A fundamental challenge of biology is to understand the vast heterogeneity of cells, particularly how cellular composition, structure, and morphology are linked to cellular physiology. Unfortunately, conventional technologies are limited in uncovering these relations. We present a machine-intelligence technology based on a radically different architecture that realizes real-time image-based intelligent cell sorting at an unprecedented rate. This technology, which we refer to as intelligent image-activated cell sorting, integrates high-throughput cell microscopy, focusing, and sorting on a hybrid software-hardware data-management infrastructure, enabling real-time automated operation for data acquisition, data processing, decision-making, and actuation. We use it to demonstrate real-time sorting of microalgal and blood cells based on intracellular protein localization and cell-cell interaction from large heterogeneous populations for studying photosynthesis and atherothrombosis, respectively. The technology is highly versatile and expected to enable machine-based scientific discovery in biological, pharmaceutical, and medical sciences.

Translation by the ribosome occurs by a complex mechanism involving the coordinated interaction of multiple nucleic acid and protein ligands. Here we have used zero-mode waveguides (ZMWs) and sophisticated detection instrumentation to allow real-time observation of translation at physiologically-relevant (μM) ligand concentrations. Translation at each codon is monitored by stable binding of tRNAs – labeled with distinct fluorophores – to translating ribosomes, allowing direct detection of the identity of tRNA molecules bound to the ribosome, and therefore, the underlying mRNA sequence. We observe the transit of tRNAs on single translating ribosomes and have determined the number of tRNA molecules simultaneously bound to the ribosome, at each codon of an mRNA. Our results show that ribosomes are only briefly occupied by two tRNAs and that release of deacylated tRNA from the E site is uncoupled from binding of A-site tRNA and occurs rapidly after translocation. The methods outlined here have broad application to the study of mRNA sequences, and the mechanism and regulation of translation.

Myosin V is a double-headed processive molecular motor that moves along an actin filament by taking 36-nm steps. Using optical trapping nanometry with high spatiotemporal resolution, we discovered that there are two possible pathways for the 36-nm steps, one with 12- and 24-nm substeps, in this order, and the other without substeps. Based on the analyses of effects of ATP, ADP and 2,3-butanedione 2-monoxime (a reagent shown here to slow ADP release from actomyosin V) on the dwell time and the occurrence frequency of the main and the intermediate states, we propose that the 12-nm substep occurs after ATP binding to the bound trailing head and the 24-nm substep results from a mechanical step following the isomerization of an actomyosin-ADP state on the bound leading head. When the isomerization precedes the 12-nm substep, the 36-nm step occurs without substeps.

Kinesin is a motor protein that transports organelles along a microtubule toward its plus end by using the energy of ATP hydrolysis. To clarify the nucleotide-dependent binding mode, we measured the unbinding force for one-headed kinesin heterodimers in addition to conventional two-headed kinesin homodimers under several nucleotide states. We found that both a weak and a strong binding state exist in each head of kinesin corresponding to a small and a large unbinding force, respectively; that is, weak for the ADP state and strong for the nucleotide-free and adenosine 5-[␤,␥-imido]triphosphate states. Model analysis showed that (i) the two binding modes in each head could be explained by a difference in the binding energy and (ii) the directional instability of binding, i.e., dependence of unbinding force on loading direction, could be explained by a difference in the characteristic distance for the kinesin-microtubule interaction during plus-and minus-end-directed loading. Both these factors must play an important role in the molecular mechanism of kinesin motility. Kinesin is a processive molecular motor that is essential for the transport of vesicles and organelles along a microtubule in various cells. Kinesin's processive movement has been explained by a mechanism that involves alternating between singleand double-headed bindings to a microtubule (1-5). Adjacent tubulin dimers of 8-nm length form consecutive binding sites (6), such that kinesin takes hundreds of 8-nm steps down a microtubule (7-10). Our recent single-molecule analysis of unbinding force (11) showed that conventional two-headed kinesin is involved in single-headed binding, both in the absence of nucleotides (nucleotide-free state) and in the coexistence of ADP and adenosine 5Ј-[␤,␥-imido]triphosphate (AMP-PNP) (ATP analogue), and double-headed binding in the presence of AMP-PNP (AMP-PNP state), which is consistent with the putative mechanism of kinesin motility.In the present study, we have measured the unbinding force of a single kinesin⅐microtubule complex under an optical microscope equipped with optical tweezers as was reported (11). To clarify the binding mode, we used one-headed kinesin heterodimers (12) in addition to conventional two-headed kinesin homodimers. Conventional two-headed homodimers or oneheaded heterodimers of kinesin molecules were attached to a polystyrene bead such that single kinesin binds to a single bead, and each bead was manipulated with optical tweezers on a microtubule that was adsorbed onto a coverslip (1, 9). An external load was imposed on the attached kinesin molecule by moving the bead toward the plus or the minus end of the microtubule. Here, we found that the two binding states exist in each head of kinesin depending on the nucleotide state. Also, we found that the dependence of the unbinding force on loading direction (where the unbinding force is smaller for the plus-end loading than for the minus-end loading) was independent of nucleotide states.We have analyzed the results for a weak and a strong bindi...