Submicron-resolution Photoacoustic Microscopy of Endogenous Light-absorbing Biomolecules

105 Pages·2016·5.05 MB·English

by  Chi Zhang

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All Theses and Dissertations (ETDs)
Spring 3-26-2014
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Chi Zhang
Washington University in St. Louis
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Zhang, Chi, "Submicron-resolution Photoacoustic Microscopy of Endogenous Light-absorbing
Biomolecules" (2014). All Theses and Dissertations (ETDs). 1275.
https://openscholarship.wustl.edu/etd/1275
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WASHINGTON UNIVERSITY IN ST. LOUIS
School of Engineering and Applied Science
Department of Biomedical Engineering

Thesis Examination Committee:
Lihong V. Wang, Chair
Gregory Lanza
Jin-Moo Lee
James Miller
Jung-Tsung Shen

Submicron-resolution Photoacoustic Microscopy of Endogenous Light-absorbing Biomolecules
by
Chi Zhang

A dissertation presented to the Graduate School of Arts and Sciences
of Washington University in partial fulfillment of the
requirements for the degree of
Doctor of Philosophy

May 2014

Saint Louis, Missouri
© 2014, Chi Zhang
Contents

List of Figures ................................................................................................................................................. iii

List of Abbreviations ...................................................................................................................................... v

Acknowledgments.......................................................................................................................................... vi

Abstract ........................................................................................................................................................... viii

1 Introduction ............................................................................................................................................... 1
1.1 Photoacoustic Imaging ..................................................................................................................... 1
1.2 Motivation .......................................................................................................................................... 2

2 High-resolution 3D Photoacoustic Microscopy.............................................................................. 3
2.1 Subwavelength-resolution Photoacoustic Microscopy in Transmission Mode ....................... 3
2.2 Submicron-resolution Photoacoustic Microscopy in Reflection Mode .................................... 9
2.3 Micron Axial Resolution Achieved with a 125 MHz Ultrasonic Transducer ........................ 16
2.4 Application in Intracellular Temperature Imaging..................................................................... 29
2.5 Conclusions...................................................................................................................................... 38

3 Endogenous Light-absorbing Biomolecules for Photoacoustic Microscopy ....................... 39
3.1 Photoacoustic Microscopy of Cytochromes ............................................................................... 39
3.2 Photoacoustic Microscopy of Myocardium ................................................................................ 46
3.3 Conclusions...................................................................................................................................... 53

4 Label-free Sectioning Photoacoustic Microscopy ........................................................................ 54
4.1 Sectioning Photoacoustic Microscopy ......................................................................................... 54
4.2 Label-free Photoacoustic Brain Histology .................................................................................. 60 3.1.2 Numbered References 14
4.3 Conclusions...................................................................................................................................... 65

5 Summary and Outlook .......................................................................................................................... 66
5.1 Summary........................................................................................................................................... 66
5.2 Outlook ............................................................................................................................................ 67

Appendix Fast and Robust Deconvolution-based Image Reconstruction
for Photoacoustic Computed Tomography in Circular Geometry .................. 69

References ....................................................................................................................................................... 82

Vita .................................................................................................................................................................... 91

ii
List of Figures

Figure 2.1: Subwavelength-resolution photoacoustic microscopy (SW-PAM)....................................... 4
Figure 2.2: Ex vivo images of cells ................................................................................................................. 6
Figure 2.3: PAM images of a black mouse ear, showing the distribution of melanin ........................... 7
Figure 2.4: Monitoring of melanoma growing on a nude mouse ear ....................................................... 8
Figure 2.5: Major forms of reflection-mode OR-PAM ............................................................................ 10
Figure 2.6: Reflection-mode submicron-resolution PAM ....................................................................... 11
Figure 2.7: Measuring the lateral resolution of the submicron-resolution PAM .................................. 12
Figure 2.8: Measurement of the axial resolution of the submicron-resolution PAM .......................... 13
Figure 2.9: Measurement of the penetration depth of the submicron-resolution PAM ..................... 14
Figure 2.10: Comparing the submicron-resolution PAM with a 2.4 µm-resolution PAM by
imaging a mouse ear in vivo ....................................................................................................... 15
Figure 2.11: Schematic of the high-axial-resolution PAM system ............................................................ 19
Figure 2.12: Experimentally measuring the axial resolution of PAM ....................................................... 21
Figure 2.13: Axial resolution of PAM enhanced by silicone oil immersion ............................................ 22
Figure 2.14: Measuring the maximum imaging depths of PAM from both the acoustic and
optical sides ................................................................................................................................. 23
Figure 2.15: Imaging of a melanoma cell ...................................................................................................... 24
Figure 2.16: Comparison of in vivo PAM images of a mouse ear acquired with 50 MHz and
125 MHz ultrasonic transducers .............................................................................................. 25
Figure 2.17: In vivo PAM images of a mouse ear with silicone oil injection ............................................ 26
Figure 2.18: FAPT system setup .................................................................................................................... 32
Figure 2.19: Calibration of PA/fluorescence ratio versus temperature ................................................... 33
Figure 2.20: 2D temperature mapping of a thin layer of Rhodamine 6G dye ........................................ 34
Figure 2.21: Intracellular mitochondrial temperature mapping by FAPT ............................................... 36
Figure 3.1: Schematic of the spectral PAM system ................................................................................... 41
Figure 3.2: Absorption spectra ..................................................................................................................... 42
Figure 3.3: PAM and fluorescence microscopy of fibroblasts ................................................................ 43
Figure 3.4: Imaging of a mouse ear section ............................................................................................... 45
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Figure 3.5: Schematic of the PAM system for myocardium imaging ..................................................... 47
Figure 3.6: Spectra of the absorption coefficient of the blood-free mouse myocardium ................... 48
Figure 3.7: Imaging of a histological section of a dog heart in the left ventricular wall
region with and without labeling ............................................................................................. 50
Figure 3.8: Imaging of a blood-free half-split mouse heart (unfixed and unstained) .......................... 51
Figure 3.9: 3D image stacks in the same area as Fig. 3.8(b) down to 150 µm in depth ...................... 52
Figure 4.1: Schematic of sectioning photoacoustic microscopy (SPAM) .............................................. 56
Figure 4.2: Resolution of SPAM .................................................................................................................. 57
Figure 4.3: Extracting cell nuclei from SPAM images .............................................................................. 58
Figure 4.4: Imaging of a paraffin section of a mouse brain ..................................................................... 60
Figure 4.5: Comparison between SPAM images of a paraffin block surface and H&E
images of the paraffin sections from the block surface ........................................................ 61
Figure 4.6: 3D SPAM image of an unstained mouse brain embedded in a paraffin block ................. 63
Figure 4.7: 3D SPAM image of an unstained mouse lung embedded in a paraffin block .................. 64
Figure A.1: Illustration of detection geometry and photoacoustic signal integration ........................... 74
Figure A.2: Experimental setup of PACT ................................................................................................... 77
Figure A.3: Received photoacoustic signals and constructed space function C(r) ................................ 78
Figure A.4: In vivo and noninvasive reconstructed images ........................................................................ 79
Figure A.5: Time costs of the back-projection algorithm and deconvolution
reconstruction algorithm ........................................................................................................... 80

iv
List of Abbreviations

1D One dimensional
2D Two dimensional
3D Three dimensional
CNR Contrast-to-noise ratio
DR Deconvolution reconstruction
ESF Edge spread function
FAPT Fluorescent-assisted photoacoustic thermometry
FFT Fast Fourier transformation
FWHM Full width at half maximum
H&E Hematoxylin and eosin
IFFT Inverse fast Fourier transformation
MAP Maximum-amplitude projection
NA Numerical aperture
OR-PAM Optical-resolution photoacoustic microscopy
PA Photoacoustic
PAI Photoacoustic imaging
PAM Photoacoustic microscopy
PSF Point spread function
R2 Coefficient of determination
SNR Signal-to-noise ratio
SPAM Sectioning photoacoustic microscopy
SW-PAM Subwavelength-resolution photoacoustic microscopy
UV Ultraviolet

v
Acknowledgments

I gratefully acknowledge the guidance, inspiration, and support from my research advisor, Dr.
Lihong Wang. I also thank him for helping me establish a high ethical standard and a positive
attitude throughout my Ph.D. study. This dissertation is based on the teamwork of many lab
members and collaborators. I appreciate the contributions, hard work, and inspiring discussions
from them, especially from Dr. Konstantin Maslov.

I would like to extend my appreciation to everyone who has helped me at Washington University.
No virtue is trivial. I cherish them.

Chi Zhang

Washington University in St. Louis
May 2014

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Dedicated to my family

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ABSTRACT OF THE DISSERTATION

Submicron-resolution Photoacoustic Microscopy of Endogenous Light-absorbing Biomolecules
by
Chi Zhang
Doctor of Philosophy in Biomedical Engineering
Washington University in St. Louis, 2014
Professor Lihong V. Wang, Chair

Photoacoustic imaging in biomedicine has the unique advantage of probing endogenous light
absorbers at various length scales with a 100% relative sensitivity. Among the several modalities of
photoacoustic imaging, optical-resolution photoacoustic microscopy (OR-PAM) can achieve high
spatial resolution, on the order of optical wavelength, at <1 mm depth in biological tissue (the
optical ballistic regime). OR-PAM has been applied successfully to structural and functional imaging
of blood vasculature and red blood cells in vivo. Any molecules which absorb sufficient light at
certain wavelengths can potentially be imaged by PAM. Compared with pure optical imaging, which
typically targets fluorescent markers, label-free PAM avoids the major concerns that the fluorescent
labeling probes may disturb the function of biomolecules and may have an insufficient density. This
dissertation aims to advance label-free OR-PAM to the subcellular scale.

The first part of this dissertation describes the technological advancement of PAM yielding high
spatial resolution in 3D. The lateral resolution was improved by using optical objectives with high
numerical apertures for optical focusing. The axial resolution was improved by using broadband
ultrasonic transducers for ultrasound detection. We achieved 220 nm lateral resolution in
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