Monitoring stem cells in their natural physiological environment is crucial to understanding stem cell differentiation and the generation of tissue. We applied high-resolution 3D multiphoton tomography in order to non-invasively visualize the stem cells of hair follicles. Single nestin GFP-expressing stem cells were tracked for up to five hours in living transgenic mice. The microenvironment was monitored by two-photon autofluorescence and second harmonic generation. Download the full article here
Multiphoton tomography visualizes collagen fibers in the tumor microenvironment that maintain cancer-cell anchorage and shape
Uchugonova A, Zhao M, Weinigel M, Zhang Y, Bouvet M, Hoffman RM, König K.
Second harmonic generation (SHG) multiphoton imaging can visualize fibrillar collagen in tissues. SHG has previously shown that fibrillar collagen is altered in various types of cancer. In the present study, in vivo, high resolution, SHG multi-photon tomography in living mice was used to study the relationship between cancer cells and intratumor collagen fibrils. Using green fluorescent protein (GFP) to visualize cancer cells and SHG to image collagen, we demonstrated that collagen fibrils provide a scaffold for cancer cells to align themselves and acquire optimal shape. These results suggest a new paradigm for a stromal element of tumors: their role in maintaining anchorage and shape of cancer cells that may enable them to proliferate.
In Vivo Multiphoton NADH Fluorescence Reveals Depth-DependentKeratinocyte Metabolism in Human Skin
Balu M, Mazhar A, Hayakawa CK, Mittal R, Krasieva TB, König K, Venugopalan V, Tromberg BJ.
Biophys J. 2013 Jan 8;104(1):258-67. doi: 10.1016/j.bpj.2012.11.3809. Epub 2013 Jan 8.
We employ a clinical multiphoton microscope to monitor in vivo and noninvasively the changes in reduced nicotinamide adenine dinucleotide (NADH) fluorescence of human epidermal cells during arterial occlusion. We correlate these results with measurements of tissue oxy- and deoxyhemoglobin concentration during oxygen deprivation using spatial frequency domain imaging. During arterial occlusion, a decrease in oxyhemoglobin corresponds to an increase in NADH fluorescence in the basal epidermal cells, implying a reduction in basal cell oxidative phosphorylation. The ischemia-induced oxygen deprivation is associated with a strong increase in NADH fluorescence of keratinocytes in layers close to the stratum basale, whereas keratinocytes from epidermal layers closer to the skin surface are not affected. Spatial frequency domain imaging optical prop-erty measurements, combined with a multilayer Monte Carlo-based radiative transport model of multiphoton microscopy signal collection in skin, establish that localized tissue optical property changes during occlusion do not impact the observed NADH signal increase. This outcome supports the hypothesis that the vascular contribution to the basal layer oxygen supply is signif-icant and these cells engage in oxidative metabolism. Keratinocytes in the more superficial stratum granulosum are either supplied by atmospheric oxygen or are functionally anaerobic. Based on combined hemodynamic and two-photon excited fluorescence data, the oxygen consumption rate in the stratum basale is estimated to be ~0.035 mmoles/106 cells/h.
Clinical coherent anti-Stokes Raman scattering and multiphoton tomography of human skin with a femtosecond laser and photonic crystal fiber
Breunig G, Weinigel M, Bückle R, Kellner-Höfer M, Lademann J, Darvin M, Sterry W and König K
Laser Phys. Lett. 10 025604 doi:10.1088/1612-2011/10/2/025604
We report on in vivo coherent anti-Stokes Raman scattering spectroscopy (CARS), two-photon fluorescence and second-harmonic-generation imaging on human skin with a novel multimodal clinical CARS/multiphoton tomograph. CARS imaging is realized by a combination of femtosecond pulses with broadband continuum pulses generated by a photonic crystal fiber. The images reveal the microscopic distribution of (i) non-fluorescent lipids, (ii) endogenous fluorophores and (iii) the collagen network inside the human skin in vivo with subcellular resolution. Examples of healthy as well as cancer-affected skin are presented.
High-resolution imaging of basal cell carcinoma: a comparison between multiphoton microscopy with fluorescence lifetime imaging
Manfredini M, Arginelli F, Dunsby C, French P, Talbot C, König K, Pellacani G, Ponti G, Seidenari S.
AIMS: The aim of this study was to compare morphological aspects of basal cell carcinoma (BCC) as assessed by two different imaging methods: in vivo reflectance confocal microscopy (RCM) and multiphoton tomography with fluorescence lifetime imaging implementation (MPT-FLIM). METHODS: The study comprised 16 BCCs for which a complete set of RCM and MPT-FLIM images were available. The presence of seven MPT-FLIM descriptors was evaluated. The presence of seven RCM equivalent parameters was scored in accordance to their extension. Chi-squared test with Fisher's exact test and Spearman's rank correlation coefficient were determined between MPT-FLIM scores and adjusted-RCM scores. RESULTS: MPT-FLIM and RCM descriptors of BCC were coupled to match the descriptors that define the same pathological structures. The comparison included: Streaming and Aligned elongated cells, Streaming with multiple directions and Double alignment, Palisading (RCM) and Palisading (MPT-FLIM), Typical tumor islands, and Cell islands surrounded by fibers, Dark silhouettes and Phantom islands, Plump bright cells and Melanophages, Vessels (RCM), and Vessels (MPT-FLIM). The parameters that were significantly correlated were Melanophages/Plump Bright Cells, Aligned elongated cells/Streaming, Double alignment/Streaming with multiple directions, and Palisading (MPT-FLIM)/Palisading (RCM). CONCLUSION: According to our data, both methods are suitable to image BCC's features. The concordance between MPT-FLIM and RCM is high, with some limitations due to the technical differences between the two devices. The hardest difficulty when comparing the images generated by the two imaging modalities is represented by their different field of view.