page 1 ao microscopy of biological systems slides thanks to prof. joel kubby, ee at ucsc

AO Microscopy of Biological AO Microscopy of Biological SystemsSystems

• Slides thanks to Prof. Joel Kubby, EE at UCSC

Wide-Field AO Correction of Green Fluorescent Sample Beads

AO Off AO On

Drosophila Embryo

Oscar Azucena, UCSC

AO in Astronomy and Biology

AO reveals a binary star AO reveals distinct structures

Wavefront Aberrations in Microscopy

na=1.000

nw=1.331

Wavefront Aberrations in Microscopy

Point Source

WavefrontAberration

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Objective Lens

Point Source

WavefrontAberration

n

Point Source

WavefrontAberration

n

Objective Lens

Wavefront aberrations due to a change in a sample’s refractive

index δn

Fluorescent Protein Guide-Stars

GFP-labeled centrosomes for biological guide-stars in adaptive optic microscopy

Prof. Roy, McGill University

Centrosome

Kinetochore

Chromosome

Guide-star Proteins can be Located in the Mitotic Apparatus

Wavefront Measurements

Oscar Azucena, UCSC

Wavefront Aberrations Measured in Drosophila Embryo

Oscar Azucena, UCSC

Confocal Images of GFP Labeled Tubulin in Drosophila Embryos

Surface 30 μm below the surface

Prof. William Sullivan, UCSC MCD Biology

AO Wide-Field Microscope

Oscar Azucena, UCSC

Injection of Fluorescent BeadReference Beacons in Drosophila Embryo

1 μm crimson beads

Oscar Azucena, UCSC

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

Wide-Field AO Correction of 1 μm Green Sample Beads

AO Off AO On

Drosophila Embryo

Oscar Azucena, UCSC

AO Confocal Microscope

L1

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L: Lens

L10

Xiaodong Tao, UCSC

AO 2P Microscope

PMT

Wavefront sensor DB1 DB2 DB3

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X Scanner

Tube Lens

Ti:Sapphire Laser 700nm – 1100nm Tunable

Helium Neon Laser(633nm)

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F2

Xiaodong Tao, UCSC

Wavefront measurements from a 1 μm fluorescent microsphere through 100 μm thick brain tissue

Xiaodong Tao, UCSC

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Confocal Images of Mouse Brain Tissue

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AO Off AO On

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Xiaodong Tao, UCSC

Fluorescent Protein Guide-Stars (YFP)

Dendrite Cell Body

Xiaodong Tao, UCSC

Drosophila Embryo

(a) (b)

PSFPSF

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 correction After correction

Cannot see the centrosomes Can see the centrosomes

Xiaodong Tao, UCSC

Depth: 83 μm

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Confocal imagingWavefront measurement Open loop control

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Live Imaging of Centrosomes in Drosophila Embryo

Improvement in Deep Tissue Imaging

Xiaodong Tao, UCSC

Conclusions

• Fluorescent proteins can be used as reference beacons for wavefront measurements in adaptive optics

• Improve relative Strehl ratio in 20 μm thick Drosophila embryo by up to 10x

• Improve relative Strehl ratio in 100 μm 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

Problems with Indirect Approachesto AO in Biological Imaging

• Too slow for live imaging

• Photo-toxicity and photo-bleaching of sample

• Information can be lost

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)

Aberrations in Biological Specimens

Mouse brain Mouse ooctye Mouse liver

Mouse heart muscle c. elegans Mouse smooth muscle

Martin J. Booth, Michael Schwertner and Tony Wilson

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Peak-Valley: 2 μm

Aberrations in Brain Tissue

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Xiaodong Tao, UCSC

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Aberrations in Brain Tissue

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Xiaodong Tao, UCSC

Spherical aberration increases with depth

page 1 ao microscopy of biological systems slides thanks to prof. joel kubby, ee at ucsc

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