Shaping light in microscopy:

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Beverley Sims
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1 Shaping light in microscopy: Adaptive optical methods and nonconventional beam shapes for enhanced imaging Martí Duocastella

3 planet detector detector sample

4 sample Aberrated wavefront Beamsplitter Adaptive optics Corrected wavefront Aberrations detector Aberration-free image

5 Outline Measuring aberrations Shaping light Adaptive optical methods Key examples Non-conventional beam shapes

6 Measuring aberrations Shack-Hartmann wavefront sensor tan wavefront slope

7 Measuring aberrations Zernike coefficients (for systems with circular pupils) coefficient Zernike mode Zernike mode 4 corresponds to defocus, 5 and 6 to astigmatism, 7 and 8 to coma

8 Shaping light Adaptive optics We need an element capable of adapting to the wavefront variations Deformable mirrors Spatial light modulator Others

9 Deformable mirrors Membranes Micromirrors

10 M. Booth et al., PNAS (2002) 99, 5789 Deformable membrane

11 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

12 Sensorless methods are preferred M. Booth et al., Optics & Photonics News (2012)

13 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

14 SLM Polarization and wavelength dependence: mostly used in two-photon microscopy

15 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

16 Beyond correcting Focusing on strongly scattering media I.M. Vellekoop et al., Opt. Lett. (2007) 32, 2309

17 Beyond correcting Simultaneous excitation of dendritic spines by using multiple foci V. Nikolenko et al., Front. Neural Circ. (2008) 2, 2309

18 Other adaptive optical methods Tradeoff between depth of field and resolution low NA High NA 1/ 1/

19 Changing focus: varifocal lenses z-focus control

20 Changing focus: varifocal lenses Optotune Varioptic C.A. Lopez et al., Nat. Photon (2008) 2, 610 TAG lens

$refraction E. McLeod et al. Opt.$

21 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

23 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, radial position

24 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

25 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

26 TAG-enabled microscope Simply place the TAG lens in the imaging path between the tube lens and the objective

27 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)

28 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

29 Electronic focus control Electronically scanned focus on histological specimens Kidney, 20x Liver, 20x Lung, 50x

30 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

31 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

32 Simultaneous capture multiple planes Focal position Two light sources Red: λ=625 nm Blue: λ=465 nm τ = 300 ns

33 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,

34 5 x objective Composite image Using Red and Blue light gives purple composite image M. Duocastella et al., J. Biom. Optics (2012) 17, μm

35 Separating color channels Blue channel: Red channel: 10 x objective Video is real time 100 μm We can image both sets of beads simultaneously

36 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

37 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:

38 Gaussian beam

39 Bessel beam The solution of the Helmholtz equation in cylindrical coordinates: J0 J1

$Non-diffracting beam Enhanced imaging in SPIM M. Duocastella et al.$

40 Non-diffracting beam Enhanced imaging in SPIM M. Duocastella et al., Laser & Photon Rev. (2012) T.A. Planchon et al., Nat. Methods (2011)

41 Self-healing properties Bessel beam Gaussian beam F.O. Fahrbarch et al., Nat. Photon (2010)

$Other non-diffracting$ Mathieu beams Airy

42 Other non-diffracting beams? Mathieu beams Airy beams Why don t you use them in microscopy?

Aberrations and adaptive optics for biomedical microscopes

Aberrations and adaptive optics for biomedical microscopes Martin Booth Department of Engineering Science And Centre for Neural Circuits and Behaviour University of Oxford Outline Rays, wave fronts and