Cell Biology Application of Microscopy Class 13: Image Processing and Manipulation Pascale Lacor, Mature rat hippocampal neurons attacked by Alzheimer s related neurotoxins (100X)
Basic Properties of Digital Images
Bit Depth 2 bit = 2 2 gray levels 4 bit = 2 4 gray levels 8 bit = 2 8 gray levels 10 bit = 2 10 gray levels http://micro.magnet.fsu.edu/primer/java/digitalimaging/processing/bitdepth/index.html http://www.micro.magnet.fsu.edu/primer/digitalimaging/digitalimagebasics.html
Basic Properties of Digital Images 4
Sampling Frequency on Image Fidelity http://micro.magnet.fsu.edu/primer/java/digitalimaging/processing/samplefrequency/index.html http://www.micro.magnet.fsu.edu/primer/digitalimaging/digitalimagebasics.html
Nyquist-Shannon Sampling Theorem To accurately preserve the spatial resolution in the resulting digital image, the sampling interval must be equal to twice the highest specimen spatial frequency Meet Nyquist Undersampling 6
Under-sampling in Video Games
Images Processing and Analysis
Image Processing and Analysis Why do this? To minimize human bias based on wish or expectation. To introduce rigor into the process of obtaining quantitative information. To make us better and more aware viewers of images. General steps involved: Correct image imperfections and defects. Enhance important details. Create a binary representation of the structures of interest. Perform measurements on features and/or overall structure. 9
Image Processing before after 10
Grayscale Histogram http://www.micro.magnet.fsu.edu/primer/digitalimaging/digitalimagebasics.html
Color Histogram http://www.micro.magnet.fsu.edu/primer/digitalimaging/digitalimagebasics.html
Image Processing Single-image pixel point operations: O(x,y) = M [I(x,y)] M: mapping function 13
Histogram Adjustment 14
Adjusting Image Brightness Original Brightness http://micro.magnet.fsu.edu/primer/java/digitalimaging/processing/histogramstretching/index.html 15
Histogram Stretching Output(x, y) = [Input(x, y) B] 255 / (W - B) 8 bit image 16
Image Manipulation or Misconduct?
The Journal of Cell Biology General Guidelines No specific feature within an image may be enhanced, obscured, moved, removed, or introduced. The grouping of images from different parts of the same gel, or from different gels, fields, or exposures must be made explicit by the arrangement of the figure (e.g., using dividing lines) and in the text of the figure legend. Adjustments of brightness, contrast, or color balance are acceptable if they are applied to the whole image and as long as they do not obscure or eliminate any information present in the original. Nonlinear adjustments (e.g., changes to gamma settings) must be disclosed in the figure legend. 18
Gross Misrepresentation Rossner and Yamada, J. Cell Biol. 2004 19
More Subtle Manipulations Lane 1 Rossner and Yamada, J. Cell Biol. 2004 20
More Subtle Manipulations Photoshop: Clone Stamp Rossner and Yamada, J. Cell Biol. 2004 21
Micrographs Manipulation Rossner and Yamada, J. Cell Biol. 2004 22
Micrographs Manipulation Misrepresentation of a microscope field Rossner and Yamada, J. Cell Biol. 2004 23
Image Resolution A pixel is a square (or dot) of uniform color in an image. The size of a pixel can vary, and the resolution of an image is the number of pixels per unit area. Although resolution is defined by area, it is often described using a linear measurement: dots per inch (dpi). 300 dpi = 300 pixels/inch * 300 pixels/inch = 90,000 pixels per inch 2 If your camera has a resolution of 1200 * 1200, it will show up as a 4 inch * 4 inch image on a 300 dpi micrograph. In Photoshop, physical size and resolution can be traded off against each other without a gain or loss in the amount of information that is, you can resize an image without altering the total number of pixels. 24
Micrographs Manipulation You should not set the total number of pixels to be greater than that in the original image; otherwise, the computer must create data for you that were not present in the original.
Data-management Issues It is crucially important to keep your original digital or analog data exactly as they were acquired and to record your instrument settings. This primary rule of good scientific practice will allow you or others to return to your original data to see whether any information was lost by the adjustments made to the images. In fact, some journal reviewers or editors request access to such primary data to ensure accuracy. Rossner and Yamada, J. Cell Biol. 2004 26
It s not enough to believe what you see, you must also understand what you see Leonardo da Vinci 27