Current State of Annotations CAA 20 th Jul 09

1. Current State of Annotations CAA 20th Jul 09

2. Current State of Annotations CAA 20th Jul 09

3. Instructions for Annotation <Demo> 1. Setup 2. Annotation 3. Cell Fusion 4. Cell Depart 5. Cell Apoptosis 6. Cell Division 7. High Density 8. Some Tricky Annotations

4. Tips for Annotation • Perform Annotations on local hard drive and if using windows vista, disable user access control • Do a pseudo cell lineage on paper as you are tracking and marked cells that have been annotated with an ‘F’ • Can untick annotations - Right bar, middle pane to remove annotations if they obscure cell • Annotate every 2-7 frames at high zoom (~225% - 800% zoom). If cells really don’t move around too much, annotate every 2-10 frames. When cells divide, u need to annotate almost every frame before and after division as cells can move a lot when they round up. When cells round up/ball up, cells are either dividing or undergoing apoptosis. • As it gets near the edge of the screen, annotate it as departed and try to annotate it as much until the centre of the cell is clearly out of view • If u have any questions, email me with an attached snapshot. Use a program called MWSnap and snap the full desktop (it will capture the frame number as well)

5. Procedure for Annotation • Annotate cell and mark the lineage paper accordingly • Take note when cells round up • Count number of cells before and after the event (adjust brightness and contrast if necessary) to figure out if cell fused, divided, etc • Track nuclei of the cell of interest • If it is hard to do so, track the surrounding cells to eliminate possibilities • Can untick annotations if annotation crosshair obscures cell (program will still annotate even though u can’t see the annotation) • Can adjust the brightness and contrast • Increased brightness makes the cell border more obvious but may obscure cell details if it is too bright • Decreased brightness makes the cell border less obvious but may reveal obscured cell details

6. Tips for Annotation – Fused Cells • To discern whether a cell fuses or not, play around with the brightness intensity to determine if u see 2 nuclei in 1 cell enclosed membrane. Also count how many cells there are before the cells fused and how many there after. • Annotating Fused Cells: Before they fuse, annotate them as separate cells. At the moment they fuse, annotate still as separate cells but make sure the cross hair occupies the same x,y coordinates (so that they have the same cell centre coordinates) and mark them as fused. At the next frame, choose one of the cells and keep annotating it (marking it as fused until u reach the end of the sequence or until it divides again). For the other one, there is no need to annotate it anymore

7. Tips for Annotation – Departed Cells • If a cell migrates out of the field of view, annotate it like this: As it gets near the edge of the screen, annotate it as departed and try to annotate it as much until the centre of the cell is clearly out of view

8. Tips for Annotation – High Cell Density Areas • When cells cannot be tracked due to high density • Try to track the cells surrounding your target cell. This will let u ‘eliminate’ possibilities • Try to track the nucleus (2-3 black spots in a circle). • Adjust the brightness and contrast. Lowered brightness allows u to see more detail but may make the cell membrane border less obvious. Increased brightness allows u to see cell borders more clearly • Count how many cells there are before and after an event (such as mitosis, apoptosis or cell fusion) to confirm that u are really seeing what u are annotating. Sometimes you may see strange events such as a single cell dividing into 3 daughter cells. • If you really spent a very long time (~45min – 1.5h) on a particular region of cells and still cannot figure out where the cell is going. It may be best to set the cell’s annotation state as ‘lost’