Abstract
A label-free microscopy method for assessing the differentiation status of stem cells is presented with potential application for characterization of therapeutic stem cell populations. The microscopy system is capable of characterizing live cells based on the use of evanescent wave microscopy and quantitative phase contrast (QPC) microscopy. The capability of the microscopy system is demonstrated by studying the differentiation of live immortalised neonatal mouse neural stem cells over a 15 day time course. Metrics extracted from microscope images are assessed and images compared with results from endpoint immuno-staining studies to illustrate the system’s performance. Results demonstrate the potential of the microscopy system as a valuable tool for cell biologists to readily identify the differentiation status of unlabelled live cells.
Highlights
There is a requirement for a non-invasive, label-free approach to discriminate distinct differentiation stages of therapeutic stem cells and to identify their differentiation potential, viability and proliferation in culture
Metrics which combine measures from microscope images obtained from both modalities are compared with results from endpoint immuno-staining studies to illustrate the performance of the microscopy system presented to evaluate stem cell differentiation status in comparison to currently used fluorescent microscopy techniques
In this work evanescent illumination was achieved through the use of an incoherent light source (Light Emitting Diode (LED)) that is spatially filtered using an annular mask and re-imaged into the back focal plane of a high numerical aperture objective lens
Summary
There is a requirement for a non-invasive, label-free approach to discriminate distinct differentiation stages of therapeutic stem cells and to identify their differentiation potential, viability and proliferation in culture. Advanced label-free light microscopy techniques are prime candidates to meet this requirement as they can be applied rapidly on both a single cell and population level, provide high resolution images and are amenable to long term time-lapsed studies of live cells To this end success has been found through the combination of non-linear optical microscopy techniques such as coherent anti-Stokes Raman scattering (CARS) and second harmonic generation (SHG) which have been shown to provide similar sensitivity as destructive biochemical methods for the analysis of stem cell differentiation, with the added benefit of providing structural microscopic www.nature.com/scientificreports/. These techniques certainly will play a role in the non-destructive and label-free analysis of stem cells, their implementation is far from straightforward and have limited throughput due to their reliance on laser scanning and low light levels to form images This approach does not satisfy the need for quick, sensitive screening methods to characterise stem cells for therapeutic use in a timely manner[14]. Metrics which combine measures from microscope images obtained from both modalities are compared with results from endpoint immuno-staining studies to illustrate the performance of the microscopy system presented to evaluate stem cell differentiation status in comparison to currently used fluorescent microscopy techniques
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