1. Page 1 AO Microscopy of Biological Systems Slides thanks to Prof. Joel Kubby, EE at UCSC

2. Page 2 Wide-Field AO Correction of Green Fluorescent Sample Beads AO OffAO On Drosophila Embryo Oscar Azucena, UCSC

3. Page 3 AO in Astronomy and Biology AO reveals a binary starAO reveals distinct structures

4. Page 4 Wavefront Aberrations in Microscopy n a =1.000 n w =1.331

5. Page 5 Wavefront Aberrations in Microscopy Wavefront aberrations due to a change in a sample’s refractive index δn

6. Page 6 Fluorescent Protein Guide-Stars GFP-labeled centrosomes for biological guide-stars in adaptive optic microscopy Prof. Roy, McGill University

7. Page 7 Centrosome Kinetochore Chromosome Guide-star Proteins can be Located in the Mitotic Apparatus

8. Page 8 Wavefront Measurements Oscar Azucena, UCSC

9. Page 9 Wavefront Aberrations Measured in Drosophila Embryo Oscar Azucena, UCSC

10. Page 10 Confocal Images of GFP Labeled Tubulin in Drosophila Embryos Surface30 μm below the surface Prof. William Sullivan, UCSC MCD Biology

11. Page 11 AO Wide-Field Microscope Oscar Azucena, UCSC

12. Page 12 Injection of Fluorescent Bead Reference Beacons in Drosophila Embryo 1 μm crimson beads Oscar Azucena, UCSC

13. Page 13 Wide-Field AO Correction of Crimson Reference Beacon Uncorrected image of a bead 40% correction The length of the bar is equal to the diffraction limit of the 40X (0.75 NA) objective lens, 0.45 µm. 10X improvement in relative Strehl ratio Correction of 1 μm microsphere 100 μm beneath surface embryo Oscar Azucena, UCSC

14. Page 14 Wide-Field AO Correction of 1 μm Green Sample Beads AO OffAO On Drosophila Embryo Oscar Azucena, UCSC

15. Page 15 AO Confocal Microscope Xiaodong Tao, UCSC

16. Page 16 AO 2P Microscope PMT Wavefront sensor DB1DB2DB3 F1 L1 L2 L4L5 L6 L7 Deformable Mirror X Scanner Tube Lens Ti:Sapphire Laser 700nm – 1100nm Tunable Helium Neon Laser (633nm) Y Scanner F2 Xiaodong Tao, UCSC

17. Page 17 Wavefront measurements from a 1 μm fluorescent microsphere through 100 μm thick brain tissue Xiaodong Tao, UCSC

18. Page 18 Confocal Images of Mouse Brain Tissue AO OffAO On 15 μm 50 μm 100 μm Xiaodong Tao, UCSC

19. Page 19 Fluorescent Protein Guide-Stars (YFP) DendriteCell Body Xiaodong Tao, UCSC

20. Page 20 Drosophila Embryo The images and PSF without (a) and with (b) correction for a cycle 14 fruit fly embryo with GFP-polo at the depth of 83 μm. Scale bars, 2 µm Before correctionAfter correction Cannot see the centrosomes Can see the centrosomes Xiaodong Tao, UCSC Depth: 83 μ m

21. Page 21

22. Page 22 Live Imaging of Centrosomes in Drosophila Embryo

23. Page 23 Improvement in Deep Tissue Imaging Xiaodong Tao, UCSC

24. Page 24 Conclusions Fluorescent proteins can be used as reference beacons for wavefront measurements in adaptive optics Improve relative Strehl ratio in 20 μ m thick Drosophila embryo by up to 10x Improve relative Strehl ratio in 100 μ m thick mouse brain tissue imaged by AO confocal microscope by up to 4.7x Currently imaging GFP (Drosophila) and YFP (mouse brain tissue) labeled samples Extending Measurements to Live Imaging

25. Page 25 Problems with Indirect Approaches to AO in Biological Imaging Too slow for live imaging Photo-toxicity and photo-bleaching of sample Information can be lost

26. Page 26 Phase Stepping Interferometry M. Schwertner, M. J. Booth, M. A. A. Neil, T. Wilson, Measurement of specimen- induced aberrations of biological samples using phase stepping interferometry, Journal of Microscopy Volume 213, Issue 1, pp. 11–19 (2004)

27. Page 27 Aberrations in Biological Specimens Mouse brainMouse ooctyeMouse liver Mouse heart musclec. elegans Mouse smooth muscle Martin J. Booth, Michael Schwertner and Tony Wilson

28. Page 28 mm mm Thickness = 50 μ mThickness = 200 μ m Peak-Valley: 2 μ m Aberrations in Brain Tissue 1.0 0.8 0.6 0.4 0.2 0.0 -0.8 -0.6 -0.4 -0.2 1.0 0.8 0.6 0.4 0.2 0.0 -0.8 -0.6 -0.4 -0.2 Xiaodong Tao, UCSC

29. Page 29 Thickness = 30 μ mThickness = 50 μ mThickness = 80 μ m Thickness = 100 μ m Thickness = 150 μ m Thickness = 200 μ m 4 6 8 10 12 14 16 18 20 22 24 Aberrations in Brain Tissue 0.05 0.00 -0.05 -0.10 -0.15 -0.20 -0.25 0.05 0.00 -0.05 -0.10 -0.15 -0.20 -0.25 0.05 0.00 -0.05 -0.10 -0.15 -0.20 -0.25 0.05 0.00 -0.05 -0.10 -0.15 -0.20 -0.25 0.05 0.00 -0.05 -0.10 -0.15 -0.20 -0.25 0.05 0.00 -0.05 -0.10 -0.15 -0.20 -0.25 μmμm μmμm μmμm μmμm μmμm μmμm # # Xiaodong Tao, UCSC Spherical aberration increases with depth