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TYPICAL SCIENTIFIC IMAGING WORKSHOP:
IMAGE INTEGRITY, POST-PROCESSING, OUTPUT AND QUANTITATION

3 DAYS

COURSE SCHEDULE AND DESCRIPTION (Classes from 9:00 am - 4:30 pm)

Day 1
Day 1 and day 2 take you through potential steps you would use in Photoshop and Image J. The emphasis is on minimally processing images using proper and acceptable methods, along with new methods shown in this course. Also, the emphasis is on making changes while using numeric information so that your changes are done on an objective (vs subjective) level. Many challenges that are particular to images in research are overcome when using methods presented in this course. Steps are repeated more than once to ensure that material is grasped. Both brightfield and darkfield (fluorescence) images are post-processed using provided images.
   
9:00 - 12:00pm

Image Integrity Lecture and Calibration Information

Setting up Photoshop: toolbar; preferences; Color Settings; Rulers; Info, Histogram & Layers palette; History Log.

Opening Images: Duplicating Image and Changing Mode of Image: Indexed Color & Color to Grayscale, Brightening 12-bit saved as 16-bit and differences in display between Photoshop and Image J.

De-Colorizing, Colorizing Grayscale images.

Opening Image Stacks: AVI files; a series of individual files.

Noise and correction of noise

Uneven Illumination: Vignetting, Uneven, Flatfield corrections.

 

1:00 - 4:30pm

Adjusting Tones of Merged images: 3 methods; and equalization

Colocalization (co-existence) by the numbers (objective)

Adjusting Tones for 3D reconstruction; Image J reconstructions

Saving Images: Photoshop, TIFF, PNG and JPEG formats discussion

Re-setting resolution (re-sampling)

Creating actions (macros) in Photoshop/Image J & Batch Processing

   
Day 2
   
9:00 - 12:00pm

Extended Focus and High Dynamic Range Images

Rotate/Crop and using guidelines

Color Correct and Adjusting Tones; correcting "neon" colors

Color Matching (Histogram Matching) and Linear Histogram matching

Gamma Corrections; Gamma correction risk

Noise: color fringing, random noise

Scale Bars

False Color: pseudocoloring, posterizing (minimizing range of tones).

 

1:00 - 4:30pm

Image stitching (montaging)

Making Figures; adding symbols and arrows; re-sizing; making inserts

Sharpening images: 2 methods

Digital Video; deinterlacing; side by side video; adding arrows and output formats.

Images to Powerpoint and posters; setting resolution; color corrections; optimizing resolution on graphs and tables; tips

Images to LaserJet and Electronic Documents: overview of Acrobat and Word; setting and maintaining resolution while minimizing file size; tips

 Images to Publication; conversion to CMYK; setting resolution; black and white limits.

Specific Problems by Class Attendees.

   
DAY 3 Using Photoshop with Quantitation Programs to separate features of interest from background (segmenting); Quantitation; Stereology Intro.
Photoshop is the most overlooked program for achieving separation of features of interest from surrounding areas (segmenting) within images, but is far and away superior to many other far more expensive programs. Almost any image can be segmented in Photoshop using very few tools, and then these images can be processed for measurement in other programs, or within the Extended versions of Photoshop itself. Segmenting of entire directories of images can be automated, reducing quantitation time into a fraction of what it would take to manually work with individual images. Learn how to use this powerful tool for segmentation and image analysis in Day 3 of this course. Some published papers are listed in which Photoshop was used for segmentation are listed after the class listing.
   
9:00 - 12:00pm

Optical density and intensity measurements discussion; masking; ways to measure.

Overview of functions available in Photoshop and measurements desired, and the penultimate importance of correcting uneven illumination and histogram matching for segmenting. Preparation of images for segmenting: correcting uneven illumination, histogram matching, linear histogram matching and equalization.

Thresholding at consistent values; determining values, eroding and dilating selections, creating binary files for measurement in quantitation programs.

 

12:00 - 4:30pm

Unbiased sampling, efficient manual measurements & stereology; stereology probes; using Excel spreadsheet for obtaining numbers for stereology.

Automating and interactive segmentation and measurement for entire directories of images.

Measurement in quantitative programs and ways to further modify measurements in Excel.

Practice on several types of images and automating; or segmenting specimens provided by attendees.

   
DAY 4 (bonus day!)

Come on Thursday to practice quantization and creating actions to apply steps automatically. Bring your own images to be worked on. Same hours as on previous days.

Papers in which Photoshop was used for segmentation and measurement that included instructor as co-author:


Co-existence:


Z-Q Zhang, D. W. Notermans, G. Sedgewick, W. Cavert, S. Wietgrefe, M. Zupancic, K.

Gebhard, K. Henry, L. Boies, Z. Chen, M. Jenkins, R. Mills, H. McDade, C. Goodwin, C.

M. Schuwirth, S. A. Danner, A. T. Haase. (1997) Kinetics of CD4+ T Cell Repopulation

of Lymphoid Tissues After Treatment of HIV-1 Infection. Proc Natl Acad Sci U S A.

Vol 95(3): 1154-9.


Segmentation of autofluorescence:


Wild R, Ramakrishnan S, Sedgewick J, Griffioen AW. (2000) Quantitative assessment

of angiogenesis and tumor vessel architecture by computer-assisted digital image

analysis: effects of VEGF-toxin conjugate on tumor microvessel density. Microvasc Res.

Vol. 59(3): 368-76.


Yokoyama Y, Sedgewick G, Ramakrishnan S. (2007) Endostatin binding to

ovarian cancer cells inhibits peritoneal attachment and dissemination. Cancer Res.

2007 Nov 15;67(22):10813-22


Segmentation of particles for FDA approval studies:


Grube E, Gerckens U, Yeung AC, Rowold S, Kirchhof N, Sedgewick J, Yadav JS,

Stertzer S. (2001) Prevention of distal embolization during coronary angioplasty in

saphenous vein grafts and native vessels using porous filter protection. Circulation.

2001 Vol. 104(20): 2436-41.


Henry M, Polydorou A, Henry I, Liasis N, Polydorou A, Polydorou V, Demesticha T,

Skandalakis P, Kotsiomitis E, Hugel M, Sedgewick J, Ruth G. (2007). New distal

embolic protection device the FiberNet 3 dimensional filter: first carotid human study.

Catheter Cardiovasc Interv. 2007 Jun 1;69(7):1026-35


Segmentation of features that border cells:


Reilly C, Wietgrefe S, Sedgewick G, Haase A. (2007) Determination of simian

immunodeficiency virus production by infected activated and resting cells. AIDS.

2007 Jan 11;21(2):163-8

 

    
     
 
 
 

 

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