| 1-HOUR LECTURE: IMAGE INTEGRITY: JPEGs, ADJUSTMENTS AND JAIL | |
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Take this course to learn 4 basic steps you can take to protect yourself against accusations of scientific misconduct. Detailed information is presented on archiving images, keeping image databases, and on what post-processing steps are appropriate, depending upon the intent of the image. Learn techniques for calibration to standards and reference materials so that you can be certain of consistent and proven results. Resource materials provided at the lecture which include guidelines for acquiring images, depending on the imaging device. This lecture provides ethics training for maintaining image integrity, required by both NIH and NSF as a subset of ethics training for lab members. |
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2-HOUR LECTURE: QUICK PHOTOSHOP: PROCESS LOTS OF IMAGES AND KEEP A RECORD |
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The demands of imaging can take enormous amounts of time, but it doesn't have to. That's because programs like Photoshop are set up for one-by-one adjustments to images. Newer imaging programs (post Photoshop CS) have opened avenues for processing large numbers of images all at once. These include Adobe Bridge, which comes with Photoshop CS2, CS3, CS4 and CS5; as well as the stand-alone Adobe Lightroom and Mac Aperture. When images require minimal adjustments, such as what is required with brightfield images (dark objects on a white background, like H&E samples), these programs are ideal. This lecture shows you how to take advantage of these programs to apply the same adjustments to an entire directory of images, how to use only linear adjustment controls (versus controls that change the gamma to make tones non-linear), how to retain a record of the adjustments for future reference, and how to save the adjustments for application to future images. Fluorescent images and more involved adjustments to brightfield images can be applied to entire directories in Photoshop as well, and the adjustments can be retained for future reference. This is done with the Auto-Photoshop (Photoshop for Research, Industry and Medicine) action buttons available at this website. These buttons are demonstrated at this lecture for colocalization, merging (overlays), pseudocolor, figures (plates), tonal adjustments, correction of uneven illumination, resolution changes, sharpening, noise removal, gamma adjustment (with warnings), high dynamic range imaging, extended focus, image stack routines, adding lettering/symbols/tick marks/scale bars, re-scaling 12-bit to 16-bit, colorizing, de-colorizing, etc. Each attendee will receive the action buttons for adding-on to their version of Photoshop, along with resource material for Bridge, Lightroom and Aperture. |
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1-DAY CORRECT PHOTOSHOP METHODS FOR IMAGING, INCLUDES METHODS IN GIMP, IMAGE J |
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| This is an intensive course for really learning to master Photoshop. Attendees receive a copy of Auto-Photoshop action buttons. The same topics are covered as in the 2-Day hands-on course, along with an introduction to methods for 3D reconstructions and quantitation. See topics for the 2-Day course below, along with the Quantitation course. GIMP (free software that mimics Photoshop) and ImageJ are covered when appropriate. | |
2-DAY CORRECT PHOTOSHOP METHODS FOR IMAGING, INCLUDES METHODS IN GIMP, IMAGE J HANDS-ON COURSE |
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Requirement: All attendees must bring along their own laptop with version 6x, 7x, CS, CS2, CS3, CS4, or CS5 installed! Attendees must have familiarity with using computers and some past experience using Photoshop. This is an intensive course for really learning to master Photoshop. Attendees receive a certificate for inclusion on CVs, a copy of Auto-Photoshop action buttons, and software (GIMP, ImageJ: attendees will be notified to download prior to course). Day 1: Post-Processing I Day 1 and day 2 take you through potential steps you would use in Photoshop and related programs (GIMP, Image J: when appropriate). The emphasis is on minimally processing images using proper and acceptable methods, along with new methods shown in this course. 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. Steps are shown in both the Windows and Mac OS environments. Both brightfield and darkfield (fluorescence) images are post-processed using provided images. 8:15-12:00pm Image Acquisition Using Adobe Bridge for processing lots of images at once. 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. Opening Image Stacks: AVI files; a series of individual files. Rotate/Crop and using guidelines Uneven Illumination: Vignetting, Uneven, Flatfield corrections. LUNCH 1:00-5:00pm 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 Extended Focus and High Dynamic Range Images False Color: pseudocoloring, posterizing (minimizing range of tones). Adjusting Tones objectively (by the numbers); using color sampling tool & Info Box; determining clipped values Day 2: Post-Processing II 8:15-12:00pm De-Colorizing, Colorizing Grayscale images & Merging 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 & Batch Processing LUNCH 1:00-5:00pm Image stitching (montaging) Making Figures; adding symbols and arrows; re-sizing; making inserts Sharpening images: 2 methods Digital Video overview; deinterlacing; side by side video; audio recording 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 presented by class attendees |
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DAY 3: Quantization, Segmenting (separating features of interest), Stereology & Excel |
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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 4 of this course. Some published papers are listed in which Photoshop was used for segmentation are listed after the class listing. 8:15-12:00pm 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 Segmenting approaches; using color modes to segment (especially useful for DAB); posterizing images; and the High Pass and median filters Thresholding at consistent values; determining values, eroding and dilating selections, creating binary files for measurement in quantitation programs LUNCH 1:00-5:00pm 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.
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