Biology subjects

Chiu, D. T.

Publications and source records attributed to Chiu, D. T..

3 recordsLinked to original sources

bioRxiv2026

High-throughput affinity profiling of antibodies against soluble and native membrane targets by digital flow cytometry

Binding affinity measurements are essential for optimizing and validating therapeutic and research antibodies, yet no high-throughput platform supports quantitative affinity measurements for both soluble and native membrane targets. Here we present a suite of digital flow cytometry (dFC) assays that determine equilibrium binding affinities by directly counting free and bound molecules in homogeneous mixtures without immobilization. The assays enable titration-free absolute KD measurements for fluorescent binding partners, affinity measurements for unlabeled antibodies against membrane receptors on cell-derived extracellular vesicles (EVs), and multiplexed assays for rapid ranking or screening of antibody variants. We demonstrate applications by quantifying the effects of degree-of-labeling on the affinity of antibody-dye conjugates, and of drug conjugation on therapeutic antibody binding to receptors on human cancer EVs. By combining single-molecule quantitation, adaptable assay designs, and high-throughput analysis, dFC provides a versatile platform for therapeutic antibody optimization and research antibody validation.

biophysics↗

bioRxiv2023

Highly Multiplexed Fluorescence Microscopy with Spectrally Tunable Semiconducting Polymer Dots

Current studies of biological tissues require visualizing diverse cell types and molecular interactions, creating a growing need for versatile techniques to simultaneously probe numerous targets. Traditional multiplexed imaging is limited to around five targets at once. Emerging methods utilizing sequential rounds of staining, imaging, and signal removal can probe tens of targets but require specialized hardware, time-consuming workflows, and face some challenges with sample distortion and artifacts. Here we present a new method for highly-multiplexed fluorescence microscopy using semiconducting polymer dots (Pdots) in a single round of staining and imaging. Pdots are small, bright, and photostable fluorescent probes with a wide range of tunable Stokes shifts (20-450 nm). Multiple series of Pdots with varying excitation wavelengths allow for fast (<1 minute) and single-round imaging of up to 21 targets in brain and kidney. This method is based on a simple immunofluorescence workflow, efficient use of spectral space, standard hardware, and straightforward analysis, making it widely applicable for bioimaging laboratories.

biochemistry↗

bioRxiv2017

In vivo multiphoton fluorescence imaging with polymer dots

Deep in vivo imaging of vasculature requires small, bright, and photostable fluorophores suitable for multiphoton microscopy (MPM). Although semiconducting polymer dots (pdots) are an emerging class of highly fluorescent contrast agents with favorable advantages for the next generation of in vivo imaging, their use for deep multiphoton imaging has never before been demonstrated. Here we characterize the multiphoton properties of three pdot variants (CNPPV, PFBT, and PFPV) and demonstrate deep imaging of cortical microvasculature in C57 mice. Specifically, we measure the two-versus three-photon power dependence of these pdots and observe a clear three-photon excitation signature at wavelengths longer than 1300 nm, and a transition from two-photon to three-photon excitation within a 1060 - 1300 nm excitation range. Furthermore, we show that pdots enable in vivo two-photon imaging of cerebrovascular architecture in mice up to 850 m beneath the pial surface using 800 nm excitation. In contrast with traditional multiphoton probes, we also demonstrate that the broad multiphoton absorption spectrum of pdots permits imaging at longer wavelengths ({lambda}ex = 1,060 and 1225 nm). These wavelengths approach an ideal biological imaging wavelength near 1,300 nm and confer compatibility with a high-power ytterbium-fiber laser and a high pulse energy optical parametric amplifier, resulting in substantial improvements in signal-to-background ratio (>3.5-fold) and greater cortical imaging depths of 900 m and 1300 m. Ultimately, pdots are a versatile tool for MPM due to their extraordinary brightness and broad absorption, which will undoubtedly unlock the ability to interrogate deep structures in vivo.

bioengineering↗