Session: 17-01-01: Research Posters

Paper Number: 149376

149376 - Uncovering Electro-Mechano-Physiological Rules of Life: A New 2d/3d All-Optical Interrogation Technology 

Increasing evidence suggests that many biological systems, including but not limited to brain and heart, can modulate their electrical membrane potential and apply mechanical forces simultaneously to regulate key functions, such as proliferation, communication, computation, and migration. However, the 4D spatial-temporal dynamics of the electro-mechano-physiological signals in most multi-cellular systems remains unknown, mainly because of the limitation of current technologies to elucidate the coupled signals. To fill this gap and reveal the general rules of life, we created a wide-area 2D/3D optical interrogation system to visualize and control the electro-mechano-physiological signals in > 50 cells simultaneously within 5 min across conditions of in vitro, ex vivo, and in vivo. Our new technologies enable mechanistic study of both electrical and mechanobiological nature of biological systems in a high-throughput, non-invasive, high-speed (> 1 kHz), high signal-to-noise ratio (SNR), cell-type-specific, and long-term (from milli-seconds to months) manner.

Neurological diseases remain a significant unmet societal need today, affecting millions of patients worldwide. However, due to the low throughput of traditional drug screening approaches (e.g., up to 200 cells per day for automated patch-clamp) and the complexity of the human nervous system (e.g., 100s of neuronal cell types and 100 billion nerve cells), neurological drugs have a much longer development time than other-organ-targeting drugs. Hence, we are in dire need of innovative approaches that can improve the throughput to discover new drugs and advance integrative understanding of neurological diseases.

Electrical activities are the “finger-printer” and “regulator” of cell states, especially in neurons. In parallel, mechanical stimuli including substrates’ mechanical stiffness, compression, and tension are demonstrated to regulate the electrical properties of neurons. However, existing publications show opposite influence of mechanics on the electrical behavior of neurons, partially due to the inconsistent methods of the measurement and control for the coupled signals. Moreover, the causal links, i.e., molecular mechanisms, among electrical, mechanical, and physiological signals in neurons are not clear, triggering the development of multidimensional systems to acquire the omics data.

In this study, we combined our customized all-optical electrophysiology interrogation (AOEI) system with mechanical-stiffness-variable hydrogel substrates to investigate the electro-mechano-physiological signaling in multiple biological systems. The AOEI system includes (1) a 2D total internal reflection fluorescence (TIRF) microscopy for in vitro primary culture and ex vivo tissue slices, (2) a 3D selective plane illumination microscopy (SPIM) for in vivo zebrafish and ex vivo tissue blocks, (3) a custormized LabVIEW interacting system to synchronize and controll hardware, (4) a molecular biology pipeline to non-invasively express genetically-encoded protein-based sensors and actuators. 

We successcully co-expressed genetically-encoded voltage indicator (GEVI) QuasArX and actuator CheRiff on the membranen of the same neuron to allow simultaneous optogenetic stimulation and readout of membrane potential, which enables the investigation of intrinsic neuronal excitability. We successcully expressed indicator QuasArX in post-synaptic neurons to allow voltage readout and actuator CheRiff in pre-synaptic neurons to allow voltage stimulation, which enables the investigation of synaptic transmission. We further expanded the FOV of the AOEI system to 4.6×0.7 mm² to enable simultaneous optical interrogation of > 50 cells, which increases the throughput by > 500-fold compared to conventional patch-clamp electrophysiology. Combining our AOEI system with hydrogel substrates, we found that primary trigeminal ganglion (TG) neurons cultured on soft polyachrylamide (PAA) gels started to fire multiple action potentials (APs) at lower intensities of the 500-msec optogenetic stimuli than those cultured on stiff glass. 

Presenting Author: Miao Huang University of Florida

Presenting Author Biography: Miao Huang was born and raised in Nanjing, China. He attended the Southeast University from 2011 to 2015 and obtained a degree of Bachelor of Science in Thermal Engineering. He obtained Master of Science in Mechanical Engineering from University of Florida at 2017. He then worked as mechanical engineer at Sunhydraulic and Cummins from 2017 to 2020. He then started his doctoral studies in the Department of Mechanical and Aerospace Engineering under the advisement of Prof. Xin Tang at the University of Florida (UF) and became a PhD candidate in 2024. He plans to graduate in May 2025. His Ph.D. projects focus on the creation and application of new technologies to elucidate previously inaccessible mechanical-biochemical-electrical nexuses, specifically in two biological systems: cancer and nervous systems. The work performed during Miao’s doctoral studies has resulted in eight peer-reviewed journal publications, plus three other manuscripts in review and in preparation, multiple honors and awards, and presentations at several conferences.

Authors:

Chenyu Liang University of Florida
Miao Huang University of Florida
Erica Hengartner University of Florida
Abygale Cochrane University of Florida
Laura Garzon University of Florida
Allison Campbell University of Florida
He Tian BioNTech SE
Urs Böhm Institute of Psychiatry and Neuroscience of Paris
Christopher Werley Vertex Pharmaceuticals
Habibeh Khoshbouei UNIVESITY OF FLORIDA
Min Lin UNIVESITY OF FLORIDA
Christopher Mccurdy University of Florida
Yuqing Li University of Florida
Lance Mcmahon Texas Tech University Health Sciences Center
Bruna Balbino De Paula University of Florida
Basak Ayaz University of Florida
Robert Caudle University of Florida
Christine Schmidt University of Florida
Dietmar Siemann University of Florida
Xin Tang University of Florida