The overall goal of this experimental method is to enable realtime study of intracellular signaling using FRET imaging of cells embedded within three-dimensional hydrogels that mimic natural tissues. This method can help answer key questions in the field of developmental biology, pharmacology, and regenerative medicine, such as development of optimum biomaterials and high throughput drug screening in engineered 3D microtissues. The main advantage of this technique is that FRET can be used on a large number of cells in numerous hydrogel types using conventional wide field microscope systems.

After fabricating molds for hydrogel casting and suspending cultured cells in hydrogel solution, according to the text protocol, in a tissue culture hood, add the cell containing precursor to the casting molds using sterile technique. Prior to crosslinking, centerfuge the cells suspended in hydrogel precursor to optimize imaging by confining cells to a single focal plane, and minimizing the out of plane signal. Next, to crosslink PC hydrogels, use a UVA light source for an exposure time equal to 3.2 joules per centimeter squared per millimeter of thickness to irradiate the cell containing precursor.

Alternately, use a less expensive hand held dental lamp. Avoid overexposing the hydrogel, because this will decrease cell viability. Physically crosslinking hydrogels are instead placed at 37 degrees Celsius.

After crosslinking, remove the PDMS hydrogel casting mold, and replace it with the previously prepared PDMS dish. Use a sterile spatula to press the PDMS down onto the glass to adhere it. Add phenol-free serum-free medium or a chemically defined medium to the well created in the PDMS dish with a coverslip to keep the hydrogel immersed.

Incubate the dish at 37 degrees Celsius for 30 minutes. Once the PDMS dish with the hydrogel has been secured on the microscope stage and oil has been applied to the objective, use transmitted light to find at least 15 cells for imaging, and verify transfection with fluorescence imaging. In the microscope software, configure the camera settings for the FRET image channels by choosing among a few cells near the center of the field of view, and optimize exposure and gain per image channel as per the text protocol.

Choose cells that are close to the center of the hydrogel, within the relatively flat illumination field that are neither too bright nor too dim, and with a FRET ratio between 00 and 1.0. Then, select multiple fields of view to acquire images for at least 15 transfected cells. Switch off the light when finished to limit the exposure.

To configure the capture rate for time lapse image acquisition, enter the periodicity of the captures. Use a rapid rate for the baseline signal capture, such as every five seconds per field of view, and a slower rate for subsequent long-term signalling kinetics. Select Run now to initiate image acquisition and capture images for five minutes to establish a baseline for probe response at the designated field of view.

After the five minutes of image acquisition, pipette in the desired agonist or antagonist, mix by pipetting, and continue imaging. After carrying out FRET calibration according to the text protocol, in the microscope software select the arrow on the background region of interest icon and choose Draw Elliptical Background Region of Interest. Draw and designate one region of interest, or ROI, per field of view near the cells to define the background level.

Ensure that Keep Updating Background Offset is selected, then activate viewing of the background region of interest by clicking the Background Region of Interest icon. Alternatively, choose the region of interest icon, and set the region of interest properties as Use as Background Region of Interest, and Regions of interest are different in each Multi Point. Using the region of interest background icon, select Subtract Background Using Region of Interest.

In the pop-up window, under Subtract Background Region of Interest, select Every Image and under Apply To, select All Frames. For each field of view, to define a region of interest around the cell using the acceptor emission channel and a binary mask, use the menu Binary, Define Threshold, and select Apply To Current Frame. Then, adjust the lower threshold to select the cells.

Next, use the Binary Close function to fill in holes in the binary mask, if necessary. Then, use the Region of Interest icon to select Copy Binary to Region of Interest to save the binary mask as regions of interest. Append the regions of interest for subsequent fields of view to the existing pool of regions of interest.

Delete any spurious regions of interest. Then, right click on the Regions of Interest and check that their properties are Use as Standard Region of Interest and Regions of Interest are different in each Multi Point. Cell region of interests can be hand-drawn, but care must be taken to remove spurious values.

The protocol contains procedures for cells that are outside the flat illumination field and for microscopes that have no flat field. Centrifugation of the hydrogel precursors before gelation maximized the number of cells at one focal plane near the coverslip. This minimized background noise and increased imaging throughput.

In this FRET experiment, for the quenched emission, or QE, FRET ratio calculation, only two images were required for the single chain binary ICUE1 probe, including QE equals CFP CFP for excitation and emission, and acceptor emission equals YFP YFP for excitation and emission. Multiple fields of view were selected in which several cells resided within the homogeneous illumination area as shown here. ROIs for background correction of channel signals and for cell analysis were designated using thresholded acceptor emission images from the start of the experiments.

In addition, as shown here, cells were selected that expressed sufficient probe. The QE and sensitized emission FRET ratios per pixel and the average FRET ratio per cell were calculated. The inverse of the SE ratio was calculated and both ratios were normalized.

Under the forskolin stimulation, both the QE and SE based Fret ratios detect the increased cyclic AMP activity in chondrocytes. Both physically crosslinked and chemically crosslinked gels showed an increase in the QE FRET ratio over time, indicating an increased cyclic AMP signalling response to the addition of forskolin. This procedure can be used with other microscopy techniques such as FRAP, flim fret, and fret and anisotropy measurement, to answer questions such as, what are the differences in permeability and cellular singling between different microtissue types.

Since crosslinking is quenched near the PDMS, it's important to remember to design the molds for the photo-crosslinking hydrogels slightly larger in diameter than the hydrogels themselves.