Biology subjects

Wazawa, T.

Publications and source records attributed to Wazawa, T..

4 recordsLinked to original sources

bioRxiv2026

Fully Two-Photon-Driven RESOLFT Microscopy for Super-Resolution Imaging inside Tissue

Two-photon excitation (2PE) microscopy reaches deep into tissue but remains diffraction-limited, and existing 2PE-STED and 2PE-RESOLFT schemes still rely on single-photon depletion or photoswitching. Here we present 2P-RESOLFT microscopy, in which excitation and reversible photoswitching are both two-photon-driven. Using the positive photoswitchable protein Padron2, we achieved 205-nm lateral resolution and resolved previously unreported membrane protrusions of {beta} cells inside pancreatic pseudoislets.

biophysics↗

bioRxiv2026

Fusion of a non-specific DNA-binding domain enhances Cas12a trans-cleavage for robust nucleic-acid diagnostics

CRISPR-Cas12a underlies powerful genome-editing and nucleic-acid detection technologies, yet its performance is limited by inefficient target engagement and low catalytic turnover, particularly at low target abundance and elevated temperatures. Here, we report a modular protein-engineering strategy to enhance Cas12a trans-activity by fusing the hyperthermophilic DNA-binding protein Sso7d to the N terminus of Lachnospiraceae bacterium ND2006 Cas12a (LbaCas12a). The resulting fusion enzyme shows a twofold improvement in detection sensitivity, a 4.6-fold increase in kcat, and substantially accelerated trans-cleavage kinetics compared with wild-type Cas12a. These enhancements are observed across multiple guide RNAs, diverse DNA substrates, and a broad temperature range from 37 to 60 {degrees}C. The engineered Cas12a enables robust detection of the intrinsic blaOXA-51 and acquired blaOXA-24 antibiotic resistance genes from multiple Acinetobacter baumannii strains with independently validated resistance profiles, whereas wild-type Cas12a produces little or no detectable signal. Signal output is increased by up to [~]30-fold, expanding the effective detection window and shortening time-to-positive. Together, these results establish fusion of an accessory DNA-binding domain as a proof-of-concept for Sso7d fusion to LbCas12a and that generalization to other CRISPR effectors or non-specific DNA-binding domains represents an exciting avenue for future investigation.

biochemistry↗

bioRxiv2024

Creating coveted bioluminescence colors for simultaneous myriad-color bioimaging

Bioluminescence, an optical marker that does not require excitation by light, allows researchers to simultaneously observe multiple targets, each exhibiting a different color. Notably, the colors of the bioluminescent proteins must sufficiently vary to enable simultaneous detection. Here, we aimed to introduce a method that can be used to expand the color variation by tuning dual-acceptor bioluminescence resonance energy transfer (BRET). Using this approach, we could visualize multiple targets with up to 20 colors through single-shot acquisition using a color CMOS camera. Overall, this method enables simple and simultaneous observation of numerous biological targets and phenomena.

biophysics↗

bioRxiv2017

In vivo brain activity imaging of interactively locomoting mice

Electrophysiological field potential dynamics have been widely used to investigate brain functions and related psychiatric disorders. Conversely, however, various technical limitations of conventional recording methods have limited its applicability to freely moving subjects, especially when they are in a group and socially interacting with each other. Here, we propose a new method to overcome these technical limitations by introducing a bioluminescent voltage indicator called LOTUS-V. Using our simple and fiber-free recording method, named \"SNIPA,\" we succeeded in capturing brain activity in freely-locomotive mice, without the need for complicated instruments. This novel method further allowed us to simultaneously record from multiple independently-locomotive animals that were interacting with one another. Further, we successfully demonstrated that the primary visual cortex was activated during the interaction. This methodology will further facilitate a wide range of studies in neurobiology and psychiatry.

neuroscience↗