Lab can be performed any time and any place (assuming that the original .lif files are copied first) using freeware ImageJ. Please refer to original page which is maintained more often:
http://confocal-manawatu.pbwiki.com/Instructions-for-Confocal-Measurements-Lab
Please install the freeware for the lab if needed:
| MBF ImageJ |
|
Free |
Multi-platform |
| LAS AF Lite |
|
Free |
Windows |
2D Measurements
- Open the image by using File/Open. In "Bio-Formats Import Options" click "OK".
- Click a check box for an Image or Series of images to be opened for analysis.
- Apply suitable LUT table for the gray sceled image: ToolBar/Select LUT Menu ("Red Hot" recommended)
- Check calibration by data from LAS Lite (Richt-click the image or series from Experiment file/Properties) and calibrate your image in ImageJ if needed: /Analyze/Set Scale
- Set up the kind of data you want to collect: Analyze/Set Measurements
- Take your measurements:
- Select "Straight Line Selection" tool from ToolBar
- /Edit/Selection/Add to Manager (Ctrl+T) -> "ROI Manager" window appeared with first measurement in the list
- Click ROI Manager/Show All -> label attached to the line
- Draw another line in direction perpendicular to the first line to define diameter in second dimension
- ROI Manager/Add -> Second measurement appeared in the list
- Continue measurements with another features (2 measurements for ellipsoid but 1 measurement for spherical shapes)
- Save file with measurements:
- ROI Manager/ Measure -> "Results" window appeared
- File/Save As... in "Results" window - save the results as "2D measurements.xls" file
- Double-click "2D measurements.xls" file to open it in MS Excel.
- Delete unrelevant data.
- Calculate volume of sphere by formula
or
(abc are radii along each of the x,y,and z axis) for ellipsoid. One dimension is not avalibale. This is in the z plane. The width is used twice (as both width and depth), assuming that the section in the middle part of the nucleolus/ nuclei is circular.
- Print out your report page that may look like that:
| |
Label |
Diameter, um |
Radius, um |
|
| 1 |
ava erin.lif |
27.8 |
13.9 |
nucleus |
| 2 |
ava erin.lif |
21.05 |
10.53 |
nucleus |
| 3 |
ava erin.lif |
12.56 |
6.28 |
nucleolus |
| 4 |
ava erin.lif |
12.12 |
6.06 |
nucleolus |
| 5 |
ava erin.lif |
2.56 |
1.28 |
nucleoleolus |
| 6 |
ava erin.lif |
2.03 |
1.02 |
nucleoleolus |
| |
|
|
|
|
| Volume of nucleus |
6449.82 |
um^3 |
|
| Volume of nucleolus |
1738.72 |
um^3 |
|
| Volume of nucleoleolus |
5.524 |
um^3 |
|
| |
|
|
|
|
| Ratios |
|
|
% |
|
| Nucleolus/nucleus |
0.2696 |
26.96 |
|
| Nucleoeiolus/nucleolus |
0.003177 |
3.18 |
|
3D Measurements
- Check calibration by data from LAS Lite and calibrate your image if needed: /Analyze/Set Scale.
- Check whether Auto-Measure option is selected from Menu: Edit/Options/Point Tool/Auto-Measure
- Calibrate distance between images in stack: /Image/ Properties/ Voxel Depth
- Select Point Selections tool from Toolbar and Shift-click points through the volume rotating mouse wheel (or clicking arrows in the bottom scroll bar) for moving through the depth
- Calculate Distance between points in Excel using formula for hypothenuses:

- Copy & Paste to Exel: (*)
=SQRT((c2-c1)^2+(d2-d1)^2+(e2-e1)^2)
- Calculate total length of segmented line: (*)
Copy & Paste to Exel: (*)
=sum(D10-D2)
* Please use relevant cell addresses in form
(Use LaTeX OnLine Equation Editor for formulas at editing this page)
-
Print out your report page that may look like that:
| |
Label |
X |
Y |
Z |
Segment length, um |
| 1 |
Brooke.lif - Series009 |
26.31 |
24.39 |
10 |
1.44 |
| 2 |
Brooke.lif - Series009 |
26.68 |
23.09 |
10.5 |
1.02 |
| 3 |
Brooke.lif - Series009 |
27.33 |
22.31 |
10.5 |
1.26 |
| 4 |
Brooke.lif - Series009 |
28.03 |
21.98 |
11.5 |
1.22 |
| 5 |
Brooke.lif - Series009 |
28.93 |
22.64 |
12 |
1.29 |
| 6 |
Brooke.lif - Series009 |
29.83 |
23.41 |
12.5 |
1.45 |
| 7 |
Brooke.lif - Series009 |
30.77 |
24.39 |
13 |
1.50 |
| 8 |
Brooke.lif - Series009 |
31.54 |
25.58 |
13.5 |
1.16 |
| 9 |
Brooke.lif - Series009 |
32.07 |
26.48 |
14 |
1.11 |
| 10 |
Brooke.lif - Series009 |
31.91 |
27.46 |
14.5 |
1.16 |
| 11 |
Brooke.lif - Series009 |
31.46 |
27.83 |
15.5 |
|
| |
|
|
|
Total |
12.61 |
- Select the most representative slice with measurements, copy to system clipboard (Alt-Print Screen)
- Create new image with content of clipboard: /File/ New/ System Clipboard (Ctrl-Shift-V)
- Save image with measurements to your folder for report.
- Create Anaglyph (stereo image) from the stack. Contact Dmitry to make it quicker with LAS AF, software for confocal microscope.
- Prepare report in ppt format including:
- 2D image with measurements.
- Excel print out with measurement data and estimation of volumes of nucleus, nucleolus and nucleoleoli if any seen from the image.
- One of the slices with points measured.
- Excel print out with points in 3D, total length of chromosome or other feature of interest.
- Anaglyph
- Well Done!!
- Please remember that You have used Your hands-on experience with following software packages: LIF Viewer to view your images and acquisition parameters, ImageJ for image analysis, MS Excel for calculations and MS PowerPoint for Lab report.
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