Abstract
Using third harmonic generation (THG) microscopy, we demonstrate that granularity differences of leukocytes can be revealed without a label. Excited by a 1230 nm femtosecond laser, THG signals were generated at a significantly higher level in neutrophils than other mononuclear cells, whereas signals in agranular lymphocytes were one order of magnitude smaller. Interestingly, the characteristic THG features can also be observed in vivo to track the newly recruited leukocytes following lipopolysaccharide (LPS) challenge. These results suggest that label-free THG imaging may provide timely tracking of leukocyte movement without disturbing the normal cellular or physiological status.
Highlights
The in vivo study of leukocytes is challenging due to their nature of fast trafficking, multiple lineages, frequent cell-cell interactions, and dynamic activation or maturation in immune response processes
With prior knowledge on the types of leukocytes, these results indicate that leukocytes with different granularities have different morphologies and contrasts in third harmonic generation (THG) microscopy
We showed that THG microscopy is sensitive to the granularity differences among leukocytes
Summary
The in vivo study of leukocytes is challenging due to their nature of fast trafficking, multiple lineages, frequent cell-cell interactions, and dynamic activation or maturation in immune response processes. Such characteristic infrared absorption bands around 1210 nm have been used for photothermolysis of lipid-rich tissues [16], and provide strong photoacoustic contrast for lipids in deep tissues [17] Given these advantages, we demonstrate in this study that the difference in granularity, which is an obvious phenotype of leukocytes, can be revealed by high resolution THG microscopy. These differences in THG features in leukocytes can be observed in lipopolysaccharide (LPS) challenge sites in mouse ears in vivo This approach can potentially be used in the study of immune cytomics, virtual optical biopsy on inflammation sites, or label-free flow cytometry for clinical use [9]
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