLICATION OF 2 PHOTON MICROSCOPY Helmchen F. & Denk W., Nature Methods (2005)

28 MULTI PHOTON MICROSCOPY SUMMARY Advantages Illumination of a small volume Non-descanned detection (better detection efficiency) Long excitation wavelength (less scattering in tissue) Ideal for deep tissue imaging (scattering samples) Disadvantage Complicated and expensive setup (Titanium Saphire Laser) Photobleaching, Sample heating