Photoshop / Illustrator Workshop November 15th 2016 Alphonse Chu alphonse.chu[at]yahoo.ca

Outline Digital Images File Formats Resolution – DPI Publication Requirement Working with images Photoshop Basics Composite images Tools Vector Graphics - Illustrator

Digital Images - Capturing

Digital Images – Image Sensor The image sensor is a matrix of small sensors named pixels Each pixel records the intensity of light Once the intensity of light is recorded, a computer generates the image

Digital Images – Color Images Color is separated into 3 basic colors (channels) with filters Red Green Blue Intensity of each channel is recorded Each pixel combines the 3 channel to create a color image Additive colors

Image Formats TIFF (Tagged Image File Format) ‘Raw’ or Lossless data format Uncompressed image Contains every bit of information (Data not discarded) Used for publications JPEG (Joint Photographic Experts Group) ‘Processed’ or Lossy data format Compressed image Contains only useful bits of information (Data is discarded) Not used for publications

Image Formats Property TIFF JPEG Acronym Tagged Image File Format Joint Photographic Experts Group Type of Format Raw or Lossless Processed or Lossy Compression No Yes Bit Information Every bit of information saved Only useful bits of information saved Loss of information No Data Loss Some data is lost Publication Use Yes, but not all journals

Lossless vs Lossy (TIFF vs JPEG) Downsampling

Resolution - DPI Resolution DPI and PPI Important for creating figures Number of pixels in the Width and Height of the image DPI and PPI Important for scanning images and final figures Dots Per Inch: Usually referred in Printing or Scanning Pixels Per Inch: Usually referred in digital images

Publication Requirements Figures PowerPoint EPS Format Final DPI / PPI settings for the final figure 300- 600 Make sure to check the journal requirements before submitting

Publication Requirements - Examples PLOS One Cell File Format TIFF or EPS Dimensions Width: 789 – 2250 pixels (at 300 dpi). Height maximum: 2625 pixels (at 300 dpi). Resolution 300 – 600 dpi File Size <10 MB Text Within Figures Arial, Times, or Symbol font only in 8-12 point File Format TIFF or PDF Dimensions Max width & height 8 x 11 inches Resolution 300 dpi (color figure) 500 dpi (black & white) 1000 dpi (line art figure) File Size <20 MB Text Within Figures Arial, Helvetica (no point size specified) Genes & Development Journal of Clinical Investigation File Format GIF, TIFF, EPS, or JPEG Dimensions N/A Resolution File Size Text Within Figures File Format TIFF, EPS, PDF, AI Dimensions 9-18 cm width Resolution 600 ppi File Size N/A Text Within Figures

Working with Images 1) Microscopes Capturing from Instrument (Microscopes) Every instrument and software combination is different Use TIFF if possible If available set highest dpi possible Capture each channel individually if possible. If the microscope software combines each channel, into a multipage TIFF you will have split them with IfranView Photoshop doesn’t do it. Demo

Working with Images 2) Scanning Film Import Directly into Photoshop File Import WIA Support Select your Scanner Scanner software should launch Capturing from Film Scan in transparency mode Use TIFF If available set dpi or ppi to 300-600 Demo

Working with Images 3) Storage/Backups As soon as the images are acquired, make a BACKUP COPY DO NOT use the acquisition computer as a backup Ideally the copy is on reliable/redundant storage (i.e. University Network Drives or Dropbox) External hard drives are ok, but can fail (i.e. dropping it, accidents with liquids, etc) ALWAYS work on COPIES of images If something goes wrong, you can go back to the original

Composite Images Each fluorophore was captured in a different channel Copy one of the images Open in Photoshop a new file in RGB mode Uses the dimensions of the copied image for the new file Paste the copied image in the correct channel Repeat for each channel/fluorophore Adjust Levels for each channel (Don’t forget the ALT button) Demo

Warning - Disclaimer Photoshop is a powerful image processing tool, however, one should be careful with the adjustments, because one can change the result or the interpretation with image adjustments! Demo

Useful Tools Rotating and Image Counting Straighten an image Cropping Image  Image Rotation Counting Image  Analysis  Count Tool You can rename the count groups with more descriptive names To add a new count group click on the folder, give it a descriptive name and a new color Straighten an image Set up a crop box Click on Straighten Draw a straight line with a reference on an image Cropping Select crop tool and select the area you want to keep Demo

Why Vector Graphics? Enlarging an image with a set resolution causes pixelation Each pixel is enlarged and causes the quality/sharpness of the image to decrease

Why Vector Graphics?

Vector Graphics Vector graphics allows you to change the size of your text, shapes without loosing details Basically a file that describes where each shape/text is located, the color etc. (Like a protocol) If you increase or decrease the size, the shape/text is changed proportionally %PDF-1.5 <</Metadata 2 0 R/OCProperties<</D<</ON[6 0 R 29 0 R 74 0 R 119 0 R 164 0 R 209 0 R]/Order 210 0 R/RBGroups[]>>/OCGs[6 0 R 29 0 R 74 0 R 119 0 R 164 0 R 209 0 R]>>/Pages 3 0 R/Type/Catalog>>

Vector Graphics – Adobe Illustrator Easy to use and intuitive Make entire figures Add excel graphs (some additional work required) If you add images to the illustrator file (i.e. IF/Staining) don’t forget to embed them! Resizing is easy! Save as EPS (Standard Specification Format)for publication. Demo

Adobe Suite & Open Source Subscription base service Discount available for students (Oct 28th exchange rate 1.3385) Annual Monthly payments 19.99 USD – 26.76 CAD Prepaid Yearly payments (No Savings if you prepay) 239.88 USD – 321.08 CAD Open Source Version of Photoshop GIMP https://www.gimp.org/ Open Source Version of Illustrator InkScape https://inkscape.org/en/