ImageJ drives primary scan (multiple fields of view tiling the sample), image analysis and secondary scan (detected targets) on a Leica SP5 confocal microscope or a widefield microscope driven by Micro-Manager.
In High-Throughput Microscopy experiment, automation makes the life of the user much easier, the microscope acquires in a controlled manner over many fields of view and the user does not need to stay at the microscope. However, depending on the type of experiment, not all cells (or specimen in general) are actually relevant to the experiment. Take a classical example, if you study mitosis but cannot synchronize your cells, how many cells will be in mitosis when you fix your sample ? And among these, how many will be in metaphase ? Issues raise if you need to perform high resolution microscopy of cells in metaphase ! A regular screening microscope will just record every position at high resolution, you will generate 100 GB of data, and in the best case only 1GB of this data will be relevant to you, while you will trash 99GB to the bin, together with 99% of the hours booked at the microscope to generate junk data.
In microscopy facilities, time at the instrument is shared among users and inefficient experiments affect everyone.
To tackle these issues, we developed an application which takes control over the microscope to teach it which specimen is relevant, and which is to be left in peace. Written in the ImageJ environment (in java and in macro language), the application communicates through the CAM interface (Leica, available add-on module for confocal and widefield microscopes), and performs image processing on a primary 'Low-resolution' scan to determine where the relevant specimen are. After positive hits detection, ImageJ commands the confocal microscope to go back to these positions for a secondary 'High Resolution' scan. Only relevant images are acquired at high resolution, hence shortening both the global duration of the experiment and the amount of data stored. The protocol also allows for more reproducible acquisition parameters.
Hence, from the side of the user, samples are not screened manually through the ocular for the right target, no analysis needs to be performed on huge datasets to select and keep the relevant data, and data transfer is faster. The length and cost of the experiments are significantly reduced for the same outcome, and finally the use of the microscope is efficiently optimized.
This poster has been presented at ISBI 2012 in Barcelona
Click here to download a high resolution version
1) An application note on the ImageJ-CAM bridge.
2) A manual on how to setup the system for a Leica SP5 microscope.
Coming soon: A manual on how to setup the system with Micro-Manager.
All the macros require this plugin to be installed to Fiji plugin folder.
Grid tiling primary scan
The macro implementing the primary scan as a grid tiling the sample. A macro containing some example analysis functions, this macro should be copied to Fiji macro folder.
The macro can be tested offline (without microscope) by using this example data set. When the macro starts select the root folder (Kidney_Glomerulus) as image folder. Make sure to un-tick "Send CAM scripts" and select the automatic pre-analysis "Glomerulus2_detector".
This is another data set to be used with the simple mitosis detection detailed in this application note. For this data set select "Metaphase_detector" as analysis function.
Block based primary scan
The macro implementing the block based primary scan. A macro containing some example analysis functions for block based primary scan, this macro should be copied to Fiji macro folder.
For an example application / data set see this page.
Micro - Manager
Coming soon: CAM emulator for Micro-manager.
Here is a movie (10 MB, downscaled by a factor 2) showing the live detection of cells entering mitosis during the primary scan. The experiment runs autonomously for about 17h30 during which 14 one hour long movies could be acquired during the secondary scan (the microscope was hence acquiring high resolution images 80% of the time). The movies have been composited, maximum intensity projected and montaged in this movie (44 MB).
Note: We decided not to distribute the live sample macros as the project setup and the image analysis is more complex than for fixed samples. Please contact us if you are interested!