This protocol describes how to measure injected volumes with the ImageJ software. The protocol requires a fluorescent load or adding of a fluorescent tracer. The method is based on the nanometer reproducibility of the nanosyringe manufacturing which allows calculating a reference intensity for a specified volume.
- Download ImageJ:
- Install the following macro (.txt file):
Install the Injection Volume Measurement Macro to ImageJ:
This description can be found in the text file of the macro:
This macro was created to semi automatically evaluate fluorescence pictures and save the data in a .csv file.
It has implemented dialogs to save the corresponding pressure, pressure duration and force settings.
It will automatically norm the intensity to an exposure time of 1s.
Press “f” to define a new target file (if you want to switch it during one session).
Press “m” to run measurements and append the data in the target file. The macro will never delete old data, just append.
The macro needs following steps from the user to work:
- Install it in ImageJ 1.46d or later.
- A reference image, and images from injected cells.
- A sample ROI defined and added to the ROI manager by the user.
- A background ROI defined and added to the ROI manager by the user.
- A mouse click on the sample ROI in the ROI manager. The background ROI is assumed to be just below (You might have many different ROI’s active and want to select).
While preparing the injection experiment, approach the FluidFM nanosyringe to the surface.
Take a reference picture of the nanosyringe with the same parameters that you will later use for cell imaging.
Exposure time can be changed as it will be normalized by the macro. However, it is important to keep the same objective, filter, and illumination intensity when imaging cells.
Record the fluorescence image of the injected cells in ARYA or any microscope software.
Export the pictures to the folder of your choice.
Open ImageJ, run the macro and press “f” (for file) on your keyboard to select where the volume estimation data should be stored. You can also select an already existing file in case you would like to append the new data to it. Use a .csv extension for the file.
Set a reference for volume calculation:
Open the reference picture in ImageJ.
Use the freehand selection tool to outline the shape of your reference area. Avoid overlapping pillars from the nanosyringe.
Add the shape to the region of interest (ROI) manager by pressing “ctrl+t”.
The ROI manager will show one entry as shown below. The ROI can be renamed to a more meaningful name.
Outline a background region that does not overlap with the nanosyringe.
Select the reference ROI, press “m” (for measure) on your keyboard. The software will ask for more information about the image properties. The pixel size is given by the ARYA software. Here, we have pixels of 146.5nm. Knowing that the height of the channel of the nanosyringe is 1µm, this gives a volume of 0.1465 x 0.1465 x 1 = 0.021462fl per pixel.
The resulting data is now saved in a text file in the .csv format (comma separated values) and will be used as a reference to calculate injected volumes.
Measure the volume injected in a specific cell:
Open a picture of injected cell(s).
Use the freehand selection tool to outline the shape of your injected cell (rather a bit more than less).
Add the shape to the region of interest (ROI) manager by pressing "ctrl+t". The ROI manager will show then the following entry:
Select and rename this ROI to a more meaningful name, for later traceability: e.g. “cell1”.
Outline a background region without injected cells. The region CAN NOT OVERLAP with "cell1" area. The ROI manager appears then as below:
If desired, the background ROI can also be renamed.
Select the cell ROI for analysis, press “m” (for measure) on the keyboard. The program will ask for more information about the image and the injection.
Only the exposure time will have an effect on the calculation of the injected volume. The other parameters are just given here to be stored as information in the .csv file.
Repeat these steps for any cell on the image and any image of your experiment.
The final .csv file can be opened with excel.
The macro calculates automatically the injected volume, according to the reference intensity obtained from the nanosyringe (indicated always in the first row of the .csv file).