Basic Properties of Digital Images

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1 Cell Biology Application of Microscopy Class 13: Image Processing and Manipulation Pascale Lacor, Mature rat hippocampal neurons attacked by Alzheimer s related neurotoxins (100X)

2 Basic Properties of Digital Images

edu/primer/java/digitalimaging/processing/bitdepth/index.

3 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

4 Basic Properties of Digital Images 4

Sampling Frequency on Image Fidelity http://micro.magnet.fsu.

5 Sampling Frequency on Image Fidelity

6 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

7 Under-sampling in Video Games

8 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.

9 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

10 Image Processing before after 10

Grayscale Histogram http://www.micro.magnet.fsu.

11 Grayscale Histogram

Color Histogram http://www.micro.magnet.fsu.

12 Color Histogram

13 Image Processing Single-image pixel point operations: O(x,y) = M [I(x,y)] M: mapping function 13

14 Histogram Adjustment 14

15 Adjusting Image Brightness Original Brightness 15

16 Histogram Stretching Output(x, y) = [Input(x, y) B] 255 / (W - B) 8 bit image 16

17 Image Manipulation or Misconduct?

18 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

19 Gross Misrepresentation Rossner and Yamada, J. Cell Biol

20 More Subtle Manipulations Lane 1 Rossner and Yamada, J. Cell Biol

21 More Subtle Manipulations Photoshop: Clone Stamp Rossner and Yamada, J. Cell Biol

22 Micrographs Manipulation Rossner and Yamada, J. Cell Biol

23 Micrographs Manipulation Misrepresentation of a microscope field Rossner and Yamada, J. Cell Biol

Image Resolution A pixel is a square (or dot) of uniform color in an image.

Although resolution is defined by area, it is often described using a linear measurement: dots per inch (dpi).

1200, it will show up as a 4 inch * 4 inch image on a 300 dpi micrograph.

24 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

the original image; otherwise, the computer must

25 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.

26 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

27 It s not enough to believe what you see, you must also understand what you see Leonardo da Vinci 27

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