Following the completion of an injection workflow, comprehensive data analysis is essential to evaluate the success and biological impact of the procedure. This section covers the key analytical approaches used to assess injection outcomes, including force curves analysis, cell viability assessment and quantification of injected volumes to ensure delivery precision, and observation of cellular behavior over extended time periods. These analytical methods provide critical quality control metrics that validate injection protocols and establish reproducibility standards across different experimental conditions and operators.
Force curves
The force curve for each injection can be evaluated in the Results history of the ARYA software.
For Constant Force injections, in a perfect injection, the Z position and the force should remain stable during the injection (as observed in figure below).
Force curve of a cell injection with Constant F workflow, showing a force overshoot at the start and stable force during the injection. In green, z position is indicated in mm. In yellow, force readout is shown. Important: exported force curves show force in Volts instead of Newtons.
For Constant Z injections, force curves can provide information on how the cell reacts to the contact of the cantilever. Depending on the force feedback, the nucleus enlargement due to the IM delivery or even a cell burst can be detected.
Force curves of a cell injection with Constant Z workflow; on the left, the increase of the deflection (in yellow) happens as a result of the injection of the nucleus, whilst in the right, a cell burst can be detected with a sudden decrease of the force readout.
Cell viability
The assessment of the viability of the cell culture is essential to determine whether the injected cells can recover after an experiment with FluidFM technology. It also allows detection of workflow issues that can interfere with the injection outcomes, including: toxic injection mix, excessive imaging exposure times or poor initial cell culture viability.
Examples of cell
death (Ethidium homodimer staining), due to injection (a), to low cell culture
viability (b) and too long exposure time (c).
Learn more about Cell Death Due to Phototoxicity
There is a large variety of assays that evaluate the viability of a cell culture. Ideally, the viability assessment readout should be tracked at a different wavelength than the tracer used for injection. Note: Some viability assays may damage the cell culture. If cells are required for subsequent experiments, use non-endpoint assays.
In this protocol, cell
damage is analyzed with ethidium homodimer staining (EthD-1). EthD-1 is a
fluorescent agent used as a DNA stain. EthD-1 cannot cross intact cell
membranes. However, in damaged or dead cells with compromised membranes, EthD-1
passes through and intercalates with the nuclear DNA. When the dye is bound to
DNA, its fluorescence is enhanced, showing an excitation/emission maximum
of 527/624nm. Therefore, the result of the assay can be easily imaged with
fluorescence microscopy.
To stain the cells:
- Wait 5 minutes after the injection.
- Without removing the plate from the system, add EthD-1 solution (2mM in DMSO) to the cell cultures to a final concentration of 4 μM.
- Incubate for 10 minutes at 37°C.
- Record images of the injected cells with multiple imaging presets:
- Brightfield image (for morphology).
- Fluorescence, filter dependent on the tracer used (for injected cells).
- Fluorescence, red filter (for dead cells).
To evaluate the viability:
- Count cells that were successfully injected (with the chosen tracer) and then count cells that are stained with EthD-1 (red).
- Calculate viability as: [1 - (dead cells/injected cells)] x 100%
Quantification of injected volumes
The injected volume can be measured after injection with the Volume Calculator tool in ARYA. The method uses the microchannel height to define a volumetric reference and requires the use of a fluorescent tracer on the injection mix.
Lateral
view of a FluidFM cantilever (SEM), indicating how the volumetric reference is defined. The volumetric
reference is defined by a cube with the side size equivalent
to the channel height.
For the calculation of the injected volume, the software will need:
- An
image of the cantilever filled with
the fluorescent solution and positioned close
to the plate surface for use as a reference.
- Images of the injected cells, grouped in a point group and obtained with Observe workflow, so they have the visualization conditions (objective used, filter and illumination intensity).
Image of the injected cells, obtained with the Observe workflow. Cell of interest is in the center of the image.
To measure the volume, open the volume calculator tool:
- 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:
- On Background, select the background
- On Cantilever, select the reference area of the cantilever, which corresponds to the area without reflective coating.
Detail of the area of interest (yellow rectangle) for volumetric reference, in brightfield and in fluorescence. Reflective gold layer is well visible in brightfield.
- Indicate Exposure time in case it appears empty
- Click on Apply Reference
Reference selector after applying reference. The background selected appears in red, and the cantilever area selected in blue. Intensity and imaging values are indicated in the lateral panel.
- In the Cell Image Selector tab, a list with all 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.
Cell Image selector after selecting images of interest. The group points appear on the top part of the window. All the pictures related to a point will be listed below.
- 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 one cell per image (with the observation workflow, the cell of interest in each picture will be in the center). When clicking on Apply, the volume will be measured for the selected area. The process can be repeated for all the selected cells.
Injected volume measurement after selecting measurement areas. The background selected appears in red, and the cell area selected in blue. Intensity, volume and imaging values are indicated in the lateral panel.
- 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.
Summary table with the selected point volume measurement.
More information can be found in the following link: https://documentation.cytosurge.com/bot/current/volume_calc/