1 Basics of Digital Imaging Digital Image Capture and Display Kevin L. Lorick, Ph.D. FDA, CDRH, OIVD, DIHD

2 Digital Imaging In the context of today’s discussion, whole slide imaging is the creation of digital images from glass slides. The creation of these images depends upon computer technologies whose purpose is to convey to the digital image the same information contained on the glass slides.

3 Digital Imaging Collection Of Images On A Pixel-Based Collector. (CCD, CMOS, PMT) Pixel=Picture element –Spatial Area (Shape, and size described in ppi) –Issues Of Color (bits/pixel) 1 bit color=pure B&W. 24 bit color =16.8 million colors. –Sensitivity To Photons (important at higher magnifications)

4 Image Resolution and Digital Capture In order to digitize a microscope specimen without losing any details available to the human eye, the dimension of the detector element (e.g. CCD pixels) should be no larger than one half the corresponding limiting resolution distance.

5 Image Resolution The limiting resolution for a particular lens is determined by its numerical aperture. For example, a 20X lens requires a numerical aperture no greater than 0.5 NA. The limiting resolution of this lens is approximately 0.8 uM (31250 resolution distances/inch x 2 pixels=62500 ppi) A camera detector would require this pixel density to avoid losing resolution OR require additional processing A high end 21.1 megapixel consumer camera contains 3873 ppi (1/16 th of the information in one dimension).

6 Three Color Chip Arrangements 9 Pixel – 3 Color Arrangements Common In Digital Cameras

7 Other Digital Color Image Approaches Beam Splitter Rotating Filter Scanning Mask Or Filter Monochrome Pixel Presented Color Sequentially

8 Black And White Different Wavelengths Of Light Appear Identical In Luminance When Converted From HSV To Black & White B&W Is 10 Logs Of Intensity Each pixel may record the luminosity of each color in terms of bits (binary digits)

9 Bit Depth Number Of Combinations Per Pixel 1 bit- (0,1) (On Or Off) 2 bit-4 (00, 01,10,11) 3 bit-8 (000,001,010,100,011,101,110,111) 4 bit-16 Old B&W 5 bit-32 6 bit-64 Common B&W, Old Color 7 bit bit-256 Old Color 9 bit bit bit bit bit bit-16,384 Current Std 15 bit-32, bit-65, bit-16 million- ”true color”

10 Image Acquistion Single Or Composite Publication Or Analysis Magnification Format File Type TIFF, BMP, PNG, JPEG, etc. Color Or B&W Single Or Multiple Resolution Image Processing Camera Settings Post-Image Processing -Compression

11 Resolution White Correction –Color Temperature Black Correction –In Combination With White Correction, Defines Dynamic Range Background Correction –Means Of Correcting Aberrations From Illumination & Other Imperfections Camera Calibration

12 Challenges In Calibration Intensity Single Point Over Extended Min - Max

13 Image Capture-Storage A typical 1.4 megapixel image requires 1.4 MB of storage space (8 bit color). A 21.1 megapixel truecolor (24 bit) image requires 63.3 MBs. To achieve “ideal” resolution a single image would require >16 GB to capture a single field of view. Alternatives may be time consuming.

14 Field Of View What The Camera Images Is A Central Region Of The Field Of View In The Ocular –Frequently Off Center Results In Shading Of The Image Field Of View, Through The Ocular 400 X Magnification Field Of View, Captured By A Camera 100 X Magnification

15 Digital Imaging Techniques Point Mapping Detector type Line Mapping Global Imaging Single -point Linear array Focal-plane array Image Acquisition Technique Tradeoffs Very Slow Image acquisition Issues Of Spatial Resolution Slow Image acquisition Better Spatial Resolution Fast Image acquisition Highest Potential Spatial Resolution

x x x x x pixels Image Size Image Display

17 Image Formats Uncompressed –Each Pixel Recorded With Full Color Depth –Large Files Lossless Compression –Color Values Of Pixels Unaltered Lossy Compression (typically Fourier Transform) –Reduction in Spatial Detail –Reduction In Number Of Colors Recorded Removal Of Colors That Are Rare/Not Present In Image Decreased Bit-Depth Of Color

18 Image Compression 678 kb JPEG 19 kb JPEG

19 Image display -It is possible to capture an image with one pixel arrangement and display with another. e.g. Monitor typically has 72 ppi and displays 1680 x 1050 pixel (3.2:2 ratio w/h) The 21.1 MP camera captures 3873 ppi on a 36 x 24 (3.0:2 ratio).

20 Summary Whole Slide Imaging is the capture, storage, and display of digital images of glass slides. There are possibilities for variability in each of these steps (image collection, storage, and display). Standardization is needed.