Shaping light in microscopy: Adaptive optical methods and nonconventional beam shapes for enhanced imaging Martí Duocastella
planet detector detector sample
sample Aberrated wavefront Beamsplitter Adaptive optics Corrected wavefront Aberrations detector Aberration-free image
Outline Measuring aberrations Shaping light Adaptive optical methods Key examples Non-conventional beam shapes
Measuring aberrations Shack-Hartmann wavefront sensor tan wavefront slope
Measuring aberrations Zernike coefficients (for systems with circular pupils) coefficient 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Zernike mode Zernike mode 4 corresponds to defocus, 5 and 6 to astigmatism, 7 and 8 to coma
Shaping light Adaptive optics We need an element capable of adapting to the wavefront variations Deformable mirrors Spatial light modulator Others
Deformable mirrors Membranes Micromirrors
M. Booth et al., PNAS (2002) 99, 5789 Deformable membrane
Where is the wavefront sensor? In microscopy: Difficult to find a guide star (a point reference) Need to exclude out of focus light Dual pass nature of the microscope
Sensorless methods are preferred M. Booth et al., Optics & Photonics News (2012)
Deformable micromirror No correction Aberration correction 3D reconstruction of a 40µm-thick slice in a whole fixed mouse embryo D. Débarre et al., Opt. Lett. (2009) 34, 2495
SLM Polarization and wavelength dependence: mostly used in two-photon microscopy
SLM Through 14 µm retina Through 25 µm retina confocal STED STED AO confocal STED STED AO T.J.. Gould et al., Opt. Exp. (2012) 20, 20998
Beyond correcting Focusing on strongly scattering media I.M. Vellekoop et al., Opt. Lett. (2007) 32, 2309
Beyond correcting Simultaneous excitation of dendritic spines by using multiple foci V. Nikolenko et al., Front. Neural Circ. (2008) 2, 2309
Other adaptive optical methods Tradeoff between depth of field and resolution low NA High NA 1/ 1/
Changing focus: varifocal lenses z-focus control
Changing focus: varifocal lenses Optotune Varioptic C.A. Lopez et al., Nat. Photon (2008) 2, 610 TAG lens
High-speed varifocal lens Acoustically-driven liquid lens: Tunable Acoustic Gradient (TAG) lens RF signal 30 mm 25 mm Periodic standing wave in the fluid density, and consequently, the index of refraction E. McLeod et al. Opt. Lett., (2006) 31, 3155
Understanding the TAG refractive index change n(r n(r,t ),t ) n Close to the lens center: n+ 2 n ωr 2 4 ccs s A 2 n J 0 + na A r sin sin ( ωt( ) ωt ) 0 0 parabolic lens 1 Optical Power = = f(t ) Ln 2c ω 2 A 2 s sin ( ωt ) M. Duocastella et al. J. PhysD., (2012) 46, 075102 radial position
Lens power versus time TAG characteristics f = 143 khz A = 10 V pp Wavefront versus time Zernike modes versus time Lens continuously changing focus at a rate given by RF signal The amplitude of the RF signal allows controlling the range of lens power
Lens power versus time TAG characteristics power time By appropriate synchronization between light pulses and TAG, it is possible to select any desired optical power within a range
TAG-enabled microscope Simply place the TAG lens in the imaging path between the tube lens and the objective
Microscope operation To capture a sharp image at each focal position: pulsed light source Change delay between light and lens to select a focal plane Focal position To increase contrast, multiple light pulses synchronized with the TAG lens C.B. Arnold, C. Theriault, M. Duocastella, Patent application 61/552,723 (2011)
Electronic focus control By simply changing the delay we can select a desired focal length 10x objective, A = 10 V pp, f=143 khz 100 µm Surface Height Time delay 30 º
Electronic focus control Electronically scanned focus on histological specimens Kidney, 20x Liver, 20x Lung, 50x
Scanning range 4 mm A=10 V Range of scanning depends on the magnification of the microscope objective Model this using geometric optics 80 µm d: distance between objective and TAG M: objective magnification δ: TAG maximum optical power f tubelens : focal length of the tube lens Scanning range = M 2 δ 2f 2 tube lens 2 TAG δ TAG ( M d f ) 2 tube lens > 4 mm for modest driving amplitude
Can we do better? Simultaneous capture of multiple and selectable focal planes Use color CCD chip + colored illumination to capture single image but split based on pixel type Simultaneous acquisition A A B
Simultaneous capture multiple planes Focal position Two light sources Red: λ=625 nm Blue: λ=465 nm τ = 300 ns
Imaging beads LOC Imaging 15 µm beads flowing through 2 channels separated by 1 mm Plane 1 Plane 2 M. Duocastella et al., J. Biom. Optics (2012) 17, 050505
5 x objective Composite image Using Red and Blue light gives purple composite image M. Duocastella et al., J. Biom. Optics (2012) 17, 050505 100 μm
Separating color channels Blue channel: Red channel: 10 x objective Video is real time 100 μm We can image both sets of beads simultaneously
About adaptive optics Powerful method to correct for aberrations in optical systems Traditional correction approach is based on direct sensing. In microscopy, indirect sensing is preferred It is possible to go beyond correction and to use AO for enhanced speed imaging, 3D microscopy
Non-conventional beam shapes One solution of the Helmholtz equation: are simply plane waves: In the paraxial approximation and using Cartesian coordinates, we can obtain:
Gaussian beam
Bessel beam The solution of the Helmholtz equation in cylindrical coordinates: J0 J1
Non-diffracting beam Enhanced imaging in SPIM M. Duocastella et al., Laser & Photon Rev. (2012) T.A. Planchon et al., Nat. Methods (2011)
Self-healing properties Bessel beam Gaussian beam F.O. Fahrbarch et al., Nat. Photon (2010)
Other non-diffracting beams? Mathieu beams Airy beams Why don t you use them in microscopy?