Time-resolved Two-photon Microscopy Research Articles

Multidimensional quantitative characterization of basal cell carcinoma by spectral- and time-resolved two-photon microscopy

Abstract Basal cell carcinoma (BCC) is a common type of skin cancer. Conventional approaches to BCC diagnosis often involve invasive histological examinations that can distort or even destroy information derived from the biomolecules in the sample. Therefore, a non-invasive, label-free examination method for the clinical diagnosis of BCC represents a critical advance. This study combined spectral- and time-resolved two-photon microscopy with a spectral phasor to extract rich biochemical information describing macroscopic tumor morphology and microscopic tumor metabolism. The proposed optical imaging technique achieved the rapid and efficient separation of tumor structures in systematic research conducted on normal and BCC human skin tissues. The results demonstrate that a combination of multidimensional data (e.g., fluorescence intensity, spectrum, and lifetime) with a spectral phasor can accurately identify tumor boundaries and achieve rapid separation. This label-free, real-time, multidimensional imaging technique serves as a complement to the conventional tumor diagnostic toolbox and demonstrates significant potential for the early diagnosis of BCC and wider applications in intraoperative auxiliary imaging.

Organic cation transport in the rat kidney in vivo visualized by time-resolved two-photon microscopy

Secretion of cationic drugs and endogenous metabolites is a major function of the kidney accomplished by tubular organic cation transport systems. A cationic styryl dye (ASP(+)) was developed as a fluorescent substrate for renal organic cation transporters. The dye was injected intravenously and continuously monitored in externalized rat kidneys by time-resolved two-photon laser scanning microscopy. To investigate changes in transport activity, cimetidine, a competitive inhibitor of organic cation transport was co-injected with ASP(+). Shortly after injection, fluorescence increased in peritubular capillaries. Simultaneously, fluorescence was transiently found at the basolateral membrane of the proximal and distal tubules at a higher intensity and shorter wavelength indicating membrane association of ASP(+). Subsequently, intracellular fluorescence increased steeply within 10 s. In the proximal tubules, intracellular fluorescence decreased by 50% within 5 min, while in the distal tubules the fluorescence decreased by only 5% within the same time frame. Intracellular uptake of ASP(+) into proximal tubules was significantly reduced by cimetidine. Our studies show that organic cation transport of the kidney can be visualized in vivo by two-photon laser scanning microscopy.