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Superresolution in Fluorescence and Diffraction Microscopies with M ultiple I lluminations

Superresolution in Fluorescence and Diffraction Microscopies with M ultiple I lluminations. - Jules Girard -. 2 December 2011. Introduction : Imaging with optics and resolution. Imaging device. Parameter of interest. Probing function. Detector. FT. Low-pass filter.

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Superresolution in Fluorescence and Diffraction Microscopies with M ultiple I lluminations

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  1. Superresolution in Fluorescence and Diffraction Microscopies with Multiple Illuminations - Jules Girard - 2 December 2011

  2. Introduction : Imaging with optics and resolution Imaging device Parameter of interest Probingfunction Detector FT Low-passfilter

  3. Introduction : Extend resolution with illumination kx → ky By using multiple and inhomogeneous illuminations, we can shift high frequency parts of the object spatial spectrum into the passband defined by the psf kx kx More generally : = W. Lukosz and M. Marchand, OpticaActa10, 241-255 (1963). W. Lukosz, JOSA 56, 1463 (1966). ky ky

  4. Introduction : Reconstruct a super-resolution image • Inversion → numerical data processing 2 cases is known is unknown • Non-linear inversion • Findboth and • with the use of constraints • « Direct » inversion • withanalyticalapproach

  5. Presentation Outline = Optical Diffraction Tomography II. Structured Illumination Fluorescence Microscopy

  6. I. Optical Diffraction Tomography

  7. II. Optical Diffraction Tomography Fourier Space = 0 for lateral frequencies > Wemeasure : (Sample dielectric permittivity contrast) (Total internal electric field) Objective • Reconstruct : quantitative microscopy of unstained sample • ≠ illuminations → ≠ → access to ≠ parts of E Wolf, Optics Communications 1, 153-156 (1969). V Lauer, Journal of Microscopy 205, 165-76 (2002).

  8. II. Optical Diffraction Tomography • Illumination with « plane waves » under ≠ incidences • Measurecomplex values of Experiment (G. Maire, F. Drsek, H.Giovannini) CCD • Calibration and normalization • → Phase modulator • Inversion : ) → Laser (λ=633nm) Sample

  9. II. Optical Diffraction Tomography 2. 1. • Low :Born Approximation • is diffraction limited→ Abbe limit = • High : Multiple Scattering Regime • depends on object and illumination • isnot diffraction limited → resolutionimprovement? ( ?)

  10. II. Optical Diffraction Tomography simulations z air • λ = 633 nm • Abbe limitwith NA = 1.5 • → 211 nm 50 nm 25 nm x 50 nm glass 50° Low High (Ge) = 10-2 = 28.8 < !

  11. II. Optical Diffraction Tomography Experimental validation z • Germanium rods • TIRF configuration (10 angles) • NA = 1.3 • → Abbe limit : 245 nm air 50 nm 25 nm x 50 nm glass (A. Talneau – LPN) 0,5 Z (µm) 0 0,5 Z (µm) 0 9/27

  12. II. Optical Diffraction Tomography • Conclusion • We achieved quantitative reconstruction of the permittivity map of unstained sample even with a multiple scattering regime • Multiple scattering : drawback way to improve the resolution of ODT far beyond diffraction limit 10/27

  13. II. Structured Illumination in Fluorescence microscopy on 2D samples

  14. III. Structured Illumination Microscopy in Fluorescence (2D) Objective Tube Lense CCD (fluorescence density) (fieldintensity) (2D and 1D) 11/27

  15. III. Structured Illumination Microscopy in Fluorescence • Use periodic pattern → R. Heintzmann and C. Cremer, SPIE, pp. 185-196.(1998) Mats G L Gustafsson, Journal of Microscopy 198, 82-7 (2000). kx • Requirements for illumination pattern : • Accurate translation→ needed for discrimination of the 3 copies • High contrast → higher SNR (no dim for shifted copies of ) ky

  16. III. Structured Illumination Microscopy in Fluorescence • Limit : Illumination pattern isdiffraction limited: • = : twicebetterthanclassical WF • How canwereachhigherfrequencies ? • Use of non-linearities :→ • (R. Heintzmann et al., JOSA A, 19, 2002 & M G L Gustafsson, PNAS, 102, 2005) • Get below diffraction limit(surface imaging) • High index substrate→ limitedn and/or absorption • Nanostructured deviceswithplasmonics • → fieldbound to the structure + difficulties to cover a large area

  17. III. Structured Illumination Microscopy in Fluorescence Grating assisted Structured Illumination Microscopy z=0 • Dielectric resonant grating ≈ 2D waveguide + 2D sub-λgrating a-Si layer @ 633nm Glass coverslip @ 633nm • Hexagonal geometry : 6 equivalent orientations → near isotropic resolution • Design optimization→numerical simulations

  18. III. Structured Illumination Microscopy in Fluorescence (J. Girard, A. Talneau, A. Cattoni LPN – CNRS) Gratings fabrication process 1. aSi deposition (PECVD) 2. Grating patterning (e-beam + RIE) 3. Planarization (A. Cattoni) A. Cattoni, A. Talneau, A-M Haghiri-Gosnet, J. Girard, A. Sentenac (oral presentation, MNE 2011)

  19. III. Structured Illumination Microscopy in Fluorescence Excitation modes of the grating substrate 2 beams excitation 1 beam excitation right left

  20. III. Structured Illumination Microscopy in Fluorescence Experimental setup • Control of orientation, phaseand incidence angle on the substrate (65°) Objective (Oil, NA 1.49) Dichroïc Mirror

  21. III. Structured Illumination Microscopy in Fluorescence Grating characterization : SNOM measurements 1 beam excitation (GeoffroyScherrer, ICB, Dijon) Stretched fiber 65° GridShifting High Frequency Pattern from the Grating simulation Theoretical simulation z= Figure IV.4.1‑1  Montage expérimental pour la mesure en champ proche optique

  22. III. Structured Illumination Microscopy in Fluorescence Grating characterization : Far field fluorescence measurements • 2 beams excitation : Low frequency component of the intensity pattern • WF Fluorescence observation with ~homogeneous layer of fluorescent beads

  23. III. Structured Illumination Microscopy in Fluorescence • Our manufacturedgratingscanproduce a grid of light with • 180 nm period (λ/3.5) (down to 147 nm, λ/4.3 with alternative design) • a highcontrast • The possibility to shift its position • According to , a final resolutionof up to 87 nm couldbereachedatλ =633 nm! = • Howeverweneed to know the illumination pattern for inversion procedure

  24. III. Structured Illumination Microscopy in Fluorescence “Blind” SIM Inversion unknowns: • Iterativeoptimizationof estimates of and through minimization of a costfunction: … (Emeric Mudry & Kamal Belkebir) +1 equations 21/27

  25. III. Structured Illumination Microscopy in Fluorescence Experimental validation • Observation of fluorescent beads (Ø 90nm) immersed in glycerinwithclassical SIM Our Result WF image Deconvolution of the WF image Optimized « analytical » algorithm Inversion by Pr. R. Heintzmann 22/27

  26. Measurement Simulation • III. Structured Illumination Microscopy in Fluorescence Speckle illumination • Speckle pattern is a perfect candidate for SIM withour ‘blind’ inversion algorithm 2. Known average illumination 1. Contains every accessible frequencies 3. Experiment far simpler than standard SIM 23/27

  27. III. Structured Illumination Microscopy in Fluorescence Speckle illumination : simulations speckles speckles object WF image Deconvolution = Deconvolution = N ≈ 80 One measured image Photon budget : average of 130 photon/pixel/image Reconstructed = 24/27

  28. III. Structured Illumination Microscopy in Fluorescence Speckle illumination : experimental results RabbitJejunum slices (150nm thick) (Cendrine Nicoletti, ISM, Marseille) TEM image of a similarsample WF image Reconstructed image from 100 speckle illuminations Deconvolution of WF image 25/27

  29. General Perspectives • Optical Diffraction Tomography : • Extend to 3D samples • Use other configuration (gratingsubstrate, mirrorsubstrate…) II. Structured Illumination in Fluorescence Microscopy • Grating-assistedSIM : • Make super-resolved images of real samples : use a priori information for inversion procedure • Speckle illumination : • Extend to 3D samples 27/27

  30. III. Structured Illumination Microscopy in Fluorescence • Conclusion • SIM with unknown illumination patterns • Extension ofSIM to the use of randomspeckle patterns • Not effective yet for grating-assisted SIM (inhomogeneousaverage illumination) 26/27

  31. Thanks… • Geoffroy Scherrer • Anne Talneau • Andrea Cattoni Eric Le Moal Kamal Belkebir Guillaume Maire Emeric Mudry Anne Sentenac • The whole MOSAIC team for advices, seminars, discussions, equipment, facilities…

  32. Thank you for your attention

  33. II. Optical Diffraction Tomography • NA = 0.7 (used up to 0.53 only for illumination) • → Abbe limit : 500 nm (450nm for full NA) z air 100 nm 110 x 300 nm AFM profile Si Reconstructed profile (linear inversion) Reconstructed map Reconstructed profile

  34. II. Optical Diffraction Tomography Multiple scattering and resolution Simulation of () =() for a plane wave illumination (incidence 50°) 100nm = 10-2 = 2(b)= 7 (c) = 14 Modulation of for the object 2 : 25nm = 28.8 (Germanium)

  35. III. Structured Illumination Microscopy in Fluorescence Grating assisted SIM : getting some images • Problem with inversion : Intensity pattern is not perfectly known • Speckle algorithmis not able to retrievefrequencies > • Add of a priori information (rough orientation and frequencies)

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