last week things shouldn't forget

-- Peter@ Surray
Made some suggestions on colocalization analysis --> improve imaging condition, such as time resolution. additionally, include MT segmentation using skeltonization routine.

-- Michael Knop
segentation of yeast shape, get long axis, measure relative position of second channel signal. Consider surface area.

Peter@Surrey lab CBB

Track microtubule end --> using the coordinates, measure the intensity at the MT tip.

For tracking

(1) Cross-Correlation
--> seems to be difficult due to large noise.

(2) Detect the position from intensity profile along microtubule.
--> edge detection

Els@CBB

For her experiments using FRAP, we did initial experiments for determining whether the recovery can be assumed reaction dominant. For this, FRAP curves with different diameters must be tested. If the recovery curves are similar regardless of the bleaching diameter, then the curve could be considered to be "reaction dominant".

We sit together and compared several curves, with bleaching diameter small, medium or large. It seems probable for the experiment she does, that the recovery is mostly reaction dominant.

Problem with DeltaVision files: Daniela@ Griffith

When image acquisition is accidentally (or intensionally) terminated before the frame number one sets at the starting of the acquisition, the file cannot be loaded using DeltaVision Opener Plugin in ImageJ. In case of Daniela, the force-termination occurred probably due to the sudden shut-down of the server network.

I modified DVreader macro I wrote two years ago for loading these "incomplete" .dv files. Details and downloads:

http://www.embl.org/cmci/downloads/DVHeaderReader.html

Perkin Elmer data --> manual Tracking 3D

Helio @CBB requested me for making interface between

tiff 4D data series after the conversion from original Perkin Elmer data

and

format readable by manual tracker plugin for 3D tracking.

--> K_convert2MPT3D.ijm
File Converter - Perkin Elmer Tiff Series to 3D stacks
http://www.embl.org/cmci/downloads/PEfileTo3Dstacks.html

Elisa @ Ellenberg GE

made some suggestions on how to set up semi-automatic macro for

saving tracking results as text files.
re-loading track coordinates from text files.
manual assignment of linking information from different channels

--> and my assignment is to give her some fragments of functions for this.

Guillaume @ CBB

Guillaume asked me for advise on quantification of signals. We made some "dream analysis and results" scheme. For three dimensional stack, intensity measurement should be done knowing how z-slice thickness is compared to the z-stepping distance. Numerical aperture, object magnification limits the theoretical thickness of the optical section so this should be kept in mind when taking images for the quantification.

Francesco @ CBB

segmentation problem:

- background is rather flat
- problem probably is the segmentation of the cavity.

--> Cihan will try his segmentation plugins on Thurseday
--> I will try Gaussian blurred image subtraction.
--> maybe also fitting ellipse.

Iva @ Karsenti

Iva wanted to use profile analyzer I made for Marianne. Macro did not work since her ImageJ was an old version.

Though the macro now works fine, the purpose of her experiment seems to be a bit different since she wants to measure the turnover rate of proteins. So I suggested her to use Time series analyzer plugin and also to read a manual I wrote for the FRAP analysis.

Katherine DB

fixed setting for the LOCI plugin
problem was multiple "bio-formats.jar" installed in several different places.
Removing these files recovered proper behavior.

Mishura @ Griffith Lab Guest

Evaluate the degree of infection of certain bacteria.

I suggested to measure

Number of bacteria inside cell / Number of all bacteria

by taking two channel images, one for bacteria and other for the cytoplasm that the bacteria counting could be carried out with/without cytoplasm channel masking.

Mishura will use 96well plates for automatic capturing, so I suggested him to talk to Arne for the use of microscope. I told Mishura to comback to me when he is ready with example images.

Elisa @ Ellenberg GE

Elisa has task to use thicker segmented line ROI, simialr to Katherine Brown's.
So I sent Elisa the same macro freshly made some days ago.

Adam @ Rorth lab

fragment of macro sent for dealing with splitted images after "RGB split" in imageJ.

Marianne @ Surray lab 4: macro ver2

Modified that
- plotting is done separately, more flexible with number of traces and increments
- profile measurement is done for all frames, printed out to the result table.
- about 3hours.

K_YprojectionV2.ijm

Carolina @ CBB Pepperkok

EM scale problem

using the scale attached to EM micrograph, same cell has different areas in different zoom (260x and 1600 x). difference is about 25% (1600x would end up in smaller appearance).

Solution is only to measure some constant scale such as grid, or beads, and plot a calibration curve for proper scales.

Marianne @ Surray lab 3: macro

A macro was written, to get profile along x-axis over generated mask from microtubule signal channel. Took about 4 hours.

K_Yprojection.ijm

Janina : Fly localization 4: done for now

A macro called
K_2chCountOverlappedPixels.ijm
was made to count the overlapped dots, took about 3 hours.

Helio @ Antony

Told how to subtract avaraged frame from a stack.

Janina : Fly localization 3

Discussed 30 minutes and Change strategy: just count the number of overlapping dots.
--> total number of dots / brain.

Marianne @ Surray lab 2

Planned for the y-axis adding up Profile mapping.

1. Thresholding
or 2. manual selection of spindle edge.

Daniela@ Griffith

cell migration activity

--> make it simple
step1: count by category for spread cells
a. stopped
b. pivot
c. migration

step2: displacement activity
take 4 frames out of 17hrs sequence, and measure the displacement during every 5 hours.

Carolina @ CBB

1. Scale problem: ImageJ area conversion might not be reliable (or maybe too complicated)
--> told how to convert pixel area to um2.

2. ROIs accumulated in the ROI manager are used sequentially and intensity is measured
--> requires macro programming.

3. EM scales --> zooming and scale adjustment --> this should be checked by EM people.

4. Photoshop scale:

Katherine's analysis 2: done

continued from
http://www.embl.de/~miura/CMCI/blg/2007/02/thicker-segmented-line-roi.html#links

So the task is to measure intensity profile along segmented line ROI with certain width in perpendicular direction. a new macro was written for this:


K_SegmentedLineWidthControl.txt

Janina : Fly localization 2

Check images from two different conditions by colocalization plugin. Initial idea to "measure the distance" between spots would be not valid since the signals are diffuse. --> design an analysis protocol by tomorrow.

Janina : Fly localization

2 channel z-stack, trying to find the degree of colocalization.
Problem is that one signal is dotty whereas the other is diffuse.

--> compare the wild type and conditioned.
--> will discuss tomorrow again, with merged z-stacks and projections. (10 each)

Celine@KnopLab

Segmentation of yeast
for Ratiometry. maybe later..

Marianne @ Surray lab

Spindle fluorescence

y-direction projected intensity (integrated)
--> intensity profile along x-axis.

(don't have time so we will discuss next week)

Martin @ Nedelec Lab

How to measure the length -severing dynamics of MT (Population, individual).
ImageJ --> use Array to label individual filaments.

Triangulation

Jerome asked for suggestion on programming triangulation of cell positioning. He has a list of position IDs, and wants to add which of the other points are connected to that point. The number of connected point is not always same, so single position should have a flexible memory allocation.

My suggestion was to make a 2D binary matrix of Number of Positions x Number of Positions, and fill 1 for direct connections while the others 0. In this way list scanning and referencing should be fast, while the memory allocation is stably defined.

Thicker Segmented Line ROI

Katherine (DB) came to me asking about getting line profile suing thick -segmented line ROI. Need to test whether the thickness control is working or not: otherwise, design a macro to do that

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From now on, I try logging all the small consulting things here.