Cell-morphodynamic phenotype classification with application to cancer metastasis using cell magnetorotation and machine-learning.

We define cell morphodynamics as the cell's time dependent morphology. It could be called the cell's shape shifting ability. To measure it we use a biomarker free, dynamic histology method, which is based on multiplexed Cell Magneto-Rotation and Machine Learning. We note that standard studies looking at cells immobilized on microscope slides cannot reveal their shape shifting, no more than pinned butterfly collections can reveal their flight patterns. Using cell magnetorotation, with the aid of cell embedded magnetic nanoparticles, our method allows each cell to move freely in 3 dimensions, with a rapid following of cell deformations in all 3-dimensions, so as to identify and classify a cell by its dynamic morphology. Using object recognition and machine learning algorithms, we continuously measure the real-time shape dynamics of each cell, where from we successfully resolve the inherent broad heterogeneity of the morphological phenotypes found in a given cancer cell population. In three illustrative experiments we have achieved clustering, differentiation, and identification of cells from (A) two distinct cell lines, (B) cells having gone through the epithelial-to-mesenchymal transition, and (C) cells differing only by their motility. This microfluidic method may enable a fast screening and identification of invasive cells, e.g., metastatic cancer cells, even in the absence of biomarkers, thus providing a rapid diagnostics and assessment protocol for effective personalized cancer therapy

Autofluorescence from NADH Conformations Associated with Different Metabolic Pathways Monitored Using Nanosecond-Gated Spectroscopy and Spectral Phasor Analysis

Cellular NADH conformation is increasingly recognized as an endogenous optical biomarker and metabolic indicator. Recently, we reported a real-time approach for tracking metabolism on the basis of the quantification of UV-excited autofluorescence spectrum shape. Here, we use nanosecond-gated spectral acquisition, combined with spectrum-shape quantification, to monitor the long excited-state lifetime autofluorescence (usually associated with protein-bound NADH conformations) separately from the autofluorescence signal as a whole. We observe that the autofluorescence response induced by two NADH-oxidation inhibitorscyanide and ethanolare similar in Saccharomyces cerevisiae when monitored using time-integrated detection but easily distinguished using time-gated detection. Results are consistent with the observation of multiple NADH conformations as assessed using spectral phasor analysis. Further, because well-known oxidation inhibitors are used, changes in spectrum shape can be associated with NADH conformations involved in the different metabolic pathways, giving bioanalytic utility to the spectral responses

Indocyanine green‐enhanced multimodal photoacoustic microscopy and optical coherence tomography molecular imaging of choroidal neovascularization

Photoacoustic microscopy (PAM) has great potential for visualization of the microvasculature with high spatial resolution and contrast. Early detection and differentiation of newly developed blood vessels named choroidal neovascularization (CNV) from normal vasculature remains a challenge in ophthalmology. Exogenous contrast agents can assist with improving PAM sensitivity, leading to differentiation of CNV. Here, an FDA‐approved indocyanine green (ICG) was utilized as a PAM contrast agent. ICG was conjugated with RGD peptides, allowing the ICG to bind to the integrin expressed in CNV. Molecular PAM imaging showed that ICG‐RGD can target CNV for up to 5 days post intravenous administration in living rabbits with a model of CNV. The PAM image sensitivity and image contrast were significantly enhanced by 15‐fold at 24 h post‐injection. Overall, the presented approach demonstrates the possibility of targeted ICG to be employed in PAM molecular imaging, allowing more precise evaluation of neovascularization.This study investigates the FDA‐approved indocyanine green (ICG) conjugated with RGD as a biocompatible fluorescent and photoacoustic microscopy (PAM) contrast agent for visualization of choroidal neovascularization (CNV). Intravenous ICG‐RGD was able to target CNV and enhance PA signal amplitude. ICG‐RGD multimodal molecular PAM, OCT and fluorescence imaging helped identify the margin and distinguish CNV. These results illustrate that ICG‐RGD demonstrates promise for multimodal imaging with high resolution and contrast.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/167806/1/jbio202000458.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/167806/2/jbio202000458_am.pd

Ion-Selective Nanosensor for Photoacoustic and Fluorescence Imaging of Potassium

Ion-selective optodes (ISOs), the optical analog of ion-selective electrodes, have played an increasingly important role in chemical and biochemical analysis. Here we extend this technique to ion-selective photoacoustic optodes (ISPAOs) that serve at the same time as fluorescence-based ISOs, and apply it specifically to potassium (K⁺). Notably, the potassium ion is one of the most abundant cations in biological systems, involved in numerous physiological and pathological processes. Furthermore, it has been recently reported that the presence of abnormal extracellular potassium concentrations in tumors suppresses the immune responses and thus suppresses immunotherapy. However, unfortunately, sensors capable of providing potassium images in vivo are still a future proposition. Here, we prepared an ion-selective potassium nanosensor (NS) aimed at in vivo photoacoustic (PA) chemical imaging of the extracellular environment, while being also capable of fluorescence based intracellular ion-selective imaging. This potassium nanosensor (K⁺ NS) modulates its optical properties (absorbance and fluorescence) according to the potassium concentration. The K⁺ NS is capable of measuring potassium, in the range of 1 mM to 100 mM, with high sensitivity and selectivity, by ISPAO based measurements. Also, a near infrared dye surface modified K⁺ NS allows fluorescence-based potassium sensing in the range of 20 mM to 1 M. The K⁺ NS serves thus as both PA and fluorescence based nanosensor, with response across the biologically relevant K⁺ concentrations, from the extracellular 5 mM typical values (through PA imaging) to the intracellular 150 mM typical values (through fluorescence imaging)