Fluorescence recovery after photobleaching (FRAP) and single particle tracking (SPT) techniques determine the diffusion coefficient from average diffusive motion of high-concentration molecules and from trajectories of low-concentration single molecules, respectively. Lateral diffusion coefficients measured by FRAP and SPT techniques for the same biomolecule on cell membrane have exhibited inconsistent values across laboratories and platforms with larger diffusion coefficient determined by FRAP, but the sources of the inconsistency have not been investigated thoroughly. Here, we designed an image-based FRAP-SPT system and made a direct comparison between FRAP and SPT for diffusion coefficient of submicron particles with known theoretical values derived from Stokes–Einstein equation in aqueous solution. The combined iFRAP-SPT technique allowed us to measure the diffusion coefficient of the same fluorescent particle by utilizing both techniques in a single platform and to scrutinize inherent errors and artifacts of FRAP. Our results reveal that diffusion coefficient overestimated by FRAP is caused by inaccurate estimation of the bleaching spot size and can be corrected by simple image analysis. Our iFRAP-SPT technique can be potentially used for not only cellular membrane dynamics but also for quantitative analysis of the spatiotemporal distribution of the solutes in small scale analytical devices.

In endoscopic submucosal dissection (ESD), the narrow gastrointestinal space can cause difficulty in surgical interventions. Tissue ablation apparatuses with high-power CO₂ lasers or Nd:YAG lasers have been developed to facilitate endoscopic surgical procedures. We studied the interaction of 808-nm laser light with a porcine stomach tissue, with the aim of developing a therapeutic medical device that can remove lesions at the gastrointestinal wall by irradiating a near-infrared laser light incorporated in an endoscopic system. The perforation depths at the porcine fillet and the stomach tissues linearly increased in the range of 2–8mm in proportion to the laser energy density of 63.7–382 kJ/cm². Despite the distinct structural and compositional difference, the variation of the perforation depth between the stomach and the fillet was not found at 808-nm wavelength in our measurement. We further studied the laser–tissue interaction by changing the concentration of the methyl blue solution used conventionally as a submucosal fluidic cushion (SFC) in ESD procedures. The temperature of the mucosal layer increased more rapidly at higher concentration of the methyl blue solution, because of enhanced light absorption at the SFC layer. The insertion of the SFC would protect the muscle layer from thermal damage. We confirmed that more effective laser treatment should be enabled by tuning the opto-thermal properties of the SFC. This study can contribute to the optimization of the driving parameters for laser incision techniques as an alternative to conventional surgical interventions.