Working with LightRoom

LightRoom (LR) is the program that keeps track of the image files for us—how they are stored, which files belong together (e.g., individual channels and the merge, tiles in a mosaic, slices in a stack, etc.) and all edits of the original. LightRoom is also going to play a big part in the analysis because it has powerful tools for selecting, grouping and comparing images. 

To get the most out of LR, here's what we need to do:

Exporting Images: Import only whole images (stitched mosaics or projections). If Volocity cannot stitch the image, assemble it using Adobe Bridge and Photoshop (PS) before importing it. Export the composite image with all channels and each channel individually. Export only one copy of each. Use an R-G-B color scheme when you can, but as long as we have the individual channels we can assign or change colors in PS as needed.

File Naming: Files should be named according to their content, or more to the point, the filename should not refer to anything that is not in the file. To denote the relationship between the composite image (an image of all components) and its component channels (multiple images of individual component), use the following scheme:


Composite:
2013_05_31_001a_DAPI, Cy2-MCH, Cy3-NOS_G04f, lvl 24,25

Channel 1:
2013_05_31_001b_DAPI_G04f, lvl 24,25

Channel 2:
2013_05_31_001c_Cy2-MCH_G04f, lvl 24,25

Channel 3:
2013_05_31_001d_Cy3-NOS_G04f, lvl 24,25


Keywords:
Keywords must come from a defined vocabulary to be useful. Most but not all keywords have already been created. Existing keywords can be found in the "Keyword List" in panel on the right. Note that that most have been organized into folders. Unfortunately the Keyword List can't be used for Keywording, but there is an autocomplete function when typing in keywords. If the keyword you are adding does not show up in the autocomplete list, check the Keyword List for the right keyword or correct spelling.

For each image, add these keywords:

• Peptide/Antibody
• Label (Cy 2, etc.)
• Counterstain (e.g., DAPI, Nissl)
• Tissue & Series (e.g., G1204a)
• Labeling method (IHC, etc.)
• Imaging method (epifluorescence, confocal)
• Image Type (Mosaic, Projection, etc.)
• Test or Data series
• Image content: Brain structures (just the obvious ones: LHA, AHN, VMH, PVH...)
• Atlas Level(s). Should know this before the image is taken (from the landmark database)

Notes:

Vocabulary: "Mosaic" and "Projection" are more accurate and we should use them instead of "Stitch" and "Stack". 

Tags:
In addition to keywords images can also be tagged with Stars, Flags, and Colors. Tags can be assigned from the menu or by right-clicking the image. 

Star Ratings:

1. Original Tile or Component
2. A complete raw image (Stitched, Projected and/or single frames). 
3. Manipulated w/out altering content (Stitched, color corrected, basic levels or curves, etc.) incl. Cropped and/rotated (part of image discarded but not altered)
4. Content Edited (for brightness/contrast, size/resolution, etc.)
5. Output version/figure component

Almost images should get a 2 star rating when first imported, or rather we should avoid adding more 1 star images. Most data series images will make a trip through PS and return with a 3 star rating. We will try to keep PS edits to a minimum but multi-channel composite images will usually get a 4 star rating.

Flags ("Rejected", "Flagged", "Unflagged)

Most images will be unflagged, which is the default. A few images will be flagged under special circumstance:

• Use the "Rejected" flag  to  hide unusable images without deleting them
• Use the "Flag" to mark images that need attention, like missing or non-standard keywords, the file format is not usable or if content is not clear, etc.

Color Labels:An image can be tagged with one of 5 different colors (R, G, B, Y, P) that we can explicitly defined. The obvious use for Color labels is to keep track of channel information (use Purple for Cy 5, Alexa 647, etc., and Yellow for Fluorogold, which isn't relevant to the LHA project).

Metadata:
Metadata opens up another realm of options. For now let's leave it at that: optional. Note though that it migh be convenient to copy the file name into the 'Title' field. Also that the 'Caption' field ccontains all of the microscope info exported with an OME Tiff, which may be useful at some point.