Label-Free Live-Cell Hyperspectral Microscopy and Analysis

Fluorescence microscopy is the established standard for imaging in most cell biology related research initiatives. Its ability to enable subcellular labeling specificity provides strong utility for many research applications. As such, the development of widefield and confocal fluorescence imaging has long been the primary focus of all major microscope companies supporting biological applications.?

However, the rapid expansion of cellular therapies into the clinical market has created a growing need to image and characterize live cells without the use of fluorescent labels. While fluorescent labels can quickly determine whether cells are viable and healthy for use in cell therapy applications, most immuno-fluorescent viability dyes cannot be utilized on cells needed for use in human therapeutic applications.??

Imaging and Characterizing Live Cells with Enhanced Darkfield Hyperspectral Microscopy

In numerous experiments, it has been demonstrated that CytoViva, Inc ’s Enhanced Darkfield Hyperspectral Microscope can be used to optically image and spectrally characterize unique elements of label-free live cell cultures based solely on the endogenous scattering spectrum produced by the cells. This is possible due to the unique combination of the CytoViva’s patented enhanced darkfield optics, which produce high signal-to- noise images of the cells, and the integrated hyperspectral imaging capability, which records the optical spectrum in each nanoscale image pixel. This integrated solution enables the resulting optical spectral data to be quickly analyzed to confirm the unique attributes of label- free live cell structures.

Label-Free Identification of Differentiating Versus Non-Differentiating Live Stem Cells

The use of CytoViva, Inc ’s system for label-free live cell analysis includes the ability to identify differentiating versus non-differentiating live stem cells. This process can be accomplished without any fluorescent labeling or other special sample preparation. In recent publications, Gartia et al. has demonstrated this ability to conduct label-free live stem cell analysis to determine differentiating versus non-differentiating cells. In the paper “Multimodal Label-Free Monitoring of Adipogenic Stem Cell Differentiation Using Endogenous Optical Biomarkers” (https://onlinelibrary.wiley.com/doi/10.1002/adfm.202103955), the authors demonstrate that when using enhanced darkfield hyperspectral microscopy, the spectral signature of stem cell adipogenesis enables differentiated cells to be identified at the single-cell level. This process is non-destructive to the cells and can allow the identified pure differentiating stem cells to be isolated and utilized in a therapeutic application.

Label-Free Healthy Versus Unhealthy Live Cell Delineation

For any use of cells in a therapeutic application, it is critical to know that the cell culture is healthy and viable and therefore capable of performing its therapeutic mission with proper efficacy.?As mentioned earlier, fluorescent viability stains can be utilized to provide this healthy-unhealthy indication, but cannot typically be used on cells targeted for human treatment.?As such, the use of CytoViva’s Enhanced Darkfield Hyperspectral Microscope has great potential utility to measure the health and viability of cells in a label-free and non-destructive manner targeted for human therapy.

Figures 1 – 4 below demonstrate the ability to identify health versus unhealthy live cells in a label-free manner using CytoViva’s Enhanced Darkfield Hyperspectral Microscope [Ref.1]. Figure 1 shows an enhanced darkfield hyperspectral image of live human dermal fibroblast (HDF) cells. These cells represent healthy control cells. Note the intact cell membrane and nuclear membrane of the cells, as well as the prominent scattering of light due to the presence of cell vacuoles in the cytoplasmic areas of the cells.?

Figure 2 illustrates an HDF cell after exposure to 1 millimolar (mM) of H2O2. ?Note the absence of both a defined cell membrane and nuclear membrane, as well as other structures observed in the healthy control cells in Figure 1. The appearance of this cell is consistent with previously observed oncoming necrosis.

In Figure 3, a mean spectral difference is shown from large areas of the healthy control cell versus the unhealthy cells exposed to the H2O2.

Figure 4 illustrates, through spectral mapping, all pixel level spectrum in the image where spectral differences exist in the unhealthy cells but are not present the healthy cells. These differences are mapped as green in the unhealthy cell.?

The ability to identify differences in live cells based on their endogenous optical spectral response creates many opportunities to advance and enhance the therapeutic use of cells in a wide range of applications. This includes stem cell therapy, immunotherapies, including NK and CAR T-cell therapy and many other developing cell therapy applications. It can also be utilized to determine cell viability for therapeutic applications in a label-free manner. To learn more about CytoViva, Inc ’s Enhanced Darkfield Hyperspectral Microscope and how it can support your need for label-free live cell imaging and analysis, please contact us at [email protected]. We will be pleased to discuss your application and conduct proof-of-concept test imaging of your samples as appropriate.

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Ref. 1: Special thanks to the Lotsch Laboratory at Auburn University for their kind assistance and expertise with the culturing of cells for the healthy versus unhealthy cell experiments. ?

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