1. Image Input Image acquisition or digitization. Sources include- Cameras Video camera w/ framegrabber Digital CCD camera w/ interface card Color or Monochrome Flatbed Scanners Photomultiplier Tubes Electron Microscopes

2. Image Quality Image Quality is critical. A good image depends on a good specimen. Optimize input conditions - Select suitable acquisition system and conditions to acquire the best possible image. Good specimens and proper imaging conditions will make image analysis much easier.

3. Limitations of Our Eyes 5 Gray Levels10 Gray Levels 20 Gray Levels40 Gray Levels

4. Instrument Setup Microscope adjusted for Koehler illumination Match magnification to object size and camera resolution Calibration- White Balance Dark Current Exposure Time Background Subtraction

5. Camera Adjustments Adjust camera for best dynamic range Gain and brightness Exposure time For intensity/density measurements turn off autoexposure For fluorescence turn off autoexposure

6. Saving Image Data Images are normally saved as TIFF (.tif) files. Do not save images as JPEG (.jpg) files! OK for e-mailing/viewing only Loses data integrity Could be impossible to analyze Does not save computer memory or processing time

7. TIFF v. JPEG TIFF JPEG