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This protocol explains how to measure the volume injected into cells by using the Volume Calculator tool in the FluidFM BIO Series systems. The method requires a fluorescent load or adding a fluorescent tracer to the injected sample.
The images used in this tool (reference and processed images) must have the same caption conditions: same objective, fluorescence filter and visualization presets (e.g. gain, gamma, attenuator). However, the software can process images with different exposure time for the reference and the cells. For the calculation of the injected volume, the software will need:
- An image of the cantilever filled with the fluorescent solution and close to the surface of the plate, so it can be used as a reference.
- Images of the injected cells, grouped in a point group and obtained with the Observe workflow, so they have the visualization conditions and show the object of interest in the center of the image.
1. Once all the images of the probe and the injected cells are done, open the Volume Calculator tool (on the left side menu). The tool will open in a new window inside ARYA.
2. In the Reference Selector tab, a list of individual images will be displayed: select the one for this experiment set. The reference image will be displayed in the window:
3. Select the background and then the reference area of the cantilever, which corresponds to the area without reflective coating, on the top of the cantilever. Click on Apply Reference. Once the background has been correctly applied, a blue mark will appear under the Reference Selector tab.
4. In the Cell Image Selector tab, a list with all the group images will be displayed. When selecting the group of interest, the list of images of this point will appear below. After selecting all the images of interest (all of them or a selection), click on Apply Selection and move to the next tab of the left side menu.
5. In the Image Processing tab, the selected images will be displayed. As for the reference, the background and the area of interest (in this case, the injected cell), must be selected. The software allows to measure one cell per image (with the observation workflow, the cell of interest in each picture will be in the center). Once one cell has been processed, click on Apply and continue with the next image on the list.
6. Once all the images from a group point have been processed, click on the Report tab: the list with the measured volumes will appear in the window. Click on Save: the results will be added to the Results history.
1. From the Results history in ARYA, click on the single display of the menu. The file with the measurement volumes will appear on the list:
2. Click on Export and define the export options:
3. The data can now be open from a .csv file.
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