Laboratory Imaging & Photography

Michael Peres is the editor-in-chief of The Focal Encyclopedia of Photography, 4th Edition, and former chair of the biomedical photographic communications department at the Rochester Institute of Technology. Since 1986, he has taught photomicrography, biomedical photography and other applications of photography used in science. Prior to joining the RIT faculty, Peres worked at Henry Ford Hospital and the Charleston Division of West Virginia University as a medical photographer. He is the recipient of the RIT Eisenhart Outstanding Teaching Award and the Schmidt Medal for Lifetime Achievement in the Field of Biocommunications.

Acknowledgements

Preface: The Beginning

Introduction: The Imaging Chain

Foundations, Fundamentals, Principles and Theory

Chapter 1
Defining a Science Image

A Frame of Reference for the Image in Science
The Science Image: a point of departure
Criteria for Good Photography
Science Photographs require a Scale
Photographer’s Intent and Subject Matter
A picture is worth a thousand words
The beginnings of permanent photographs and scientific photography
Making the Invisible visible
Historical images and Contemporary Point of View
Standardized Approaches and Repeatability
Father of Standardized Imaging
Innovators and technological progress
Instrumentation
Microscopy and Carl Zeiss
The Invisible Spectrum
Advancement of Film Technology – Kodak. Agfa, Ilford and Polaroid
Short Duration Light, Electric Flash and Stroboscopes
Modern Technologies - Digital and Electronic Photography
Scanning Electron Microscopy
Confocal Microscope
Duality of Images
Science Images as Art

Chapter 2
Human Vision and Perception

The Human Visual System
The Imaging Room
Seeing
Basic Structure of the Human Visual System
Optics of the Eye and Image Formation
Physiology of Seeing
Dominant Eye
Visual Perception and the Physiological of Sight
Perception of color
Persistence of Vision
Afterimage
Perception of Depth
Adaption
More on Perception
Illusions

Chapter 3
Applied Physics and Image Formation for the Scientific Photographer

Visibility requires Contrast, Magnification, and Resolution
Light & Illumination
Sources and Spectrums
Continuous and Discontinuous Spectrums
Color temperature
Light behaviours
Reflection
Refraction
Dispersion
Interference
Lenses
Lenses for Scientific Applications
Fundamental optics
Teleconverters
Working Distance
Close up Lenses
Supplementary Lenses
Mirror Lenses
Telecentric Lenses
Photographic Filters
Polarizing Filters
Neutral Density Filters
Aberrations
Curvature of the Field
Chromatic Aberrations
Depth of Field
Diffraction

Chapter 4
Digital Cameras, Digital Images, and Strategies

The role of the camera
Camera Components
Shutters
Modes of Operation
Manual
Automatic
Secondary Modes of Operation
Photographic Exposure
Light measurement
Types of Shutters
Focal Plane Shutters
Syncing with electronic flash
Electronic shutters
Shutter affects on subjects
Vibration affects
Mirrorless cameras
Sensors
Pixels
Single shot cameras
Scanning arrays
Multi-shot arrays
Sensor sensitivity ISO, Binning, Gain
Noise production, dark, shot, sensor and evaluating
Sensor evaluation
Bit depth
Color space
Gamma
White balance
Spectral sensitivity
Capture file formats
Other file formats
Filters
Sharpening
Color reproduction
Noise reduction
Digital Artifacts
Connecting devices
Memory cards

Applications, Best Practices and Methods

Chapter 5
The Sample and its Role in Laboratory Photography

Laboratory Photography Overview
The Sample and Treatment
Treatment
Preparation
Selecting a Sample
Isolating the sample
Objects and characteristics
Isolating the subject
Composition
Handling samples, preparation and treatments
Staining and other contrast producing factors
Wet samples and immersion methods
Making chambers
Specimen Tables
Surface replicas
White, Black or Gray Backgrounds
Use of Scales to indicate size

Chapter 6
Basic Laboratory Photography Methods:
Close-Up Photography, Photomacrography, and Stereomicroscopy

Overview of Close-up Photography
Close- Up Methods
Lenses for Close-Up Photography
Supplementary Lenses
Extension Tubes
Focusing, Depth of Field, and Diffraction
Creating Camera to Subject Alignment
Selecting the Aperture
Exposure in Close-Up applications
Photomacrography
Introduction
Bellows and Laboratory Set-Ups
True Macro Lenses and Optical Considerations
Other lenses that can be used for magnifications 2:1 and higher
Setting up a Macro System
Exposure Compensation
Exposure Factor equations
Depth of Field
Stereo Microscopes
Photographing with a stereomicroscope

Chapter 7
Advanced Laboratory Photography Methods – Making Things Visible

Introduction
I- Fluorescence
Jablonski diagram
Ultraviolet and Short Wave Blue Excitation
The Fluorescence System
II - Photographing with the Invisible spectrum
Basic Problems
Energy Sources
Cameras
Lenses
Filters
Focusing
Live View or Auto-focus
Exposure Determination
Increasing the ISO
Noise Reduction Filters
Work tethered
Multiple Discharges for Electronic Flash
Other Strategies
III - Polarized light
Seeing internal structure
The System
IV - Schlieren
Photographing Schlieren Images
V - Scanners as Cameras
Scanner Settings
Using Descreen
Unsharp Masking
Imaging Objects on a Scanner
VI - Peripheral Photography
VII - Stereo and Anaglyphs
Making a Stereo Pair
Making an Anaglyph
VIII - Stroboscopy

Chapter 8
A Primer for Lighting Small Laboratory Subjects

There is light and then there is lighting
Making good light
White and Neutral Backgrounds
Making Contrast
Reducing Contrast
Axial lighting
Glassware
Metal and tent lighting
Immersion methods
A Working Summary

Chapter 9
Light Microscopy
I - Foundations and brightfield methods

Introduction
Fundamentals of Magnified Images
Optical Magnification
Optical Elements on a Light Microscope

Eyepieces
Prism
Photo or Imaging System Lenses
Substage Condendsers
Objectives
Numerical Aperture

Forming Images - Diffraction and Resolution
More on Numerical Aperture
Objective Corrections

Fundamentals useful in Operating a Light Microscope
Setting the Eyepieces
Focusing
Very Small Working Distances
Interpupillary Distances
Looking into the Body Tube
Nosepiece or Turrets
Adjusting the Substage Condenser
Setting the Field Diaphragm
Lamp
Setting the Aperture Diaphragm

Establishing Proper Brightfield or Kohler Illumination
More on Kohler

Photographing using a Light Microscope
Instrument Cameras
DSLR cameras
Attaching a Camera to a Microscope

II: Advanced Methods
Darkfield
Differential Interference Contrast
Fluorescence
Phase Contrast
Polarized light
Rheinberg Differential Colorization Technique



Chapter 10
Confocal Microscopy
by James Hayden

Introduction
Why Confocal ?
Types of Confocal Microscopes
Fluorescence Microscopy and Confocal Methods
Fluorescent Markers
Choosing and Working with Fluorophores
How a Confocal Microscope Works
Balance and Compromises required for forming a Good 2D image
Hardware Considerations
Lasers
Detectors
Overview of Instruments Controls and Software
Laser Power
Detector Settings
Simultaneous of Sequential Acquisition
Gain and Offset
Pinhole Size and Resolution
Spatial Resolution and Format
Scanning Speed

Bidirectional Scanning
Digital Zoom
Bit depth
Averaging / Signal to Noise
Accumulation
3D imaging
Considerations for Creating an Effective Z stack

Consideration for Live Cell Imaging
Advanced techniques

Chapter 11
Scanning Electron Microscopy
by Ted Kinsman

Introduction
History
Modern Machines
Theory and Design of Instruments
The Nature of an Electron in a Vacuum
Electron Source
Electron Microscopy Optics
Astigmatization
The Electron Aperture
Resolution in a SEM
Signal to Noise Ratio
Scan Rotation
Specimen Charging
Maximizing Resolution
Sample Preparation
Critical Point Drying
Sputter Coating

Chapter 12
Ethical Considerations in Scientific Photography: Why Ethics?
by James Hayden

The Need for Protocols
The Image as Data
Manipulation and Disclosure
Manipulation by Specimen Selection
Manipulation by Hardware Settings
Manipulation by Imaging Technique
Manipulation by Software
Manipulation by Presentation
Forensic Examination
Uncovering Digital Image Fraud
Industry Oversight
Consequences
Conclusions

Chapter 13
Considerations and Methods for Image Processing in Science
by Staffan Larsson

Introduction
Terminology: Manipulation, Enhancement, Clarification
Software
Image J
GIMP
Adobe Photoshop
Basic Color Theory
Fundamental Digital Color Models
Channels
Layers
Fundamental Image Editing Methods in Science
Monitor Calibration
Selection tools and tools overview
Image Size
Image Editing Tools Overview
Selection Image editing tools
Pixel Adjustment Tools
Image Processing
I - Contrast and Color Balance Corrections
Method: Setting a white and black point
Method: Changing contrast using Levels
Method: Using Curves
II - Converting RGB files to Grayscale
Method: Grayscale
Method: Split Channels
Method: Channel Mixer
Method: Black and White Adjustment Layer
III – Sharpening
Method: Unsharp Masking
Method: High Band Pass Filter
Noise reduction using Adobe Camera RAW
Method: Eliminating Luminance Noise
Method: Despeckle
Method: Smart Blur Filter
Method: Reducing Noise using the Reduce Noise Filter
V – Noise Reduction using the Camera Raw Convertor Software
Method: Using the Camera RAW Module
VI - Combining fluorescent images
VII - Pseudo-coloring B & W images
VIII - Making composite images
Method: Making a Composite File
IX- Type and the Text Tool
X - Shapes
XI - Preparing files for Publication
Method: converting RGB to CMYK
Method: Evaluating a CMYK images Black Point
Profiles
Proofing
Gamut Warning

Chapter 14
Applications of Computational Photography for Scientist Photographers

Image editing and Batch Processing
Making actions

Increased DOF
Making Image Slices
Global Image Processing
Z Stack file processing using Adobe® Photoshop
Z Stack file processing using Helicon Focus®
Z Stack file processing using Zerene Stacker

Wide field high resolution
Methods
Global Image Processing
Creating the Image Map

High Dynamic Range Images
Making Photographic Exposures for HDR
Blending the Images

Time based imaging
Photographic Considerations
Intervalometers
Making the Photographs

Chapter 15
Best Practices

Introduction
More Thoughts about Best Practices and Workflow
The Laboratory and Environmental conditions
Cleanliness is imperative
Optimizing Camera’s Settings
Cleaning A Lens
Monitors and video displays
Color Management
Software, upgrades and Optimizing a Computer
Image Workflow, Folders, and Naming Files
Archiving, Data Redundancy, and Backing Up
Planning for Data loss and Disk Failure
Digital housekeeping
Keeping things Tuned Up
Smart phone photography
Social Media
Conclusion

Best Practices Cheat Sheet