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Scientific Imaging with Photoshop: Methods, Measurement, and Output

Book Description

Adobe Photoshop is one of the more powerful tools available to scientists today. It is indispensable in the preparation of digital images of specimens for measurement, especially for separating relevant features from background detail. Scientific Imaging with Photoshop is the authoritative guide to the use of Photoshop in scientific research, with a special emphasis on the ethical ramifications of the use of image-enhancement software to extract data from digital images. Beginning Photoshop users will benefit from its tutorials in the basics of image processing, and more sophisticated users will appreciate the sections on automating Photoshop operations with actions. In addition, the book lays out procedures in straightforward language for acquiring digital images as well as outputting processed images in digital and hard-copy formats.

Scientific Imaging with Photoshop provides all this and more:
• Little-known methods separating features of interest from the background for subsequent quantification
• How to make dense colors and subtle visual detail reproduce properly in publication
• Correct Photoshop methods and techniques for all user levels
• Procedures that are usable in legacy versions of Photoshop as well as Photoshop Elements and Photoshop Extended

Table of Contents

  1. Copyright
  2. Register this Book!
  3. Acknowledgments
  4. 1. Ethics and Background Information
    1. 1. Visual Data and Ethics
      1. Accurate Representation of Visual Data
        1. When and Where Misrepresentation Takes Place
        2. Further Division of Areas into Categories
        3. Author Guidelines
        4. Using Standards and References
    2. 2. General Guidelines for All Images
      1. Guidelines
      2. Acquisition
        1. Specimen Preparation
        2. Optimize Imaging System
        3. Turn Off Auto-filtering
        4. Bit Depth
          1. Detecting Color
          2. 8-Bit vs. 12- or 16-Bit
        5. Clipping
        6. Color: White Balancing for Brightfield Images
          1. Color Temperature
          2. Auto White Balancing vs. Manual
        7. Noisy Image: Frame Averaging
          1. Non-Lossy Images
        8. Archiving
        9. Controls
          1. Bleed Through
      3. Post-processing
        1. Global Changes and Application
        2. Cropping and Straightening
        3. Color Mode Changes
          1. Grayscale, Indexed Color, RGB Color Modes
          2. Color to Grayscale
          3. Pseudocoloring
          4. Colorizing
        4. Changing Bit Depth
        5. Color Correction
        6. Color Noise: Digital Cameras
        7. Merging and Image Stack Functions
          1. Warping, Spatial Repositioning
        8. Symbols, Lettering, Scale Bars
      4. Conformance
        1. Bit Depth Decrease
        2. White/Black Limits
        3. Image Size Changes
          1. Pixel Resolution
          2. Output Dimensions
        4. Color Changes: RGB to CMYK
        5. File Format
        6. Documentation
      5. What Can’t Be Done in Post Processing
        1. Spot Changes
        2. Transfer of Features from One Image to Another
        3. Intentional Manipulation of Visual Data
        4. Image Size Changes (Subsampling)
        5. Brightness/Contrast Tool
        6. Copying and Pasting
    3. 3. Guidelines for Specific Types of Images
      1. Images Intended for OD/I Measurements
        1. Electrophoretic Specimens on Flatbed Scanners
        2. Acquisition
          1. Use Internal Standards in Specimens
          2. Include an External Calibration Standard or a Reference
          3. Choose Adequate Pixel Resolution
          4. Keep Gamma at 1
          5. Use Consistent Imaging System Settings
          6. Use a Higher Bit Depth
          7. Measure Areas within the Dynamic Range of the Substrate
        3. Post-processing
          1. Eliminate Dust and Scratches
        4. OD/I Measurements Using Camera and Scanned Beam Systems
        5. Acquisition
          1. Use Calibration and Consistent Imaging System Settings
          2. Use Adequate Pixel Resolution and Magnification
          3. Keep Gamma at 1
          4. Flatfield Correct the Image
          5. Use Background Subtraction
          6. Do not Measure when Labeling Fades
        6. Post-processing
          1. Flatfield Correction
          2. Masking
          3. Deconvolution
          4. Brightfield (Including Environmental Scenes): Color and Histogram Matching
          5. Use Filters to Reduce Noise
      2. Representative Images
        1. Acquisition
          1. Consistent Imaging System Methods
          2. Gamma can be Nonlinear
          3. Use a Reference Image
          4. Use a Higher Bit Depth
          5. Flatfield/Background Correct
          6. Use Controls
        2. Post-processing
          1. Sharpening
          2. Color Toning
          3. Contrast, Brightness, and Gamma
          4. Desaturation: Digital Cameras
        3. Conformance
          1. Color Changes from Colorized Images
      3. Images Intended for Quantification and Visualization
        1. Visualization
        2. Acquisition
          1. Use Thick Sections for Volume Data
          2. Use a Reference Image
          3. Flatfield Correct and Background Subtract
          4. Choose Adequate Resolution and Magnification
          5. Bit Depth
        3. Post-processing
          1. Histogram Matching
          2. Using Filters
          3. Artifact Removal and Grouping of Visual Data
          4. Isolating Features of Interest Manually
          5. Contrast, Brightness, and Gamma
          6. Thresholding and Clipping
  5. 2. Input, Corrections, and Output
    1. 4. Getting the Best Input
      1. Acquiring Images from Standard (Compound) Microscopes
        1. Accurate Representations
        2. Even Illumination
        3. Noise Reduction
        4. Setting Up the Microscope
        5. Capturing Images on Camera or in Acquisition Software
          1. Automatic Exposure
          2. Manual Exposure: Setting Dynamic Range Limits
          3. Color, Brightfield: White Balance
          4. Camera Settings
          5. Acquire the Image
          6. Flatfield Correction
          7. Noise Reduction
          8. Grayscale: Filters
      2. Laser Scanning Confocal Systems
        1. Confocal Imaging Depending on Intent
        2. Steps for Imaging on a Confocal System
          1. Phase 1: Find Brightest Plane and Set Up
          2. Phase 2: Set Dynamic Range
          3. Phase 3: Set Z Planes and Frame Averaging
          4. Phase 4: Documentation
      3. Flatbed Scanners
        1. Prescan Settings for Flatbed Scanners
        2. General Procedure for Scanning
          1. Tips for Scanning
      4. Imaging on Stereo Microscopes
        1. Controlling Glare and Lighting
        2. Tips for Imaging Challenging Specimens
      5. Environmental Imaging
        1. Exposure Time and Aperture
        2. Lighting
          1. Manual White Balancing for Consistent Results
          2. Reference Standards
          3. JPEG, TIFF, and Raw
        3. Calibrating Cameras
      6. Images from PowerPoint and Other Applications
        1. Copy and Paste as a (Poor) Solution
        2. Best Methods for Retention of Resolution
    2. 5. Photoshop Setup and Standard Procedure
      1. Approaches to Color and Contrast Matching
      2. Color Settings
        1. Unsynchronized
        2. Working Spaces
        3. Color Management Policies
        4. Conversion Options
      3. Standard Procedure
        1. 1. Output levels and the Color Sampler tool
        2. 2. Finding black and white reference points
        3. 3. Setting white and black output levels
    3. 6. Opening Images and Initial Steps
      1. Image Correction Flowchart
      2. Opening Images
        1. Bridge Database Application (CS2 and CS3)
        2. Smart Object or Duplicate Image
          1. Smart Objects
          2. Duplicate Image
        3. Trouble Opening Files
          1. Open As
          2. Dark or All Black Images
        4. Opening Images in Adobe Camera Raw
        5. Opening Multiple Images to Blend into a Single Image
          1. Frame Averaging to Reduce Noise
          2. Creating an HDR Image (CS2 and CS3 Only)
        6. Image Stacks for Blending or Layering
          1. Multiple Images to Layers (CS3 Extended)
          2. Image Statistics Method (CS3 Extended)
          3. Multiple Images to Layers (Pre-CS3 Extended)
        7. Opening Multiple Images for Photomerging (Photostitching)
      3. Precorrection Changes
        1. Indexed Color (to RGB Color)
        2. Uneven Illumination Correction
          1. Uneven Illumination Correction Using a Flatfield Image
          2. Uneven Illumination Correction Using the Image
          3. Photoshop’s Vignette Correction (CS2 and CS3)
          4. Uneven Illumination Correction Using the High Pass Filter
        3. Problem Images
          1. Low Contrast Images
          2. Clipped Images
          3. Graphic Images
          4. Aliased Images
        4. Noise Reduction
          1. Hot Pixels Removal Methods
          2. Dust and Specks
          3. Low Noise, Grayscale
          4. High Noise, Grayscale
          5. De-Interlacing for Video
    4. 7. Color Corrections and Final Steps
      1. Brightfield Color Corrections and RGB Color to CMYK
        1. Precise Color Correction
        2. Reference Areas
        3. Hue and Saturation
        4. Color Noise
        5. White or Gray Eyedropper Method
        6. Color Matching to a Reference Image
          1. Automated Match Color to Reference Method
          2. Manual Match Color to Reference Method
        7. Manual or Auto Color Corrections: Other Methods
          1. Color Correct the Whole Image
          2. Color Correct Part of the Image: Selection Steps
          3. Color Correct Part of the Image: Correction Steps
        8. Reduce Saturation, Change Hue, and Make Image CMYK Ready
        9. Noise Reduction: Color Fringing and Color Noise
      2. Brightfield: Color to Grayscale
      3. Single Color, Darkfield Images
        1. Setting Black and White Limits and Brightness Matching
        2. Single Color Image to Grayscale
        3. Colorize Grayscale Images
        4. Change Existing Colors
        5. Show Colocalization/Coexistence
        6. Make Images CMYK Ready
          1. Steps to Make Images CMYK Ready
          2. Final Steps: Sharpen the Image and Change Gamma
        7. Colorizing, Decolorizing Actions
      4. Grayscale to Color: Pseudocolor and Colorizing
        1. Pseudocolored Images Along a Color Table
        2. Posterizing
      5. Grayscale Toning
      6. Sharpening
        1. Unsharp Mask Sharpening Method
        2. High Pass Sharpening Method
      7. Gamma
    5. 8. Making Figures/Plates and Conforming to Outputs
      1. Making a Figure or Plate
        1. Retain Resolution Method for Figures: Automated
        2. Retain Resolution Method: Manual
        3. Publication Resolution Method: Automated
        4. Publication Resolution Method: Manual
          1. Aligning Images
          2. Lining Up Lanes in Electrophoretic Samples
        5. Matching Backgrounds of Images
        6. Add Lettering to Figures
          1. Augmenting Lettering to Make it Visible
        7. Aligning Text, Numbering, and Symbols
          1. Lettering at Angles
          2. Tic Marks
          3. Brackets for Text and Numbers
        8. Flattening Text and Line Layers into Single Layers
        9. Symbols, Shapes, and Arrows
      2. Working with Graphs
      3. Image Insets
      4. Resample for Output (Image Size)
      5. Sharpening, Gamma, CMYK Color, and Saving Figures
      6. Output
        1. Inkjet Printing
          1. Poster Printing from Powerpoint
          2. Poster Printing from Illustrator
          3. Proof Printing
        2. Laser Printing
        3. Electronic Documents
          1. To Acrobat from Microsoft Word
          2. To Acrobat from Photoshop
  6. 3. Segmenting and Quantification
    1. 9. Separating Relevant Features from the Background
      1. Segment Images with Photoshop or Use Stereology?
        1. Stereology
        2. Computer-Aided Image Measurement
        3. Mixing Both Methods
        4. Manual Measurement
      2. Computer-Aided Measurement: Procedure for Segmenting Images
        1. Check Image for Corrections Needed
          1. High Pass Filtering for Scattered Bright Areas
        2. Group Together or Average Features
        3. Color Images: Finding a Grayscale Channel with the Highest Contrast or Selecting by Color
          1. Grayscale Channel with Highest Contrast
          2. Using Color Range and Posterize
        4. Differentiating the Edges of Features from the Background
        5. Applying a High Pass Filter
        6. Binarizing with Threshold
        7. Modifying a Binarized Image
          1. Eliminate or Include Binarized Features
          2. Eliminate Features Touching Image Borders
          3. Creating a Mask
          4. Select Areas Surrounding Features (Orbits)
        8. Reference Area
        9. Testing Segmentation Procedure with Related Images
        10. Histogram and Linear Histogram Matching
        11. Creating an Action (or Script) to Automate Steps
      3. Manual Segmentation
        1. Dividing Images with Grids
        2. Creating Small, Fixed Selections
        3. Manual Selections Using the Lasso or Magic Wand Tools
    2. 10. Measuring Images
      1. Measuring Selected Features
        1. Measuring in Photoshop CS3 Extended from Selected Features
          1. Selecting Data Points
          2. Making and Exporting Measurements
          3. Transfer Selections to Related Images
          4. Optical Density/Intensity Measurements (OD/I)
          5. Measuring with the Ruler and Count Tools
      2. Taking Measurements in Legacy Versions of Photoshop
      3. Measuring Colocalization/Coexistence in Photoshop
      4. Using a Database/Spreadsheet Program to Distinguish Features
  7. Bibliography