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Basics of Digital ImagingDigital Image Capture and Display

Kevin L. Lorick, Ph.D.FDA, CDRH, OIVD, DIHD

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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.

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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)

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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.

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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 (1/16th of the information in one dimension).

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Three Color Chip Arrangements9 Pixel – 3 Color Arrangements

Common In Digital Cameras

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Other Digital Color Image Approaches

Beam Splitter

Rotating Filter

Scanning Mask Or Filter

Monochrome Pixel Presented

Color Sequentially

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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)

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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-128• 8 bit-256 Old Color

• 9 bit-512• 10 bit-1024• 11 bit-2048• 12 bit-4096• 13 bit-8192• 14 bit-16,384 Current Std

• 15 bit-32,768• 16 bit-65,536• 24 bit-16 million-”true color”

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Image AcquistionSingle 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

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• 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

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Challenges In Calibration

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1 0

2 0

3 0

4 0

5 0

6 0

7 0

8 0

9 0

1 00

0 1 2 3 4 5 6 7 8 9 1 0

Intensity

Intensity

Sin

gle

Poin

t

Over Extended

Min

- Max

10 -

90

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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.

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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 ImageField Of View,

Through The Ocular

400 X Magnification

Field Of View,

Captured By A Camera

100 X Magnification

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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

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232 x232

464 x464

1360 x 1360

2060 x 2060

3090 x 3090

200 pixels

200

pixe

ls

Image SizeImage Display

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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

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Image Compression678 kb JPEG 19 kb JPEG

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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).

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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.