Quadrant darkfield for label-free imaging of intracellular puncta.

SIGNIFICANCE

Imaging changes in subcellular structure is critical to understanding cell behavior but labeling can be impractical for some specimens and may induce artifacts. Although darkfield microscopy can reveal internal cell structures, it often produces strong signals at cell edges that obscure intracellular details. By optically eliminating the edge signal from darkfield images, we can resolve and quantify changes to cell structure without labeling.

AIM

We introduce 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.

APPROACH

Using a programmable LED array as the 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 levels of directional scattering produced by features based on their sizes relative to the wavelength of light used.

RESULTS

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.

CONCLUSIONS

QDF imaging enhances the study of subcellular structures in living cells, offering improved quantification of organelle content compared with 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.