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Calculation of injected volumes

By
Paul Monnier
on 11/20/18, 6:41 AM 48 views


This protocol describes how to measure injected volumes with the ImageJ software. This is limited to the estimation of injected volume of fluorescent molecules. This is based on the remarkable reproducibility in the nanosyringe manufacturing which allows to calculate a reference intensity for a specified volume.

Prerequisites

  • Download ImageJ:

https://imagej.net/Downloads

  • Install the macro:

Get the injection_measurement_macro (a text file) and install it to ImageJ:

Screenshot

Macro description

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 the measurements and append the data in the target file. The macro will never delete old data, just append.

The macro needs following steps from you to work:

  1. Install it in ImageJ 1.46d or later.
  2. A reference image, and images from injected cells.
  3. A sample ROI defined and added to the ROI manager by you.
  4. A background ROI defined and added to the ROI manager by you.
  5. 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).

Measurement procedure

  • 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 use later for imaging the cells.

Screenshot

Note

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 the cells.

  • Perform the injection experiment.
  • 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 existing file if you would like to append the new data to it. Use the .csv extension for the file.

Screenshot

  • 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 below. You can rename the ROI to a more meaningful name.

Screenshot

  • Outline a background region which does not overlap with the nanosyringe.

Screenshot

  • Select the reference ROI, press “m” (for measure) on your keyboard. You will be asked to add some information about the image. 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 give a volume of 0.1465 x 0.1465 x 1 = 0.021462fl per pixel.

Screenshot

  • The resulting data is now saved in a text file in the .csv (comma separated values) format and will be used as a reference to calculate injected volume.

  • 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 one entry as below.

Screenshot

  • 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 MUST NOT overlap with “cell1” . The ROI manager will look like below:

Screenshot

  • You can also rename the background ROI if you like.
  • Select the cell ROI you want to analyse, press “m” (for measure) on your keyboard. You will be asked to add some information about the image and the injection.

Screenshot

Note

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.

  • The resulting data is now saved in a text file in the .csv (comma separated values) format.
  • Repeat for any cell on the image and any image of your experiment.
  • The .csv file can be opened with excel.

Screenshot

  • The macro automatically calculated the injected volume, according to the reference intensity obtained from the nanosyringe (Always first row in the .csv file).

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Asked: 11/20/18, 6:41 AM
Seen: 48 times
Last updated: 11/20/18, 6:41 AM