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Single-molecule localization microscopy (SMLM) has allowed the observation of various molecular structures in cells beyond the diffraction limit using organic dyes. In principle, the SMLM resolution depends on the precision of photoswitching fluorophore localization, which is inversely correlated with the square root of the number of photons released from the individual fluorophores. Thus, increasing the photon number by using highly bright fluorophores, such as quantum dots (QDs), can theoretically fundamentally overcome the current resolution limit of SMLM. However, the use of QDs in SMLM has been challenging because QDs have no photoswitching property, which is essential for SMLM, and they exhibit nonspecificity and multivalency, which complicate their use in fluorescence imaging. Here, we present a method to utilize QDs in SMLM to surpass the resolution limit of the current SMLM utilizing organic dyes. We confer monovalency, specificity, and photoswitchability on QDs by steric exclusion via passivation and ligand exchange with ptDNA, PEG, and casein as well as by DNA point accumulation for imaging in nanoscale topography (DNA-PAINT) via automatic thermally driven hybridization between target-bound docking and dye-bound complementary imager strands. QDs are made monovalent and photoswitchable to enable SMLM and show substantially better photophysical properties than Cy3, with higher fluorescence intensity and an improved resolution factor. QD-PAINT displays improved spatial resolution with a narrower full width at half maximum (FWHM) than DNA-PAINT with Cy3. In summary, QD-PAINT shows great promise as a next-generation SMLM method for overcoming the limited resolution of the current SMLM., Microscopy: Seeing individual molecules inside cells with improved resolution A new microscopy method called QD-PAINT will allow researchers to see inside cells with nanoscale precision. The method combines two existing technologies, DNA-PAINT and steric exclusion, to show a proof-of-concept of a proper utilization of quantum dots (QDs) in single-molecule localization microscopy (SMLM) with improved resolution. In the last decade, SMLM using organic dyes allowed observation of previously invisible biological phenomena, such as individual proteins, but resolution was limited to around 20 nm. QDs emit much brighter fluorescence than organic dyes and could theoretically improve the resolution of SMLM. Sung Ho Ryu and Do-Hyeon Kim at Pohang University of Science and Technology in South Korea adapted QDs for SMLM, conferring them with photoswitchability and specificity, and improved SMLM resolution to about 8 nm. The researchers suggest the method could be fine-tuned for even higher resolution.

Dimerization of beta 2-adrenergic receptor (β

A current-mode hysteretic buck converter is described in which inductor current is sensed by a resistor-capacitor (RC) network. The inductor current sensing RC network is reset multiple times per switching clock period, which allows it to have time constant much smaller than switching clock period and therefore occupy small silicon area. A frequency regulator ensures the switching frequency to be constant regardless of the operating condition of the buck converter and also integrates comparator delay compensator as its loop filter. The current-mode hysteretic buck converter implemented in a 65 nm complementary metal-oxide-semiconductor (CMOS) process provides 0.6–2.0 V output from 3.3 V input with 1 MHz switching frequency. The maximum load current is 1.5 A and the measured peak power efficiency is 96.3%.

The controller of a current-mode buck converter is realized by only time-domain circuits such as voltage-controlled oscillator (VCO), voltage-controlled delay line (VCDL), and phase detector (PD). The inductor current is sensed by a VCO, which helps improving power efficiency and eliminates the need for the slope compensation preventing the sub-harmonic oscillation. The type-II frequency compensation network is realized by a combination of VCO and VCDL without an error amplifier (EA) and RC network which may consume large power and occupy large silicon area. Instead of voltage comparator, a PD detects the error of the output voltage, which allows the switching duty cycle and thus the output voltage to be controlled in a wide range. With the proposed time-domain current-mode controller, a buck converter has been implemented in a 65-nm CMOS process. The output voltage can be regulated from 0.15 to 1.69 V from a 1.8-V input and the maximum load current is 0.6 A. The peak power efficiency is 94.9% when the output is 1.5 V and the load current is 250 mA. The load transient speed is better than 3.5 $\mu \text{s}$ for both the step-up and step-down changes of the load current by 480 mA in 0.1 $\mu \text{s}$ .

Multicolor fluorescence imaging is a powerful tool visualizing the spatiotemporal relationship among biomolecules. Here, we report that commonly employed organic dyes exhibit a blue-conversion phenomenon, which can produce severe multicolor image artifacts leading to false-positive colocalization by invading predefined spectral windows, as demonstrated in the case study using EGFR and Tensin2. These multicolor image artifacts become much critical in localization-based superresolution microscopy as the blue-converted dyes are photoactivatable. We provide a practical guideline for the use of organic dyes for multicolor imaging to prevent artifacts derived by blue-conversion., Blue-conversion, a photooxidative conversion leading to the hypsochromic shift of absorption and emission spectra, occurs in popular organic dyes under conventional laser illumination and produces severe artifacts in multicolor fluorescence imaging.

Various repertoires of membrane protein interactions determine cellular responses to diverse environments around cells dynamically in space and time. Current assays, however, have limitations in unraveling these interactions in the physiological states in a living cell due to the lack of capability to probe the transient nature of these interactions on the crowded membrane. Here, we present a simple and robust assay that enables the investigation of transient protein interactions in living cells by using the single-molecule diffusional mobility shift assay (smDIMSA). Utilizing smDIMSA, we uncovered the interaction profile of EGFR with various membrane proteins and demonstrated the promiscuity of these interactions depending on the cancer cell line. The transient interaction profile obtained by smDIMSA will provide critical information to comprehend the crosstalk among various receptors on the plasma membrane., Cell membrane: assaying protein interactions in cancer cells A new method for analyzing transient interactions between proteins in the outer membrane of living cells could help researchers better understand how cancer cells develop resistance to targeted drug therapies. A research team led by Do-Hyeon Kim and Sung Ho Ryu from Pohang University of Science and Technology, South Korea have developed a technique for investigating weak protein–protein interactions on the basis of changes in their rate of diffusion when bound to each other. Using this “single-molecule diffusional mobility shift assay,” the researchers showed that a cancer-linked protein called EGFR tends to bind with a range of other proteins, depending on the type of cancer cell studied. EGFR is the target of several drug therapies, and the variability of its interactions may account for its ability to circumvent drug-induced inhibition in resistant cancer cells.

A switching battery charger integrated circuit (IC) for a mobile device is described which can regulate the system supply voltage and charge a Li-ion battery simultaneously. The charging current level is adaptively controlled according to the system load current and a battery can be forced to supply current to the system load when the input power is not sufficient. In order to allow a mobile device to supply power to peripheral devices, the battery charger IC can also be configured to operate as a boost dc–dc converter with the reversed direction of power flow. The operation mode of the battery charger IC is smoothly switched by adopting a diode-based mode selection scheme. Implemented in a 130-nm BCDMOS process, the battery charger IC occupies 12.25 mm2 and has been verified to provide the appropriate charging profile for a Li-ion battery and regulate the system supply voltage under various operating conditions. The maximum charging current is 1.5 A and the output voltage ranges from 2.5 to 4.2 V. The maximum power efficiency of dc–dc conversion is 90% and 92%, respectively, for the buck and boost modes.

A myriad of receptor types crowds the plasma membrane of cells. Here, we revealed that clathrin-coated pits (CCPs) presort receptors to modulate receptor activation on the plasma membrane. We visualized individual molecules of receptors, including epidermal growth factor receptor (EGFR), ErbB2, transferrin receptor (TfR), and beta2-adrenergic receptors (β2-AR), inside a single CCP in a living cell using single-molecule diffusivity-based colocalization analysis. The spatially distinct subsets of CCPs selectively allocated for these receptors were observed in a resting state. The EGFR pre-allocated CCP subset was partially shared with that for ErbB2, whereas the EGFR and TfR pre-allocated CCP subsets were mutually exclusive. PICALM was necessary for the pre-allocation of CCPs for EGFR. Furthermore, EGFR dimerization was markedly elevated inside the pre-allocated CCP subset after dynamin recruitment. The pre-sorting function of CCPs provides an efficient mechanism to control competition and cooperation between receptors on the crowded plasma membrane.One Sentence SummaryClathrin-coated pits are small-sized organelles pre-sorting receptor kinds to modulate receptor activation on the crowded plasma membrane.

Interactions between membrane proteins are poorly understood despite their importance in cell signaling and drug development. Here, we present a co-immunoimmobilization assay (Co-II) enabling the direct observation of membrane protein interactions in single living cells that overcomes the limitations of currently prevalent proximity-based indirect methods. Using Co-II, we investigated the transient homodimerizations of epidermal growth factor receptor (EGFR) and beta-2 adrenergic receptor (β2-AR) in living cells, revealing the differential regulation of these receptors’ dimerizations by molecular conformations and microenvironment in a plasma membrane. Co-II should provide a simple, rapid, and robust platform for visualizing both weak and strong protein interactions in the plasma membrane of living cells., Author summary Protein–protein interactions govern cellular processes. The majority of these physical interactions previously identified are strong/permanent interactions, which typically remain unbroken even after purification. The weak/transient interactions between proteins have been implicated in the control of dynamic cellular process that maintain cellular homeostasis and trigger signaling cascades upon environmental changes. However, these interactions are poorly investigated, mainly due to the methodological limitations. Here, we have developed a co-immunoimmobilization assay called Co-II that enables the direct visualization of protein–protein interactions in the membrane of living cells at the single-molecule level. Co-II is based on the intuitive concept that if the protein of interest is immobilized, the interacting protein must be co-immobilized. The use of intrinsic protein diffusivity fundamentally overcomes the limitations of proximity-based methods. Using Co-II, we study the transient homodimerizations of EGFR and β2-AR in living cells, which have been implicated in several types of cancers and heart diseases. We show that the dimerization of these receptors is differently regulated by molecular conformations and the microenvironment in the plasma membrane.

Current-mode DC-DC converters offer various advantages over voltage-mode DC-DC converters such as much simpler frequency compensation, automatic over-current protection, and faster transient response [1,2]. For current-mode control, however, an accurate inductor current sensor is required which can be very sensitive to noise. Another concern in designing a current-mode DC-DC converter is the instability under certain operating conditions known as subharmonic oscillation. A peak-current-mode buck converter, for example, may become unstable when its switching duty cycle is larger than 50% and slope compensation is required to ensure stable operation. While both current-mode and voltage-mode DC-DC converters are conventionally controlled by voltage-domain controllers that use voltage signals as control variables, the works in [3] and [4] have shown that voltage-mode DC-DC converters can also be controlled by time-domain controllers consisting of only time-domain circuits such as voltage-controlled oscillators, voltage-controlled delay lines, and phase detectors (PD). Because time-domain controllers do not use any wide-bandwidth error amplifier, voltage comparator, and passive RC filter required for conventional voltage-domain controllers, they consume much less power and occupy smaller silicon area.

Reaction progress kinetic analysis utilizing single molecule trajectories revealed the comprehensive molecular mechanisms of cetuximab induced EGFR endocytosis., Cellular processes occur through the orchestration of multi-step molecular reactions. Reaction progress kinetic analysis (RPKA) can provide the mechanistic details to elucidate the multi-step molecular reactions. However, current tools have limited ability to simultaneously monitor dynamic variations in multiple complex states at the single molecule level to apply RPKA in living cells. In this research, a single particle tracking-based reaction progress kinetic analysis (sptRPKA) was developed to simultaneously determine the kinetics of multiple states of protein complexes in the membrane of a single living cell. The subpopulation ratios of different states were quantitatively (and statistically) reliably extracted from the diffusion coefficient distribution rapidly acquired by single particle tracking at constant and high density over a long period of time using super-resolution microscopy. Using sptRPKA, a series of molecular mechanisms of epidermal growth factor receptor (EGFR) cellular processing induced by cetuximab were investigated. By comprehensively measuring the rate constants and cooperativity of the molecular reactions involving four EGFR complex states, a previously unknown intermediate state was identified that represents the rate limiting step responsible for the selectivity of cetuximab-induced EGFR endocytosis to cancer cells.