In Vivo Two-Color 2-Photon Imaging of Genetically-Tagged Reporter Cells in the Skin

Our protocol allows us to visualize how fluorescently tagged cells respond to an inflammatory peripheral stimulus in a live animal in real time. 2-Photon imaging allows visualization deep into the tissue of a live specimen, preserving the integrity of their cells and their microenvironment and providing an accurate representation of the biological system. Breathing moves the paw over time, causing blurriness and a loss of the focal plane.

Be sure to affix the paw firmly with tape to a stable surface before imaging. In this you must be able to find appropriate plane of interest, make sure that objective is close to the paw without touching and is directly above injection site. Before beginning the procedure, turn on the multi-photon system and select the 25X subjective.

Place a stereotaxic apparatus onto the stage of the multi-photon microscope and connect the apparatus to an anesthesia delivery machine. Place a piece of matte black paper on the surface of the apparatus as a connection point for the mouse paw. Set the resonance scanner with a fixed scan area of 512 by 512 micrometers.

Tune the excitation lasers to the 930 and 1, 100 nanometer excitation wavelengths for green and red fluorescent protein signals respectively. Use a dichroic mirror of 690 to 1, 050 nanometers to direct the light path of both excitation lasers to the single objective allowing the 930 nanometer tuned excitation laser to be reflected to the main scanner and the 1, 100 nanometer tuned laser to pass directly into the main scanner. Then set the laser power of FITC to 5%and green fluorescent protein to 20%and turn off the overhead lights.

For in vivo imaging, anesthetize the mouse and outlined in the text protocol. Confirm a lack of response to toe pinch in the anesthetized mouse and place the mouse in the stereotaxic apparatus with access to a nose cone. Use black tape to firmly affix the hind paw to the piece of black paper on areas both proximal and distal to the area of interest, making sure the plantar surface of the paw is unobstructed and facing up toward the objective.

Place a generous amount of water based lubricant onto the plantar surface of the paw. Touch the objective to the lubricant to create a column of liquid between the paw and the objective. Use the FITC excitation light to focus into the dermal layer of the paw, confirming that the tandem dimer Tomato tagged fibroblasts can be visualized.

Image the area of cells located just below the plantar surface of the hind paw with both lasers. Acquire a 15 minute time lapse of about five to 10 Z-slices at approximately one micrometer per slice to establish a baseline representation of the environment. When the baseline imaging has been obtained, load a 25 microliter glass Hamilton syringe with five micrograms of FITC conjugated lipopolysaccharide, or LPS, per 20 microliters of PBS.

Administer the solution by intraplantar injection into the experimental hind paw without disturbing the paw position. Then image an area of cells located just below the plantar surface of the hind paw with both lasers. Acquire a 60 to 120 minute time lapse of about five to 10 Z-slices at approximately one micrometer per slice to identify the cell mediated intraplantar uptake of LPS FITC.

As there is no inherent fluorescence by the cells within the dermal layer, a myriad of cells in the dermal layer of the hind paw can be observed taking fluorescently tagged LPS after intraplantar injection in a wild type mouse. After LPS FITC injection, only fibroblast specific protein one positive fibroblasts expressing toll like receptor four bind and uptake the injected protein with a high level of co-localization with a tandem dimer Tomato tag expressed by these cells. In contrast, mice that have toll like receptor four knocked out of the entire body do not bind and uptake LPS after injection.

Indeed, cell silhouettes are visible after LPS FITC injection indicating that the drug is dispersing in the interstitial fluid around cells, but is not actually being bound by a receptor. The most important thing to remember in this protocol is to ensure that the paw's immobilized so that there are no distortions in the video due to movement or respiration. Using this procedure, the recruitment of fluorescently tagged cells to an area after injury and the morphological changes in a cell's response to a stimulus over time can be tracked.

So 2-Photon imaging allows researchers to combine genetic reporter mice and fluorescently tagged compounds to assess what happens in a live animal after an injection.