A Ratiometric Two-Photon Fluorescent Probe for Tracking Lysosomal ATP: Direct In†Cellulo Observation of Lysosomal Membrane Fusion Processes.

Vesicles exchange their contents through membrane fusion processes, kiss-and-run and full-collapse fusion. Indirect observation of these fusion processes using artificial vesicles enhanced our understanding on the molecular mechanisms involved. Direct observation of the fusion processes in a real biological system, however, remains a challenge owing to many technical obstacles. We report a ratiometric two-photon probe offering real-time tracking of lysosomal ATP with quantitative information for the first time. By applying the probe to two-photon live-cell imaging, the lysosomal membrane fusion process in cells has been directly observed and the concentration of its content, lysosomal ATP, has been measured. Results show that the kiss-and-run process between lysosomes proceeds through repeated transient interactions with gradual content mixing, whereas the full-fusion process occurs at once. Furthermore, it is confirmed that both the fusion processes proceed with conservation of the content. Such a small-molecule probe exerts minimal disturbance and hence has potential for studying various biological processes associated with lysosomal ATP.

Fluorescent Labeling of Protein Using Blue-Emitting 8-Amino-BODIPY Derivatives.

8-Amino-BODIPY (boron-dipyrromethane) dyes show bright blue fluorescence. Disclosed here are synthesis and characterization of the photophysical properties of a series of functionalized 8-Amino-BODIPY (BP1-4) for protein labeling. The compact structure and solvent-insensitive absorption property of the dye are desirable features for protein labeling. For the model protein, bovine serum albumin (BSA), the labeling proceeds under mild condition via amide bond formation or thiol-ene conjugation with maintaining the bright blue fluorescence. The chromatography and mass spectroscopy analysis clearly support the labeling of the BODIPY dye on the BSA. The protein labeling with blue-emitting BODIPY would be applicable for studying protein dynamics and fluorescence resonance energy transfer (FRET) with intrinsic biomolecules.

Simultaneous Evaluation of Thermal and Non-Thermal Effects of High-Intensity Focused Ultrasound on a Tissue-Mimicking Phantom.

Physiologically relevant phantoms with high reliability are essential for extending the therapeutic applications of high-intensity therapeutic ultrasound. Here we describe a tissue-mimicking phantom capable of quantifying temperature changes and observing non-thermal phenomena by high-intensity therapeutic ultrasound. Using polydiacetylene liposomes, we fabricated agar-based polydiacetylene hydrogel phantoms (PHPs) that not only respond to temperature, but also have acoustic properties similar to those of human liver tissue. The color of PHPs changed from blue to red depending on the temperature in the range 40°C-70°C, where the red/blue ratio of PHP had a good linearity of 99.06% for the temperature changes. Furthermore, repeated high-intensity focused ultrasound led to histotripsy on the PHP with liquefied and damaged areas measuring 0.7 and 4.0 cm2, respectively, at the signal generator amplitude setting voltage of 80 mV. Our results indicate not only the usability of the thermochromic phantom, but also its potential for evaluating non-thermal phenomena in various high-intensity focused ultrasound therapies.