Quadrant darkfield (QDF) for label-free imaging of intracellular puncta.

Abstract

Measuring changes in cellular structure and organelles is crucial for understanding disease progression and cellular responses to treatments. A label-free imaging method can aid in advancing biomedical research and therapeutic strategies. This study introduces a computational darkfield imaging approach named quadrant darkfield (QDF) to separate smaller cellular features from large structures, enabling label-free imaging of cell organelles and structures in living cells. Using a programmable LED array as illumination source, we vary the direction of illumination to encode additional information about the feature size within cells. This is possible due to the varying level of directional scattering produced by features based on their sizes relative to the wavelength of light used. QDF successfully resolved small cellular features without interference from larger structures. QDF signal is more consistent during cell shape changes than traditional darkfield. QDF signals correlate with flow cytometry side scatter measurements, effectively differentiating cells by organelle content. QDF imaging enhances the study of subcellular structures in living cells, offering improved quantification of organelle content compared to darkfield without labels. This method can be simultaneously performed with other techniques such as quantitative phase imaging to generate a multidimensional picture of living cells in real-time.