1. 1 METHOD FOR DEFOCUS CORRECTION IN OPTICAL COHERENCE MICROSCOPY Dmitry Lyakin 1, Anton Sdobnov 2, Vladimir Ryabukho 2,1 1 Institute of Precision Mechanics and Control, RAS, Russia 2 Saratov State University, Russia SARATOV FALL MEETING – SFM’2015 International Symposium “Optics and Biophotonics – III” September 22 – 25, 2015 Saratov, Russia

2. 2 CONTENT OF PRESENTATION Optical Coherence Microscopy Defocus Problem Known Methods for Defocus Correction Main Idea of Presented Method Experimental Setup How Does It Work Experimental Results Conclusions

3. 3 OPTICAL COHERENCE MICROSCOPY (OCM) Single-point OCM Full-field OCM OCM = Optical Coherence Tomography (OCT) + + High-Numerical Aperture (High-NA) Optics [1] J.A.Izatt, M.R.Hee, G.M.Owen, E.A.Swanson, J.G.Fujimoto, Optical coherence microscopy in scattering media // Optics Letters,1994, Vol.19, No.8, p.590-592. [2] A. Dubois, L. Vabre, A.-C. Boccara, E. Beaurepaire, High-resolution full-field optical coherence tomography with a Linnik microscope // Applied Optics, 2002, Vol.41, No.4, P.805-812. Low temporal coherence (broadband) light High spatial coherence (point) source Point photodetector En-face imaging by point-by-point object scanning Low temporal coherence (broadband) light Low spatial coherence (extended) source Area camera as photodetector En-face imaging by single z-scan Fig.1. Fiber single-point optical coherence microscope (from [1]). Fig.2. Linnik-type full-field optical coherence microscope (from [2]).

4. 4 DEFOCUS PROBLEM Optical Coherence Tomography (OCT) Signal Confocal Microscopy (CM) Signal d – geometrical thickness; n group ≈n(λ 0 ) - λ 0 δn/δλ – group refractive index n=n(λ 0 ) – phase refractive index of object medium; n im =ni m (λ 0 ) – phase refractive index of immersion Fig.3. Degradation of the signal of an Otical Coherence Microscope from the depth of the two layer object (microscope cover glass + air gap between glass and metal mirror) at increasing NA

5. 5 KNOWN METHODS FOR DEFOCUS CORRECTION Extending Depth of Focus Numerical Correction J. Ojeda-Castaneda, L.R. Berriel-Valdos, Zone plate for arbitrarily high focal depth // Applied Optics, 1990, Vol.29, No.7, P.994-997. E.R. Dowski, Jr., W.T. Cathey, Extended depth of field through wave-front coding // Applied Optics, 1995, Vol.34, No.11, P.1859-1866. Z. H. Ding, H. W. Ren, Y. H. Zhao, J. S. Nelson, and Z. P. Chen, High-resolution optical coherence tomography over a large depth range with an axicon lens // Optics Letters. 2002, Vol.27, No.4, P.243–245. R.A. Leitgeb, M. Villiger, A.H. Bachmann, L. Steinmann, T. Lasser, Extended focus depth for Fourier domain optical coherence microscopy // Optics Letters, 2006, Vol.31, No.16, P.2450-2452. K. S. Lee and J. P. Rolland, Bessel beam spectral-domain high-resolution optical coherence tomography with micro-optic axicon providing extended focusing range // Optics Letters, 2008, Vol.33, No.15, P.1696–1698. A. Zlotnik, Y. Abraham, L. Liraz, I. Abdulhalim, Z. Zalevsky, Improved extended depth of focus full field spectral domain Optical Coherence Tomography // Optics Communications, 2010, Vol.283, P.4963-4968. S. Labiau, G. David, S. Gigan, A.C. Boccara, Defocus test and defocus correction in full-field optical coherence tomography // Optics Letters, 2009, Vol.34, No.10, p.1576-1578. A.A. Grebenyuk, V.P. Ryabukho, Numerical correction of coherence gate in full-field swept-source interference microscopy // Optics Letters, 2012, Vol.37, P.2529-2531. Mechanical Adjustment of Optical Elements J.M. Schmitt, S.L. Lee, K.M. Yung, An optical coherence microscope with enhanced resolving power in thick tissue // Optics Communications, 1997, Vol.142, P.203–207. A. Dubois, G. Moneron, C. Boccara, Thermal-light full-field optical coherence tomography in the 1.2 μm wavelength region // Optics Communications, 2006, Vol.266, Iss.2, P.738-743. J. Binding, J. Ben Arous, J.-F. Léger, S. Gigan, C. Boccara, L. Bourdieu, Brain refractive index measured in vivo with high-NA defocus-corrected full-field OCT and consequences for two-photon microscopy // Optics Express, 2011, Vol.19, No.6, P.4833-4847.

6. 6 Fig.4. Adjustment of axial position of microscope objective in object arm of an optical coherence microscope to overlap coherence and focal planes (gates) (from[3]) [3] A. Dubois, G. Moneron, C. Boccara, Thermal-light full-field optical coherence tomography in the 1.2 μm wavelength region // Optics Communications, 2006, Vol.266, Iss.2, P.738-743.

7. 7 MAIN IDEA OF PRESENTED METHOD USAGE OF ILLUMINATING INTERFEROMETER AS LIGHT SOURCE FOR AN OPTICAL COHERENCE MICROSCOPE

8. 8 EXPERIMENTAL SETUP

9. 9 HOW DOES IT WORK Interference peak (2) from the rear plane of a single-layer object in the signal of an interference microscope arising at and posses its maximal value when the shift Δz 1 of the mirror M1 of illuminating interferometer from its zero path length position becomes equal to the value δz comp

10. 10 EXPERIMENTAL RESULTS Fig.5. Experimental results obtaned at single-laer object measurement SLD: λ 0 =0.831 μm, Δλ=16 nm Microscope objective lenses: LOMO® SHP-OPA-20-50, 20x, NA=0.50, ∞/0 Object: Menzel-Gläser® microscope cover slip #1 (Shott ® D263 glass), d=145±1 μm, n(λ 0 )=1.517, n im (λ 0 )=1

11. 11 CONCLUSIONS THANK YOU FOR YOUR ATTENTION!!! We have presented the method for correction of defocus in an optical coherence microscope caused by mismatch of the refractive index of a layered object and immersion and leading to degradation of the interference signal from the depth of the object. This method is based on the usage of an illuminating low-coherence interferometer as a light source for the optical coherence microscope. We have demonstrated experimentally that we are able to compensate this index-induced defocus creating corresponding optical path difference in arms of illuminating low-coherence interferometer. This method can be applied for defocus correction in commercially available interference microscopes which construction does not allow moving optical parts within microscope, for example such as Mirau microscopes. This method can also be used to simultaneous determination of geometrical thickness and refractive index of an object becouse the two quantities δz CM and δz comp are obtained experimentally.