Click3D: Click reaction across deep tissues for whole-organ 3D fluorescence imaging.

Click chemistry offers various applications through efficient bioorthogonal reactions. In bioimaging, pretargeting strategies have often been used, using click reactions between molecular probes with a click handle and reporter molecules that make them observable. Recent efforts have integrated tissue-clearing techniques with fluorescent labeling through click chemistry, allowing high-resolution three-dimensional fluorescence imaging. Nevertheless, these techniques have faced a challenge in limited staining depth, confining their use to imaging tissue sections or partial organs. In this study, we introduce Click3D, a method for thoroughly staining whole organs using click chemistry. We identified click reaction conditions that improve staining depth with our custom-developed assay. The Click3D protocol exhibits a greater staining depth compared to conventional methods. Using Click3D, we have successfully achieved whole-kidney imaging of nascent RNA and whole-tumor imaging of hypoxia. We have also accomplished whole-brain imaging of hypoxia by using the clickable hypoxia probe, which has a small size and, therefore, has high permeability to cross the blood-brain barrier.

Transient sensorimotor projections in the developmental song learning period.

Memory recall and guidance are essential for motor skill acquisition. Like humans learning to speak, male zebra finches learn to sing by first memorizing and then matching their vocalization to the tutor's song (TS) during specific developmental periods. Yet, the neuroanatomical substrate supporting auditory-memory-guided sensorimotor learning has remained elusive. Here, using a whole-brain connectome analysis with activity-dependent viral expression, we identified a transient projection into the motor region, HVC, from neuronal ensembles responding to TS in the auditory forebrain, the caudomedial nidopallium (NCM), in juveniles. Virally induced cell death of the juvenile, but not adult, TS-responsive NCM neurons impaired song learning. Moreover, isolation, which delays closure of the sensory, but not the motor, learning period, did not affect the decrease of projections into the HVC from the NCM TS-responsive neurons after the song learning period. Taken together, our results suggest that dynamic axonal pruning may regulate timely auditory-memory-guided vocal learning during development.

Heterogenous expression of endoglin marks advanced renal cancer with distinct tumor microenvironment fitness.

Intratumoral heterogeneity, including in clear cell renal cell carcinoma, is a potential cause of drug resistance and metastatic cancer progression. We specified the heterogeneous population marked by endoglin (also known as CD105) in a preclinical model of clear cell renal cell carcinoma progression. Highly malignant derivatives of human clear cell renal cell carcinoma OS-RC-2 cells were established as OS5Ks by serial orthotopic inoculation in our previous study. Expression of both ENG (encoding endoglin) mRNA and protein were heterogeneously upregulated in OS5Ks, and the endoglin-positive (ENG+ ) population exhibited growth dependency on endoglin in anchorage-independent cultures. Despite the function of endoglin as a type III receptor, transforming growth factor ß and bone morphogenetic protein-9 signaling were unlikely to contribute to the proliferative phenotype. Although endoglin has been proposed as a marker for renal cancer-initiating cells, the OS5K-3 ENG+ population did not enrich other reported cancer-initiating cell markers or differentiate into the ENG- population. Mouse tumor inoculation models revealed that the tumor-forming capabilities of OS5K-3 ENG+ and ENG- cells in vivo were highly dependent on the microenvironment, with the renal microenvironment most preferable to ENG+ cells. In conclusion, the renal microenvironment, rather than the hypothesized ENG+ cell-centered hierarchy, maintains cellular heterogeneity in clear cell renal cell carcinoma. Therefore, the effect of the microenvironment should be considered when evaluating the proliferative capability of renal cancer cells in the experimental settings.

Simple method to measure and analyze the fluctuations of a small particle in biopolymer solutions.

We developed a simple method to investigate the motion of a small particle in biopolymer solutions. Using optical tweezers with low stiffness, a trapped probe particle fluctuates widely for a long time along the light axis, which reflects the rheological properties of the surrounding environment. We present a convenient technique for three-dimensional position tracking and the analysis focused on the distribution of particle positions and its variance in a given time interval. It allows us to obtain useful information about the dynamics of a small particle in a wide range from a free diffusive motion to a constrained motion with statistical significance. We applied this method to investigate the dynamics in collagen and DNA solutions; it was found that a collagen solution behaves as a simple viscous liquid and a DNA solution has apparent elasticity due to the slow relaxation of the configuration of molecules.

Experimental realization of directed percolation criticality in turbulent liquid crystals.

This is a comprehensive report on the phase transition between two turbulent states of electroconvection in nematic liquid crystals, which was recently found by the authors to be in the directed percolation (DP) universality class [K. A. Takeuchi, Phys. Rev. Lett. 99, 234503 (2007)]. We further investigate both static and dynamic critical behaviors of this phase transition, measuring a total of 12 critical exponents, 5 scaling functions, and 8 scaling relations, all in full agreement with those characterizing the DP class in 2+1 dimensions. Developing an experimental technique to create a seed of topological-defect turbulence by pulse laser, we confirm in particular the rapidity symmetry, which is a basic but nontrivial consequence of the field-theoretic approach to DP. This provides a clear experimental realization of this outstanding truly out-of-equilibrium universality class, dominating most phase transitions into an absorbing state.

Directed percolation criticality in turbulent liquid crystals.

We experimentally investigate the critical behavior of a phase transition between two topologically different turbulent states of electrohydrodynamic convection in nematic liquid crystals. The statistical properties of the observed spatiotemporal intermittency regimes are carefully determined, yielding a complete set of static critical exponents in full agreement with those defining the directed percolation class in 2+1 dimensions. This constitutes the first clear and comprehensive experimental evidence of an absorbing phase transition in this prominent nonequilibrium universality class.