Recent Advances of Optical Tweezers–Based Dynamic Force Spectroscopy and Mechanical Measurement Assays for Live-Cell Mechanobiology

Abstract

Cells sense and respond to mechanical stimuli for activation, proliferation, migration, and differentiation. The associated mechanosensing and biomechanical properties of cells and tissues are significantly implicated in the context of cancer, fibrosis, dementia, and cardiovascular diseases. To gain more mechanobiology insights, dynamic force spectroscopies (DFSs), particularly optical tweezers (OT), have been further advanced to enable in situ force measurement and subcellular manipulation from the outer cell membrane to the organelles inside of a cell. In this review, we first explain the classic OT-DFS rationales and discuss their applications to protein biophysics, extracellular biomechanics, and receptor-mediated cell mechanosensing. As a non-invasive technique, optical tweezers’s unique advantages in probing cytoplasmic protein behaviors and manipulating organelles inside living cells have been increasingly explored in recent years. Hereby, we then introduce and highlight the emerging OT rationales for intracellular force measurement including refractive index matching, active–passive calibration, and change of light momentum. These new approaches enable intracellular OT-DFS and mechanical measurements with respect to intracellular motor stepping, cytosolic micro-rheology, and biomechanics of irregularly shaped nuclei and vesicles. Last but not least, we foresee future OT upgrades with respect to overcoming phototoxicity and system drifting for longer duration live-cell measurements; multimodal integration with advanced imaging and nanotechnology to obtain higher spatiotemporal resolution; and developing simultaneous, automated, and artificial intelligence–inspired multi-trap systems to achieve high throughput. These further developments will grant unprecedented accessibility of OT-DFS and force measurement nanotools to a wider biomedical research community, ultimately opening the floodgates for exciting live-cell mechanobiology and novel therapeutic discoveries.

Fluorescence-coupled micropipette aspiration assay to examine calcium mobilization caused by red blood cell mechanosensing

“…This advance enables measurements of calcium signaling under fine-tuned mechanical stimuli and improves the efficacy. More importantly, micropipette assays have the potential to be combined with emerging dynamic force spectroscopy methods, such as the biomembrane force probe (Chen et al 2015 ) and optical tweezers (Wang et al 2022a ), and be used to measure other types of forces, including tension, compression and shear stress, to study mechanosensing.…”

Robust single membrane protein tweezers

“…This approach of obtaining repetitive (un)folding or (un)binding of single molecules can determine the bond strength, kinetics, free energy landscape, and intermediate states 40,41 . The accuracy of the physicochemical quantities depends on the number of data points that are obtained from the single-molecule stretching.…”

“…These results suggest that our high-throughput method can expedite membrane protein studies using single-molecule tweezers, which have been largely hindered by weak molecular tethering. This approach of obtaining repetitive (un)folding or (un)binding of single molecules can determine the bond strength, kinetics, free energy landscape, and intermediate states 40,41 .…”

Investigation of Soft Matter Nanomechanics by Atomic Force Microscopy and Optical Tweezers: A Comprehensive Review

“…Coated plastic beads with soft matter materials can be subjected to the underlying attractive or repulsive forces at the piconewton range [ 73 ]. OT can quantitatively evaluate the assembly of protein droplets and their characteristics that lead to the pathological solidification [ 74 ], biomolecular folding and unfolding events that undergo conformational changes to reach their biological functions [ 75 ], decode the molecular mechanisms related to DNA and RNA organization, translation, repair and replication processes [ 76 ], hydrodynamic forces involved in endocytosis processes of eukaryotic cells [ 77 ], cell migration probed in engineered environments [ 78 ] and mechanical response of soft matter systems [ 79 ]. Recently, the integration of OT, label-free microscopy, fluorescence spectroscopy and advanced microfluidic systems has allowed the visualization of dynamic interactions in real time [ 80 ].…”