Micro-Femto Energetics Lab Wiki-Page

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===== Analysis of Hyperspectral Microscopy Images Using ImageJ (EDIT IN PROGRESS)===== 

==== Warnings ====

Some of the image stacks acquired may be very large in size (multiple gigabytes), ImageJ will load this file completely into the memory. Additionally image calculations will generate new images that will also be loaded into the memory. If too many images are loaded simultaneously it can utilize the maximum memory of the computer at which point the computer may become unresponsive. As such it is a good idea to load the computer's task manager and keep an eye on the utilization of RAM (Random Access Memory) under the performance tab.

=== Loading Files ===

To load files into the software you can navigate through the menus (File>Open) and select the image file or Lightfield .spe file.

Alternatively you can choose to drag and drop the file onto the ImageJ main window at which point ImageJ will initialize the file. 

File initialization can take a few moments depending upon the file size.

For any given measurement you should acquire a scan of the sample, a corresponding scan of the substrate, and a single frame of the dark current (acquired with shutter closed).

=== Calculations (Differential Reflection) ===

Once you have loaded your image stacks for the sample and substrate as well as the single frame of dark current you will perform calculations as follows: 

1. Measure the mean value of the dark current image (Analyze>Measure) 

2. Subtract the dark current mean value from the sample and substrate stacks (Process>Math>Subtract) 

3. Perform image calculation to subtract the substrate image from the sample image (Process>Image Calculation)
 
4. Perform another image calculation to divide the result of step 3 by the substrate image. 

The resultant image from step 4 will be your subtracted differential reflection image for the sample. From here you can select regions of interest (ROI) and perform various analysis measurements.

=== Time Series Analysis ===

Once you have a subtracted image you can then analyze regions within the image for variations through the stack. By selecting a region of interest (ROI) and averaging the value within that region you can then plot the values corresponding to various wavelengths. This plot is our differential reflection plot for that region.

In order to do this time series analysis we must have the Time Series Analyzer plugin for ImageJ installed, if it is not please do so now: http://rsb.info.nih.gov/ij/plugins/time-series.html

Once that is installed it should be available under the Plugins menu. Navigating there opens the Time Series and ROI Manager windows.

Using the selection tool of your choice, select a region that you would like to analyze in the image. Then in the ROI manager hit the "Add[t]" button to add the selection to your list of ROIs. Once you have selected all regions that you wish to analyze you can then click the "Get Average" button in the Time Series window. This will then measure the average value of each region of interest for each slice in your stack. When it is complete it will output the results in a new window called Time Trace(s). In this window click File>Save As and save the output.