Two-colour single-molecule photoinduced electron transfer fluorescence imaging microscopy of chaperone dynamics

Many proteins are molecular machines, whose function is dependent on multiple conformational changes that are initiated and tightly controlled through biochemical stimuli. Their mechanistic understanding calls for spectroscopy that can probe simultaneously such structural coordinates. Here we present two-colour fluorescence microscopy in combination with photoinduced electron transfer (PET) probes as a method that simultaneously detects two structural coordinates in single protein molecules, one colour per coordinate. This contrasts with the commonly applied resonance energy transfer (FRET) technique that requires two colours per coordinate. We demonstrate the technique by directly and simultaneously observing three critical structural changes within the Hsp90 molecular chaperone machinery. Our results reveal synchronicity of conformational motions at remote sites during ATPase-driven closure of the Hsp90 molecular clamp, providing evidence for a cooperativity mechanism in the chaperone’s catalytic cycle. Single-molecule PET fluorescence microscopy opens up avenues in the multi-dimensional exploration of protein dynamics and allosteric mechanisms.

Anti-Melanogenesis, Antioxidant and Anti-Tyrosinase Activities of Scabiosa columbaria L.

Scabiosa columbaria is a plant traditionally used to treat skin ailments, such as scabies, wound bruises, sores and hyperpigmentation. To find a novel skin depigmenting agent, the present study was investigated to determine the possible anti-melanogenesis, antioxidant and anti-tyrosinase effects of methanol extract of S. columbaria leaves. Cytotoxicity towards human dermal fibroblast (MRHF) cells was assessed using the live-cell fluorescence imaging microscopy. The inhibitory effects of the extract on tyrosinase, collagenase and melanin synthesis were also investigated using standard in vitro method, while ferric reducing power (FRAP) was used to determine the antioxidant potential of the plant extract. The effect of the extract on collagen content in MRHF cells was also investigated. The plant extract displayed no meaningful cytotoxicity towards MRHF cells and no significant cell death was recorded at all the tested concentrations. The extract (25–100 µg/mL) effectively decreased melanin content in B16F10 (mouse melanoma) cells with moderate inhibition of tyrosinase enzyme in a dose-dependent manner. However, the extract also demonstrated no significant effect on collagenase and collagen content in MRHF cells, but showed strong antioxidant activity at the concentrations tested. The results suggest that S. columbaria could be a promising candidate in the treatment of skin hyperpigmentation disorders

Resolution enhancement through microscopic spatiotemporal control

Operating at biologically benign conditions, multi-photon fluorescence imaging microscopy has benefitted immensely from recent developments in microscopic resolution enhancement. Fluorescence microscopy continues to be the best choice for experiments on live specimens, however, multi-photon fluorescence imaging often suffers from overlapping fluorescence of typical dyes used in microscopy, limiting its scope. This limitation has been the focus of our research where we show that by making simple modifications to the laser pulse structure, it is possible to resolve these overlapping fluorescence complications. Specifically, by using pairs of femtosecond pulses with variable delay in place of single pulse excitation, we show controlled fluorescence excitation or suppression of one of the fluorophores over the other through wave-packet interferometry. Such an effect prevails even after the fluorophore coherence timescale, which effectively results in a higher spatial resolution. Here we extend the effect of our pulse-pair technique to microscopic axial resolution experiments and show that such pairs of pulses can also ‘enhance’ axial resolution.