Location and identification of berberine in spores.

(A) DIC images and fluorescence images of individual dormant spores (panes 1, 3), or germinated spores that were incubated in 10 mM L-valine at 23°C for 60 min as described in Methods plus 50 μg/mL of berberine (panes 2, 4). Note that a single spore appears as two bright spots in the DIC image due to the differential interference of illumination light. Berberine fluorescence at 520 nm was acquired with excitation at 473 nm. Germinated spores (dashed arrows) appeared dim in DIC microscopy but bright in fluorescence microscopy, and ungerminated spores (solid arrows) were bright in DIC microscopy but dim in fluorescence microscopy. (B) Average Raman spectra of 30 individual spores that were: curve 1—germinated as described in panel (A) with 200 μg/mL berberine; curve 2—germinated as described in panel (A) without berberine; curve 3—ungerminated after incubation at 37°C for 60 min in 25 mM K-HEPES buffer (pH 7.4) and 200 μg/mL berberine; and curve 4—dormant and incubated as described for curve 3 without berberine. Curve 5 is a Raman spectrum of 200 μg/mL berberine in 25 mM K-HEPES buffer (pH 7.4). The peak intensities in curve 5 were multiplied by 20.