Assessment of burn wound tissue in situ by multiphoton microscopy employing fluorescence and second harmonic generation contrasts in live animals

Current assessment of burn wound depth and progression of healing for proper choice of treatment is based on time-consuming and invasive techniques that may interfere with the healing process. One way to overcome these problems is to use noninvasive techniques. We use multiphoton microscopy (MPM) that employs fluorescence and second harmonic generation (SHG) contrasts to noninvasively follow the burn healing process in situ as healing progresses in live animals. Healing progression was followed in a partial thickness burn wound made on the dorsum of anesthetized Sprague-Dawley rats with a 2.8 cm diameter brass cylinder heated at 80oC for 60 seconds and pressed against the shaved rat skin for 6 seconds. During the first five days, burn healing was characterized by changes in the organization of collagen into a compact, mat-like assembly, suggesting progressive degradation of collagen within the injury site.We also detected increased follicular cell damage and a strong auto-fluorescence signal from cellular debris. Deposition of new collagen was seen after the scab fell off and gradually increased thereafter as detected with an increase in the SHG signal intensity. A great increase in the density of cells was observed as well. From day 21 on, a network of capillaries with blood flow was observed. The epidermal cell layer and the blood vessel network became progressively more organized. At day 29, fibrillar collagen had SHG signal levels and morphology of the nearly preburn state. These findings were corroborated by histology/histochemistry. In conclusion, MPM technology that employs fluorescence and SHG contrasts is instrumental in following the healing process, in particular during the early stages of healing. The degree and rate with which these events occur early after burning could help clinicians make treatment decisions. The outcomes of healing at later times would be indicative of the effectiveness of the treatment applied

In situ multiphoton optical tomography of hair follicles in mice.

We report multiphoton in situ optical sectioning of hair follicles in mice and a preliminary investigation of the pathological hair follicles in a transgenic mouse model. Using this imaging technology, we rapidly obtain detailed three-dimensional (3-D) reconstructions of individual hair follicles. No staining or mechanical sectioning is involved, since multiphoton microscopy coregisters two-photon excited fluorescence (TPF) from cells and second harmonic generation (SHG) signals from the extracellular matrix (ECM). These signals are ideally suited for estimating molecularly encoded hair follicular 3-D geometries, including sizes of the follicular orifices and their angles relative to the skin surface. In the normal hair follicles, spectral separation of SHG signals generated by the ECM of the hair follicle from that of intrinsic cellular fluorescence revealed intricate spatial interaction of the cellular components with the surrounding connective tissue. In the pathological hair follicles, these were clearly modified. In particular, in the transgenic mice, we observed lack of cellular fluorescence and significantly shallower angles of follicular orifices with respect to the skin surface. The combination of TPF with SHG is sensitive to structural changes in cells and extracellular matrix brought on by normal hair follicle physiology and specific gene alterations

Multiphoton imaging of actin filament formation and mitochondrial energetics of human ACBT gliomas.

We studied the three-dimensional (3D) distribution of actin filaments and mitochondria in relation to ACBT glioblastoma cells migration. We embedded the cells in the spheroid form within collagen hydrogels and imaged them by in situ multiphoton microscopy (MPM). The static 3D overlay of the distribution of actin filaments and mitochondria provided a greater understanding of cell-to-cell and cell-to-substrate interactions and morphology. While imaging mitochondria to obtain ratiometric redox index based on cellular fluorescence from reduced nicotinamide adenine dinucleotide and oxidized flavin adenine dinucleotide we observed differential sensitivity of the migrating ACBT glioblastoma cells to femtosecond laser irradiation employed in MPM. We imaged actin-green fluorescent protein fluorescence in live ACBT glioma cells and for the first time observed dynamic modulation of the pools of actin during migration in 3D. The MPM imaging, which probes cells directly within the 3D cancer models, could potentially aid in working out a link between the functional performance of mitochondria, actin distribution and cancer invasiveness