3D spectral imaging with synchrotron Fourier transform infrared spectro-microtomography.

We report Fourier transform infrared spectro-microtomography, a nondestructive three-dimensional imaging approach that reveals the distribution of distinctive chemical compositions throughout an intact biological or materials sample. The method combines mid-infrared absorption contrast with computed tomographic data acquisition and reconstruction to enhance chemical and morphological localization by determining a complete infrared spectrum for every voxel (millions of spectra determined per sample).

Cell death, non-invasively assessed by intrinsic fluorescence intensity of NADH, is a predictive indicator of functional differentiation of embryonic stem cells.

BACKGROUND INFORMATION: Continued advances in stem cell biology and stem cell transplantation rely on non-invasive biomarkers to characterise cells and stem cell aggregates. The non-invasive quality of such biomarkers is essential because exogenous labels, probes or reporters can unintentionally and dramatically alter stem cell state as can disruption of cell-cell and cell-matrix interactions. Here, we investigate the utility of the autofluorescent metabolite, nicotinamide adenine dinucleotide (NADH), as a non-invasive, intrinsic biomarker of cell death when detected with multi-photon optical-based approaches. To test this possibility, cell death was induced in murine embryoid bodies (EBs) at an early stage (day 3) of differentiation using staurosporine, an ATP-competitive kinase inhibitor of electron transport. Several hours after staurosporine treatment, EBs were stained with a single-colour, live/dead probe. A single-cross-sectional plane of each EB was imaged to detect the fluorescence intensity of the live/dead probe (extrinsic fluorescence) as well as the fluorescence intensity of NADH (intrinsic fluorescence). EBs were assessed at subsequent time points (days 6-12) for the formation of beating areas as an indicator of functional differentiation. RESULTS: Statistical comparison indicated a strong positive correlation between extrinsic fluorescence intensity of the live/dead stain and intrinsic fluorescence of NADH, suggesting that the intensity of NADH fluorescence could be used to reliably and non-invasively assess death of cells of EBs. Furthermore, EBs that had high levels of cell death soon after aggregate formation had limited ability to give rise to functional cardiomyocytes at later time points. CONCLUSIONS: We demonstrate the utility of NADH fluorescence intensity as a non-invasive indicator of cell death in stem cell aggregates when measured using multi-photon excitation. In addition, we show that the degree of stem cell death at early stages of differentiation is predictive for the formation of functional cardiomyocytes.

Multiphoton flow cytometry to assess intrinsic and extrinsic fluorescence in cellular aggregates: applications to stem cells.

Detection and tracking of stem cell state are difficult due to insufficient means for rapidly screening cell state in a noninvasive manner. This challenge is compounded when stem cells are cultured in aggregates or three-dimensional (3D) constructs because living cells in this form are difficult to analyze without disrupting cellular contacts. Multiphoton laser scanning microscopy is uniquely suited to analyze 3D structures due to the broad tunability of excitation sources, deep sectioning capacity, and minimal phototoxicity but is throughput limited. A novel multiphoton fluorescence excitation flow cytometry (MPFC) instrument could be used to accurately probe cells in the interior of multicell aggregates or tissue constructs in an enhanced-throughput manner and measure corresponding fluorescent properties. By exciting endogenous fluorophores as intrinsic biomarkers or exciting extrinsic reporter molecules, the properties of cells in aggregates can be understood while the viable cellular aggregates are maintained. Here we introduce a first generation MPFC system and show appropriate speed and accuracy of image capture and measured fluorescence intensity, including intrinsic fluorescence intensity. Thus, this novel instrument enables rapid characterization of stem cells and corresponding aggregates in a noninvasive manner and could dramatically transform how stem cells are studied in the laboratory and utilized in the clinic.

Multiparametric flow cytometry using near-infrared fluorescent proteins engineered from bacterial phytochromes.

Engineering of fluorescent proteins (FPs) has followed a trend of achieving longer fluorescence wavelengths, with the ultimate goal of producing proteins with both excitation and emission in the near-infrared (NIR) region of the spectrum. Flow cytometers are now almost universally equipped with red lasers, and can now be equipped with NIR lasers as well. Most red-shifted FPs of the GFP-like family are maximally excited by orange lasers (590 to 610 nm) not commonly found on cytometers. This has changed with the development of the iRFP series of NIR FPs from the protein family of bacterial phytochromes. The shortest wavelength variants of this series, iRFP670 and iRFP682 showed maximal excitation with visible red lasers. The longer wavelength variants iRFP702, iRFP713 and iRFP720 could be optimally excited by NIR lasers ranging from 685 to 730 nm. Pairs of iRFPs could be detected simultaneously by using red and NIR lasers. Moreover, a novel spectral cytometry technique, which relies on spectral deconvolution rather than optical filters, allowed spectra of all five iRFPs to be analyzed simultaneously with no spectral overlap. Together, the combination of iRFPs with the advanced flow cytometry will allow to first image tissues expressing iRFPs deep in live animals and then quantify individual cell intensities and sort out the distinct primary cell subpopulations ex vivo.