Table Of ContentA Laboratory Manual
in Biophotonics
A Laboratory Manual
in Biophotonics
Vadim Backman
Adam Wax
Hao F. Zhang
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Library of Congress Cataloging-in-Publication Data
Names: Backman, Vadim, author. | Wax, Adam, author. | Zhang, Hao (Hao F.), author.
Title: A laboratory manual in biophotonics / Vadim Backman, Adam Wax, and Hao Zhang.
Description: Boca Raton : Taylor & Francis, 2017. | Includes bibliographical references and index.
Identifiers: LCCN 2017037476| ISBN 9781439810514 (hardback : alk. paper) |
ISBN 9781498744331 (ebook)
Subjects: | MESH: Light | Tissues--physiology | Phototrophic Processes |
Optics and Photonics--methods | Optics and Photonics--instrumentation | Laboratory Manuals
Classification: LCC R857.T55 | NLM QT 162.L5 | DDC 616.07/548--dc23
LC record available at https://lccn.loc.gov/2017037476
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Contents
Preface ..............................................................................................................................................xi
Authors .........................................................................................................................................xiii
1. General Introductory Topics ................................................................................................1
Part 1: Fundamental Mathematics .........................................................................................1
Dirac Delta Pulse .................................................................................................................1
Kronecker Delta Pulse ........................................................................................................3
Kronecker Comb .................................................................................................................3
sinc Function ........................................................................................................................3
Convolution ..........................................................................................................................4
Cross-Correlation ................................................................................................................4
Autocorrelation ....................................................................................................................5
Fourier Transform ...............................................................................................................6
Discrete Fourier Transform ...............................................................................................8
Least-Squares Fit .................................................................................................................9
Method of Least-Squares Fit for Multiple Parameters ............................................10
Nyquist–Shannon Sampling Theorem ..........................................................................11
Basic Statistics ....................................................................................................................11
Student’s t-Test ...................................................................................................................13
Speckle Contrast ................................................................................................................14
Statistical Distribution Functions ...................................................................................15
Part 2: Fundamental Biology ................................................................................................16
Basic Biological Concepts .................................................................................................16
Cell Structure ................................................................................................................17
Tissue Structure ............................................................................................................34
References ...............................................................................................................................37
2. Optics Components and Electronic Equipment .............................................................39
Lenses ......................................................................................................................................40
Convex Lenses ...................................................................................................................40
Concave Lenses .................................................................................................................41
Compound Lenses and Arrays .......................................................................................41
Maintenance and Care of Optical Lenses ..........................................................................43
Handling ............................................................................................................................44
Storage ................................................................................................................................44
Cleaning and Maintenance .............................................................................................44
Waveplate ...........................................................................................................................45
Linear Polarizers ....................................................................................................................46
Mirrors .....................................................................................................................................46
Plane Mirror .......................................................................................................................47
Convex Mirror ...................................................................................................................47
Concave Mirror .................................................................................................................48
Optical Coating .............................................................................................................49
Metal Coatings ..............................................................................................................49
v
vi Contents
Antireflection Coatings ...............................................................................................50
High-Reflection Coatings ............................................................................................50
Filters .......................................................................................................................................50
Prisms ......................................................................................................................................51
Combination Prisms .........................................................................................................52
Optical Fibers .........................................................................................................................53
Structure .............................................................................................................................53
Single-Mode and Multimode Fibers ...............................................................................55
Fiber Connectors ...............................................................................................................55
Optomechanical Equipment ................................................................................................56
Optical Table ......................................................................................................................56
Translational Stages ..........................................................................................................56
Rotation Stages ..................................................................................................................57
Mirror Mounts ...................................................................................................................58
Lasers ..................................................................................................................................58
Photodetectors ...................................................................................................................62
Photodiodes .......................................................................................................................62
Phototransistors.................................................................................................................62
Photomultiplier Tubes ......................................................................................................63
Charge-Coupled Devices ......................................................................................................63
Power Meter .......................................................................................................................64
Spectrometers ....................................................................................................................64
Electrical Equipment .............................................................................................................65
Data Acquisition System .......................................................................................................66
Amplifiers...........................................................................................................................66
Digitizer ..............................................................................................................................67
Signal Generation ..............................................................................................................69
Power Supply .....................................................................................................................69
Function Generator ...........................................................................................................70
Electrical and Light Safety....................................................................................................71
Electrical Safety .................................................................................................................71
Light Safety ........................................................................................................................71
3. Fundamental Light–Tissue Interactions: Light Scattering and Absorption ............73
Principles of Light Scattering and Absorption ..................................................................73
Refractive Index of Biological Tissue ..................................................................................74
Basics of Theory of Light Scattering and Absorption ......................................................78
Scattering and Absorption of Light in Tissue by Small Particles ...................................85
Lab Discussion .......................................................................................................................99
Laboratory 1: Absorption: Measurement of Hemoglobin Concentration ...............100
Laboratory Protocol ........................................................................................................100
Instrumentation ..........................................................................................................100
LED Sources ................................................................................................................101
Lenses...........................................................................................................................103
Detectors ......................................................................................................................103
Mechanical Design .....................................................................................................104
Sample Preparation .........................................................................................................105
Experiment ..................................................................................................................105
Data Analysis ..............................................................................................................106
Contents vii
Laboratory 2: Biomedical Application of Absorption Measurements .....................106
Laboratory 3: Scattering: Measurement of Structure .................................................109
Attenuation and Anisotropy of Scattering .............................................................109
Instrumentation ..........................................................................................................110
Sample Preparation ....................................................................................................110
Experiment ..................................................................................................................110
Data Analysis ..............................................................................................................111
Backscattering Cross-Section Spectra .....................................................................111
Instrumentation ..........................................................................................................113
Sample Preparation ....................................................................................................114
Experiment ..................................................................................................................114
Data Analysis ..............................................................................................................115
Laboratory 4: Biomedical Application of Scattering Measurements .......................115
References .............................................................................................................................117
4. Microscopic Tissue Imaging ............................................................................................119
Principles of Optical Microscopy ......................................................................................119
Types of Contrast in Microscopy .......................................................................................124
Phase Contrast .................................................................................................................124
Spectral Contrast: Scattering .........................................................................................130
Spectral Contrast: Fluorescence ....................................................................................131
Spectral Contrast: Nonlinear Scattering ......................................................................133
Depth Sectioning in Microscopy .......................................................................................134
Laboratory Exercises ...........................................................................................................138
Laboratory 1: Overview of the Microscope: Kohler Illumination ...........................138
Laboratory Protocol ...................................................................................................138
Materials and Sample Preparation ..........................................................................140
Experiment ..................................................................................................................140
Data Analysis ..............................................................................................................141
Laboratory 2: Contrasts in Microscopy ........................................................................142
Instrumentation ..........................................................................................................142
Phase Contrast Settings .............................................................................................142
DIC Settings ................................................................................................................142
Fluorescence Settings ................................................................................................142
Digital Camera Settings ............................................................................................143
Materials and Sample Preparation ..........................................................................143
Experiment ..................................................................................................................144
Analysis .......................................................................................................................145
Laboratory 3: Build Your Own Microscope ................................................................145
Discussion ...................................................................................................................145
Materials ......................................................................................................................146
Exercises ......................................................................................................................147
Laboratory 4: Interference-Based Imaging ..................................................................150
Instrumentation ..........................................................................................................151
Materials and Sample Preparation ..........................................................................152
Experiment ..................................................................................................................152
Data Analysis ..............................................................................................................154
Confocal Light Absorption and Scattering Spectroscopic Microscopy .......................155
Introduction .....................................................................................................................155
viii Contents
Light Scattering Spectroscopy for the Determination of Particle Size ....................156
Effect of Objective Lens on LSS ....................................................................................157
Laboratory 5: Experimental Observation of LSS and Effect of Finite
NA on LSS Spectrum ......................................................................................................158
Laboratory 6: Spectroscopic Microscopy and Thin-Film Interference ....................159
Background .................................................................................................................160
Thin-Film Interference Basics: Recovering Film Properties from
Spectrum (Normal Incidence) ..................................................................................161
Slab with Inhomogeneous Refractive Index ..........................................................162
Backscattering in Three-Dimensional Media ........................................................165
Laboratory Protocol .............................................................................................................166
Instrumentation...............................................................................................................166
Measurement Protocol ...................................................................................................166
Experiment 1: Thin-Film Interference Observed from Microspheres ................167
Experiment 2: Interference Spectrum from an Inhomogeneous Film
of Nanospheres ...........................................................................................................168
Experiment 3: Interference Spectrum from a Biological Cell as an
Inhomogeneous Film .................................................................................................169
Answers and Hints .........................................................................................................170
Experiment 1: Data Analysis ....................................................................................170
Experiment 2: Sample Preparation ..........................................................................170
Data Analysis ..............................................................................................................171
Laboratory 7: Measurement of Real and Imaginary Refractive
Index of Cells ...................................................................................................................171
Background .................................................................................................................171
Relationship between the Real and Imaginary Parts of the
Refractive Index ..........................................................................................................172
Laboratory Protocol .............................................................................................................173
Instrumentation...............................................................................................................173
What Is Needed ..........................................................................................................173
Sample Preparation .........................................................................................................173
Experiment .......................................................................................................................174
Data Analysis ...................................................................................................................174
References .............................................................................................................................175
5. Tissue Spectroscopy ...........................................................................................................177
Light Transport in a Medium with Continuously Varying Refractive Index .............178
Discussion ........................................................................................................................178
Differential Scattering Cross-Section ...........................................................................179
Modeling Continuous Media with a Whittle–Matérn Correlation Function ........179
Range of Validity .............................................................................................................182
Phase Function–Corrected Diffusion Approximation ...................................................182
Introduction .....................................................................................................................182
Transport Equation for the Phase Function–Corrected Radiance ...........................183
Derivation of the Phase Function–Corrected Radiance, Fluence Rate,
and Reflectance ................................................................................................................188
Accuracy of the Phase Function–Corrected Diffusion Approximation .................190
Physical Interpretation of the Phase Function–Corrected Radiance .......................191
Summary and Concluding Remarks............................................................................191
Contents ix
Laboratory 1: Building a Spectrometer .............................................................................193
Suggested Lab Components ..........................................................................................194
Laboratory Protocol ........................................................................................................194
Analysis ............................................................................................................................195
Discussion ........................................................................................................................196
Questions ..........................................................................................................................196
Laboratory 2: Diffuse Reflectance Spectroscopy .............................................................196
Measurement of Optical Properties .............................................................................196
Materials ...........................................................................................................................197
Lab Protocol .....................................................................................................................197
Analysis ............................................................................................................................198
Questions ..........................................................................................................................199
Learning Objectives ........................................................................................................200
Laboratory 3: Measurement of Optical Properties of Tissue Using
Spectroscopic Optical Coherence Tomography ...............................................................200
Background ......................................................................................................................200
Laboratory Protocol ........................................................................................................204
Suggested Lab Components ..........................................................................................204
Exercises ...........................................................................................................................204
Discussion ........................................................................................................................206
Questions ..........................................................................................................................206
Laboratory 4: Depth-Selective Tissue Spectroscopy: Measuring Reflectance
Impulse Response Function by Using Enhanced Backscattering ................................207
Enhanced Backscattering: An Introduction ................................................................207
The Scalar Theory of EBS ...............................................................................................207
Vector Theory of EBS ......................................................................................................210
Effects of Partial Spatial Coherence Illumination and Finite Illumination
Spot Size on EBS ..............................................................................................................215
Full EBS Equation............................................................................................................220
Penetration Depth and Measurement of Optical Properties Using
Low-Coherence EBS ........................................................................................................220
Laboratory .............................................................................................................................223
Materials ...........................................................................................................................223
Exercises ...........................................................................................................................225
Laboratory 5: Depth-Selective Tissue Spectroscopy: Polarization-Gated
Spectroscopy .........................................................................................................................227
Laboratory .............................................................................................................................230
Materials ...........................................................................................................................230
Protocol .............................................................................................................................231
Analysis ............................................................................................................................231
Questions ..........................................................................................................................232
Learning Objectives ........................................................................................................232
References .............................................................................................................................232
6. Computational Biophotonics............................................................................................237
Overview of Computational Biophotonics Methods ......................................................237
Laboratory 1: Designing a Spherical Particle Suspension to Mimic Scattering
Properties ..............................................................................................................................244
Discussion ........................................................................................................................244
