Two-Photon Microscopy for Non-Invasive, Quantitative Monitoring of Stem Cell Differentiation

BACKGROUND: The engineering of functional tissues is a complex multi-stage process, the success of which depends on the careful control of culture conditions and ultimately tissue maturation. To enable the efficient optimization of tissue development protocols, techniques suitable for monitoring the effects of added stimuli and induced tissue changes are needed. METHODOLOGY/PRINCIPAL FINDINGS: Here, we present the quantitative use of two-photon excited fluorescence (TPEF) and second harmonic generation (SHG) as a noninvasive means to monitor the differentiation of human mesenchymal stem cells (hMSCs) using entirely endogenous sources of contrast. We demonstrate that the individual fluorescence contribution from the intrinsic cellular fluorophores NAD(P)H, flavoproteins and lipofuscin can be extracted from TPEF images and monitored dynamically from the same cell population over time. Using the redox ratio, calculated from the contributions of NAD(P)H and flavoproteins, we identify distinct patterns in the evolution of the metabolic activity of hMSCs maintained in either propagation, osteogenic or adipogenic differentiation media. The differentiation of these cells is mirrored by changes in cell morphology apparent in high resolution TPEF images and by the detection of collagen production via SHG imaging. Finally, we find dramatic increases in lipofuscin levels in hMSCs maintained at 20% oxygen vs. those in 5% oxygen, establishing the use of this chromophore as a potential biomarker for oxidative stress. CONCLUSIONS/SIGNIFICANCE: In this study we demonstrate that it is possible to monitor the metabolic activity, morphology, ECM production and oxidative stress of hMSCs in a non-invasive manner. This is accomplished using generally available multiphoton microscopy equipment and simple data analysis techniques, such that the method can widely adopted by laboratories with a diversity of comparable equipment. This method therefore represents a powerful tool, which enables researchers to monitor engineered tissues and optimize culture conditions in a near real time manner

Photogeneration and quenching of reactive oxygen species by urocanic acid

Urocanic acid, UCA, is characterized by two electronic transitions in the UV-B (280−320 nm) which comprise its broad absorption spectrum and give rise to wavelength-dependent isomerization quantum yields. The absorption spectrum of UCA extends into the UV-A (320−400 nm). Given the UV-A component of sunlight is significantly greater than the UV-B component it is hypothesized even weak UV-A photochemistry of UCA could be important for in vivo responses to UV radiation. Degenerate pump−probe experiments performed on t-UCA at several wavelengths in the UV-A reveal an excited-state absorption that undergoes a rapid, 1 ps decay. Photoacoustic experiments performed on both the cis and trans isomers reveal the formation of a long-lived intermediate following UV-A excitation. The efficiency and action spectra for this latter photoactive process are presented and are similar for both isomers of UCA. Cholesterol hydroperoxide assays designed to investigate the nature of the UV-A photoreactivity of t-UCA confirm the production of reactive oxygen species. The bimolecular rate constant for the quenching of singlet oxygen by t-UCA is determined to be 3.5 × 106 M-1 s-1. Taking into consideration recent theoretical calculations and jet expansion studies of the electronic structure of gas-phase t-UCA, a model is proposed to explain the isomerization and photoreactivity of t-UCA in solution over the UV-A region

Probing the spatial dependence of the emission spectrum of single human retinal lipofuscin granules using near-field scanning optical microscopy

The emission spectra of single lipofuscin granules are examined using spectrally resolved confocal microscopy and near-field scanning optical microscopy (NSOM). The emission spectrum varies among the granules examined revealing that individual granules are characterized by different distributions of fluorophores. The range of spectra observed is consistent with in vivo spectra of human retinal pigment epithelium cells. NSOM measurements reveal that the shape of the spectrum does not vary with position within the emissive regions of single lipofuscin granules. These results suggest that the relative distribution of fluorophores within the emissive regions of an individual granule is homogeneous on the spatial scale ∼150 nm

Suppression of different phases of systemic contact hypersensitivity by urocanic acid oxidation products

On exposure to UV-B, the epidermal component trans-urocanic acid (UCA) is not only photoisomerized into cis-UCA but will also, at least in part, be photooxidized into UCA oxidation products (UOPs). We hypothesized that UOPs can mimic UV-induced systemic immunosuppression comparable to the suppressive properties already established for cis-UCA. A crude mixture of UOPs showed a significant suppression of the sensitization phase of the systemic contact hypersensitivity (CHS) response to picryl chloride (PCl). Three of the UOPs were selected for this study: imidazole-4-carboxylic acid (ImCOOH), imidazole-4-carboxaldehyde (ImCHO) and imidazole-4-acetic acid (ImAc). Effects on the sensitization, elicitation and postelicitation phases of CHS to PCl in BALB/c mice were studied and compared with the effects of cis-UCA. ImCHO was equally effective at suppressing the sensitization phase as cis-UCA. The triplet combination of the imidazoles (1:1:1) showed more pronounced suppression than that induced by cis-UCA. The most effective compounds for the suppression of the elicitation phase appeared to be ImAc and cis-UCA. Significant suppression of the postelicitation phase was only obtained with the triplet combination of ImCHO, ImCOOH and ImAc, the combination that appeared to be effective at all three tested phases. Because these three UOPs are present in UV-B-exposed human stratum corneum, these compounds may play a role in UV-B-induced immunosuppression