Name: Direct Force Measurements of Subcellular Mechanics in Confinement using Optical Tweezers (Video) | JoVE Journal
Uploaded: 2021-08-31T13:00:00.000+00:00
Duration: 9 min 56 s
Description: Learn to measure subcellular mechanics using optical tweezers on isolated embryonic zebrafish cells. This protocol enables direct force measurement of nuclear properties without disrupting the cell membrane or cytoskeleton.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

MK acknowledges financial support from the Spanish Ministry of Economy and Competitiveness through the Plan Nacional (PGC2018-097882-A-I00), FEDER (EQC2018-005048-P), Severo Ochoa program for Centres of Excellence in R&#38;D (CEX2019-000910-S; RYC-2016-21062), from Fundaci&#243; Privada Cellex, Fundaci&#243; Mir-Puig, and from Generalitat de Catalunya through the CERCA and Research program (2017 SGR 1012), in addition to funding through ERC (MechanoSystems) and HFSP (CDA00023/2018). V.R. acknowledges support from the Spanish Ministry of Science and Innovation to the EMBL partnership, the Centro de Excelencia Severo Ochoa, MINECO&#39;s Plan Nacional (BFU2017-86296-P, PID2020-117011GB-I00) and Generalitat de Catalunya (CERCA). V.V. acknowledges support from the ICFOstepstone PhD Programme funded by the European Union&#39;s Horizon 2020 research and innovation program under Marie Sk&#322;odowska-Curie grant agreement 665884. We thank Arnau Farr&#233; for critical reading of the manuscript; Maria Marsal for the help with the imaging and mounting of the 24 hpf embryo and; Senda Jim&#233;nez-Delgado for support with zebrafish micronjections.

The authors have nothing to disclose.

In this protocol, we describe a unique method to interrogate the mechanical properties of the cell nucleus inside the living cells. Different to other force spectroscopy techniques, non-invasive optical trapping allowed us to decouple the contribution of the cell membrane and cytoskeleton from the cell nuclear stiffness. Importantly, optical micromanipulation is compatible with multimodal microscopy, which will allow the experimenter to study different processes involved in cell nuclear mechanobiology. As a representative result, we used DNA-Hoechst staining to measure the nucleus deformation upon indentation performed by forces of the order of several hundreds of picoNewton.
Potential applications of our method beyond the examples outlined in this protocol 
The possibility to extract quantitative mechanical information from measurements inside living cells without external perturbations enable a plethora of unprecedented opportunities that are just beginning to be explored. Thus, the presented protocol of our optical micromanipulation platform can be extended to more complex experiments with great versatility. Acousto-optic deflectors (AOD) can generate multiple optical traps for synchronous force measurements across different cell locations, as well as can be used for active microrheology in a wide frequency range51,61. As has been mentioned, the force response upon indentation can overcome the maximum trapping force, leading to an escape of the bead from the optical trap. In this case, a force feedback can be configured with the AOD in order to clamp the optical force. All in all, multiple microrheological approaches, such as the stress relaxation described in this protocol, but also active microrheology or creep compliance, can be experimentally obtained with this platform and thoroughly analyzed by novel software packages61,62,63,64,65. Furthermore, the application of forces is not limited to the nucleus but could in principle be carried out to measure diverse intracellular structures and in complex tissues as demonstrated for trapping flowing red blood cells inside intact blood vessels66,67 or trapping and deforming chloroplasts and mitochondria68. Light-momentum calibration is independent of the shape and size of the trapped object, hence enabling direct force measurements on any force probe with arbitrary shape38,39. The use of injected microspheres allowed us to apply high forces onto the nucleus with relatively low laser power as compared to direct manipulation of cellular structures69,70,71. However, given a high enough refractive index difference, no externally applied force probe is necessary and intracellular organelles can be manipulated directly without injected beads (unpublished observations and reference70).
Potential modifications of our method to extend the applications 
Different sizes of microbeads can be injected depending on the experiment, but the relative controls must be done. For example, to study cells at later stages smaller beads can be injected. This will reduce the maximum force that can be exerted by the optical trap (such as shown in reference55). Bigger beads can be injected to exert higher forces, but these might affect embryo development depending on their size or stage of interest. In experiments where microbead injection is not an option, various organelles displaying refractive indices differences compared to that of the cytoplasm can still be optically manipulated, giving rise to optical forces measurable from light momentum changes42. As mentioned above, these methods have been employed by Bambardekar et al. to deform cell-cell junctions in the Drosophila embryo70. Likewise, the cell&#39;s nucleus has a lower refractive index than the surrounding medium44, which allows for bead-free indentation (unpublished observations and reference72) even though with a lower trapping strength. Thus, the nucleus cannot be trapped easily and escapes the trap.
The spin-coated PDMS spacer is fabricated via a convenient and fast method but might be out of reach for labs without access to a micro-/nanofabrication facility or engineering labs. Thus, the spacer can be easily assembled from lab tape or parafilm (step 4). The protocol can also be adapted by manufacturing microfluidic channels that automate the delivery of single cells into predefined measurement wells or into a chamber with a defined height to estimate the confinement effect within the same specimen. However, such microfluidic devices must be designed so that they fit the space between the microscope objective and the collecting lens of the optical force sensor, of around 2 mm (see step 3). Note that the optical force sensor must be positioned at the appropriate height so that no optical aberrations from defocusing affect the photon momentum measurement.
Other modifications could include the change of biological reporters. We found that Hoechst fluorescence spectrally bleeds into the GFP channel and we thus favor the combination with mCherry-tagged histone&#160;as a nuclear marker for simultaneous measurement in two fluorescent channels. Alternatively, nuclear deformation can easily be tracked with a label targeted to the inner nuclear membrane such as Lap2b-GFP (Figure 2).
Indentation onto the cell nucleus was of the order of 2-3 microns, which we could accurately measure by image analysis of diffraction-limited spinning-disk confocal microscopy. For the case of stiffer nuclei or smaller forces, indentation will be barely measurable using this approach. However, the&#160;absolute force-calibrated optical tweezers can be also calibrated for position measurements of the trapped bead in situ using BFP interferometry with nanometer accuracy51. Using this approach, the voltage signal and the optical force sensor can be translated into the position of the trapped probe through parameter&#160;&#946; [nm/V], while the invariant parameter&#160;&#945; [pN/V] yields force values through the aforementioned light-momentum calibration41 (see below for details).
Troubleshooting 
We found that the following challenges could occur during the experiment:
No stable trap is formed and the microsphere escapes easily 
Any dirt on the microscope objective or a misaligned correction collar could lead to a failure of a stable trap. If an immediate solution is not found, measure the point-spread function of the objective lens. If the specimen of interest is deep inside an optically dense tissue, the laser focus might experience severe optical aberrations leading to unstable trapping (this effect is usually negligible in isolated cells but becomes more evident in thicker tissues). For high stiffness, the restoring force of the nucleus could exceed the escape force of the trap, such that the microsphere is lost and the indentation routine fails. Initially, the nuclear membrane edge proximal to the optical trap gets hardly indented (Figure S2A). When this occurs, the trapping laser is no longer affected by force and Brownian motion, which leads to a force drop to zero and a decrease of the signal noise (Figure S2B). In case this happens, laser power can be increased to have a stronger trap, the amplitude of the trapezoidal trajectory pushing the bead into the nucleus can be reduced, or the initial position of the trapped microbead can be set further off the nucleus.
The cell is moving during the stimulation 
If cells are not sufficiently attached, the optical gradient trap will move the cells while performing the intracellular indentation routine, such that the forces and underlying mechanics of the nucleus are artefactual. To prevent displacement of the entire cell, we recommend increasing the concentration of cell adhesion molecules on the surface, e.g., ConA.
Initial momentum compensation 
If an initial momentum compensation routine is not available in the OTs platform (step 6.5), an artificial, force-independent baseline signal needs to be corrected for. This is visible as a slope on the force curve even with no bead trapped (Figure S1E). To do the correction, the same trajectory needs to be performed without a bead, outside of the cell at exactly the same position. For this, move the cell away from the trap using the stage control. As a reference, the force offset changes 5 pN across the FOV at 200 mW in our system; thus, it becomes negligible for short trajectories. Alternatively, a piezo scan stage can be used to move the cells on the sample, leaving the laser position constant.
Critical steps of the presented protocol 
Microspheres should be injected at the right, 1-cell stage to ensure maximal distribution over the embryo. Beads should not be fluorescent so that no light leaks into the fluorescent channels used for imaging. For example, even typical red-fluorescent beads are clearly visible in the blue channel used for imaging the cell nucleus after Hoechst staining due to their brightness (excitation: 405 nm; emission: 445 nm). Stable attachment of the cell to the substrate is critical to prevent lateral displacement during the indentation routine. If the cell moves during the routine, forces are underestimated. Should this happen frequently, optimize the attachment protocol. For tissue culture cells, other cell adhesion proteins, such as fibronectin, collagen, or poly-L-lysine lead to satisfactory attachment (unpublished observations). During the confinement, cells are subjected to a sudden and severe mechanical stress. This can cause damage to the cells and frequently the experimenter will encounter bursted cells if the procedure is not carried out carefully. Also, if the confinement height is too small, all the cells will suffer from nuclear envelop breakage or irreversible damage. To mitigate these, lower the upper coverslip more slowly and/or increase the spacing between the coverslip.
Limitations of the technique and suggestions to overcome them 
A clear limitation of the technique is the penetration of the laser light into deep sections of the tissue, which leads to aberrations and unstable trapping. Thus, a lower limit of penetration depth depends on the clarity of the sample, the aberration correction that can be employed73 and the applied laser power. It should be taken into account that a higher laser power leads to thermal excitation of the sample in the vicinity of the microsphere. However, heating of the sample originated by the 1064 nm wavelength laser spot is minimized to avoid plausible heat-related stress onto our biological samples74.
Another limitation is the maximum force that can be measured. Even though direct light-momentum detection enables force measurements far beyond the linear response regime of the optical trap40,41, the maximum applied force is in the order of a few hundred picoNewtons. This is limited by laser power and the consequential damage threshold of soft biological material and the refractive index differences, which are normally not larger than 0.1 or 0.344. Several methods have been proposed to increase the force detection limit, e.g., using structured light75, anti-reflective coated microspheres76, high-refractive index particles77 or highly doped quantum dots78.
OTs can be used for nanometer-scale position measurements through BFP interferometry, such that the position of the bead within the trap is&#160;&#916;x =&#160;&#160;&#946;Sx, where Sx is the voltage signal of the sensor, and&#160;&#946; [&#181;m/V] can be calibrated on-the-fly following different protocols35,54. For an optical force sensor, it can be proved that the voltage-to-force invariant conversion factor&#160;&#945; [pN/V] directly relates to&#160;&#946; and the trap stiffness, k [pN/&#181;m], through&#160;&#945; = k&#946; 37) In experiments with bead displacements that are too small to be detected from optical imaging, this strategy can be used to complement force measurements with small position detection. An example is the application of the experimental routines presented here onto very stiff nuclei, for which forces at reasonable laser powers (200-500 mW) are not sufficient to induce indentation values large enough. In that case, the bead needs to be brought in contact with the nucleus and the trapping stiffness must be calibrated prior to the measurement (step 8.6). The indentation d of the nucleus as a function of force can be indirectly determined as:
d = xtrap - F/k
where xtrap is the trap position. Different to the invariant light-momentum factor&#160;&#945; [pN/V], factor &#946; [&#181;m/V] needs to be calibrated prior to each experiment since it depends on many local variables determining the trapping dynamics, such as the particle size, optical trap spot size, and relative refractive indices.

Tissue morphogenesis is a complex process in which biochemical signals and physical forces are spatiotemporally coordinated. In the developing embryo, gradients of biochemical signaling factors dictate fate specification and ensure correct tissue patterning1,2. At the same time, intrinsic and extrinsic forces play a role in building the architecture of the embryo3,4. The influence of cell cortex mechanics in this context has been studied extensively5,6. The tight interconnection between mechano-chemical processes during morphogenesis relies on the properties of single cells to sense and respond to mechanical forces in their tissue microenvironment. Cells, thereby decode mechanical signals via the presence of force-sensitive subcellular and molecular elements that transduce mechanical information into specific signaling pathways controlling cell behavior, cell fate, and cell mechanics.
A hallmark of developmental processes is that cells organize as groups to build multicellular structures. As such, single cells rarely rearrange and move alone but are associated in a tight sociotope in which they show collective behavior such as supracellular migration7, (un)jamming transitions8,9 or blastocyst compaction10. Mechanical forces generated within and between cells serve as important cues to instruct collective cell dynamics7,11. But even when cells move alone, such as progenitor cells that squeeze their way between tissue sheets or narrow tissue niches, they experience extensive anisotropic mechanical forces when navigating a three-dimensional environment. These mechanical stresses on cells have profound consequences on cellular behavior12,13. Several mechanisms have been investigated that converge on the nucleus as a major mechanotransduction element14,15, as passive or active mechanical element during migration within a dense 3D tissue environment15,16.
We recently proposed a mechanism that equips cells to measure shape deformations using the nucleus as an elastic intracellular mechano-gauge12. The nucleus, being the largest organelle in a cell, undergoes large deformations when cells polarize, migrate, or change their shape under mechanical stretch, confinement, or osmotic stress16,17,18,19. We found that nuclear envelope stretch along with the intracellular positioning of the nucleus provides cells with information on the magnitude and type of cell deformation (such as cell compression versus cell swelling). Stretching of the nucleus is associated with an unfolding of the inner nuclear membrane (INM), which promotes calcium-dependent cPLA2 (cytosolic phospholipase A2) lipase activity at the INM followed by the release of Arachidonic Acid (AA) and rapid activation of myosin II at the cell cortex. This leads to increased cell contractility and amoeboid cell migration above a threshold of cortical contractility6. The mechanosensitive response to cell deformation occurs in less than a minute and is reversible upon confinement release, suggesting that the nucleus acts as a strain gauge for cellular proprioception regulating adaptive cell behavior under mechanical stress conditions. This mechanosensitive pathway is shown to be active in progenitor stem cells derived from zebrafish embryos, both in pluripotent and lineage-committed cells12 and is conserved in different species and cell lines20.
In addition to the nuclear properties as a cell-mechanosensor, nuclear architecture and mechanics are intrinsically regulated during development and in response to cell fate specification21, hence tuning cellular mechano-sensitivity22,23. The consequence might be a change in nuclear compliance that allows for morphological changes and transitions from a premigratory to a migratory state and vice versa8.
Several techniques to measure cell nucleus mechanics have been applied, such as atomic force microscopy24,25, micropipette aspiration26,27, microfluidic technology28, and microneedles29. However, many of these techniques are invasive in the sense that the entire cell must be deformed, limiting the measurement of mechanical characteristics and force-dependent responses of the nucleus itself. To circumvent the simultaneous deformation of the cell surface and its mechanosensitive cell cortex30, isolated nuclei were studied in various contexts31,32. However, it cannot be ruled out that nuclear isolation is associated with a change in mechanical nucleus properties and their regulation (reference24 and own unpublished observations).
Optical tweezers (OTs) are a versatile technology that has allowed a plethora of experiments in cell mechanobiology and have been instrumental in our understanding of how molecular machines convert chemical&#160;into mechanical energy33,34. Optical tweezers use a tightly focused laser beam to exert optical forces onto dielectric particles that have a refractive index higher than the surrounding medium33. Such forces can be of the order of hundreds of pico-Newtons and result in effective confinement of the particle within the laser trap focus, enabling manipulation of the trapped particle in three dimensions. The use of light has an important advantage in that the measurement can be performed non-invasively inside living cells. Optical manipulations are further limited to the trap focus of the laser beam. Hence, the manipulation can be performed without stimulating the surrounding cellular membranes and does not perturb the actin cortex or mechanosensitive processes at the plasma membrane, such as the force-dependent activation of ion channels.
The difficulty of the optical tweezer approach is to precisely determine the forces applied to the microsphere using classical approaches that rely on indirect force calibration based on the equipartition theorem or the use of defined Stokes-drag forces to measure a laser-power dependent escape force35. Whereas these methods are straightforward to implement in an in vitro experiment, they usually cannot be translated into a cellular environment. Several strategies have been introduced into the field that rely on a direct force calibration, derived from the first principles of momentum conservation36,37. Unlike other force spectroscopy approaches, force measurements are deduced from a local interchange of light momentum with the arbitrarily-shaped trapped particle38,39. In our experimental set-up, changes in light momentum arising from optical forces are directly measured without the need for in situ trap calibration40,41,42,43. Thus, the measurements become possible in a viscous environment such as the interior of the cell or even within a tissue, and forces can be readily quantified down to the pN level.
In this protocol, we describe an assay to mechanically manipulate intracellular organelles or structures and quantitatively assess their mechanical properties by an optical tweezer set-up. This set-up is integrated into a spinning disk fluorescent microscope enabling parallel imaging of cellular behavior or intracellular dynamics. The assay allows for the characterization of the mechanical properties of specific cellular compartments, such as the nucleus, while simultaneously studying the possible mechanoresponse and activation of molecular signaling pathways as a result of the deformation itself. Furthermore, optical trapping of injected microbeads within cells allows for an increase in the indentation force thanks to a considerably higher refractive index of the polystyrene bead (n = 1.59) compared to the intrinsic refractive contrast44 of the nucleus (n ~ 1.35) versus cytoplasm (n ~ 1.38). The presented strategy can be easily adapted to the study of other intracellular structures and organelles, as well as other approaches involving active microrheology, the use of multiple optical traps to probe the same/different sub-cellular structures simultaneously, and measurements targeting cell mechanobiology in the live embryo.

<table><tbody><tr><td>#1.5 22 mm cover glasses</td><td>Ted Pella</td><td>260148</td><td></td></tr><tr><td>#1.5 22x60 mm Coverglasses</td><td>Ted Pella</td><td>260152</td><td></td></tr><tr><td>#1.5H glass bottom dishes</td><td>Willco</td><td>GWST-5040</td><td></td></tr><tr><td>10-um beads</td><td>Supelco</td><td>72986</td><td></td></tr><tr><td>1-mm glass capillaries</td><td>Harvard Apparatus</td><td>30-0020 GC100F-15</td><td></td></tr><tr><td>1-um polystyrene microbeads</td><td>Sigma</td><td>89904</td><td></td></tr><tr><td>1-um red-fluorescent beads</td><td>ThermoFisher</td><td>F8816</td><td></td></tr><tr><td>Agar</td><td>ThermoFisher</td><td>16500500</td><td></td></tr><tr><td>Aqcuisition cameras sCMOS</td><td>Andor</td><td>Sona-4BV11-UNI</td><td></td></tr><tr><td>Auxiliary camera (Figure 3, AUX)</td><td>Blackfly, FLIR</td><td>BFS-U3-200S6M-C</td><td></td></tr><tr><td>Calbryte 520</td><td>AAT Bioquest</td><td>520 AM</td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>5453000011</td><td></td></tr><tr><td>Concanavalin A</td><td>Sigma</td><td>C5275</td><td></td></tr><tr><td>DMEM</td><td>Sigma</td><td>D2906</td><td></td></tr><tr><td>DNA-Hoechst</td><td>ThermoFisher</td><td>33342</td><td></td></tr><tr><td>Double scotch tape</td><td>Biesse Adesivi</td><td></td><td></td></tr><tr><td>E3</td><td>5 mM NaCl. 0.17 mM KCl. 0.33 mM CaCl2. 0.33 mM MgSO4</td><td></td><td></td></tr><tr><td>Eclipse Ti2</td><td>Nikon</td><td></td><td></td></tr><tr><td>Forceps</td><td>Fine Science Tools</td><td>11252-20</td><td></td></tr><tr><td>GPI-GFP</td><td></td><td></td><td></td></tr><tr><td>H2A-mCh</td><td></td><td></td><td></td></tr><tr><td>Image acquisition software</td><td>Fusion-Andor</td><td></td><td></td></tr><tr><td>Immersion Oil</td><td>Cargille</td><td>Type B: 16484</td><td></td></tr><tr><td>IR protection googles</td><td>Thorlabs</td><td>LG1</td><td></td></tr><tr><td>Lap2b-eGFP</td><td></td><td></td><td></td></tr><tr><td>Micro loader pipette</td><td>Eppendorf</td><td>GELoader</td><td></td></tr><tr><td>Microinjector</td><td>World Precision Instruments</td><td>SYS-PV820</td><td></td></tr><tr><td>MicroManager 2.0</td><td></td><td></td><td></td></tr><tr><td>Micromiter slide</td><td>ID5243 GXMGRAT-5 5mm/100 divisions</td><td></td><td></td></tr><tr><td>Mineral oil</td><td>Sigma</td><td>M3616</td><td></td></tr><tr><td>Motorized stage</td><td>ASI</td><td></td><td></td></tr><tr><td>Needle puller</td><td>Sutter instrument Co.</td><td>Model P-97</td><td></td></tr><tr><td>Optical tweezers platform</td><td>Impetux Optics</td><td>Sensocell</td><td></td></tr><tr><td>OTs software (LightAce)</td><td>Impetux Optics</td><td></td><td></td></tr><tr><td>PDMS</td><td>Sigma</td><td>Sylgard 184</td><td></td></tr><tr><td>PDMS Curing agent</td><td>Sigma</td><td>Sylgard 184</td><td></td></tr><tr><td>Post processing software (Matlab)</td><td>Mathworks</td><td></td><td></td></tr><tr><td>RNAse free water</td><td>Thermofisher</td><td>AM9937</td><td></td></tr><tr><td>Short-pass dichroic mirror (Figure 3, IR-F)</td><td>Semrock</td><td>FF01-950/SP-25</td><td></td></tr><tr><td>Spin-coater</td><td>Specialty Coating Systems</td><td>Spincoat G3P-8</td><td></td></tr><tr><td>Spinning-disk confocal microscope</td><td>Andor</td><td>DragonFly 502</td><td></td></tr><tr><td>Stereomicroscope</td><td>Leica M80</td><td></td><td></td></tr><tr><td>Triangular microinjection mold</td><td>Adaptive Science Tools</td><td>TU1</td><td></td></tr><tr><td>Universal oven</td><td>Memmert</td><td>UNB 200</td><td></td></tr><tr><td>Water immersion objective</td><td>Nikon</td><td>MRD07602</td><td></td></tr></tbody></table>

direct force measurements of subcellular mechanics in confinement using optical tweezers

Direct Force Measurements of Subcellular Mechanics in Confinement using Optical Tweezers

During the development of a multicellular organism, a single fertilized cell divides and gives rise to multiple tissues with diverse functions. Tissue morphogenesis goes in hand with molecular and structural changes at the single cell level that result in variations of subcellular mechanical properties. As a consequence, even within the same cell, different organelles and compartments resist differently to mechanical stresses; and mechanotransduction pathways can actively regulate their mechanical properties. The ability of a cell to adapt to the microenvironment of the tissue niche thus is in part due to the ability to sense and respond to mechanical stresses. We recently proposed a new mechanosensation paradigm in which nuclear deformation and positioning enables a cell to gauge the physical 3D environment and endows the cell with a sense of proprioception to decode changes in cell shape. In this article, we describe a new method to measure the forces and material properties that shape the cell nucleus inside living cells, exemplified on adherent cells and mechanically confined cells. The measurements can be performed non-invasively with optical traps inside cells, and the forces are directly accessible through calibration-free detection of light momentum. This allows measuring the mechanics of the nucleus independently from cell surface deformations and allowing dissection of exteroceptive and interoceptive mechanotransduction pathways. Importantly, the trapping experiment can be combined with optical microscopy to investigate the cellular response and subcellular dynamics using fluorescence imaging of the cytoskeleton, calcium ions, or nuclear morphology. The presented method is straightforward to apply, compatible with commercial solutions for force measurements, and can easily be extended to investigate the mechanics of other subcellular compartments, e.g., mitochondria, stress-fibers, and endosomes.

Here, we present a protocol to investigate the intracellular mechanical properties of isolated embryonic zebrafish cells in three-dimensional confinement with direct force measurement by an optical trap.

During the development of a multicellular organism, a single fertilized cell divides and gives rise to multiple tissues with diverse functions. Tissue ...

direct-force-measurements-subcellular-mechanics-confinement-using

Research

JoVE Journal

Developmental Biology

Here we describe a quick and simple method to measure cell stiffness. The general principle of this approach is to measure membrane deformation in response to well-defined negative pressure applied through a micropipette to the cell surface. This method provides a powerful tool to study biomechanical properties of substrate-attached cells.

Micropipette Aspiration of Substrate-attached Cells to Estimate Cell Stiffness

We measured the tension release in an axon that was partially lesioned with a laser dissector by simultaneous force spectroscopy measurement performed on an optically-trapped probe adhered to the membrane of the axon. The developed experimental protocol evaluates the axon adhesion to the culture substrate.

Measurement of Tension Release During Laser Induced Axon Lesion to Evaluate Axonal Adhesion to the Substrate at Piconewton and Millisecond Resolution

We present a setup of optical tweezers coupled to a light sheet microscope, and its implementation to probe cell mechanics without beads in the Drosophila embryo.

Probing Cell Mechanics with Bead-Free Optical Tweezers in the Drosophila Embryo

Here, we present a protocol for the simultaneous use of Förster resonance energy transfer-based tension sensors to measure protein load and fluorescence recovery after photobleaching to measure protein dynamics enabling the measurement of force-sensitive protein dynamics within living cells.

Measurement of Force-Sensitive Protein Dynamics in Living Cells Using a Combination of Fluorescent Techniques

Using a previously designed device to apply mechanical strain to adherent cells, this paper describes a redesigned substratum geometry and a customized apparatus for high-resolution single-cell imaging of strained cells with a 100x oil immersion objective.

High-resolution Imaging of Nuclear Dynamics in Live Cells under Uniaxial Tensile Strain

Presented here is a comprehensive protocol to perform ultrafast force-clamp experiments on processive myosin-5 motors, which could be easily extended to the study of other classes of processive motors. The protocol details all the necessary steps, from the setup of the experimental apparatus to sample preparation, data acquisition and analysis.

Dissecting Mechanoenzymatic Properties of Processive Myosins with Ultrafast Force-Clamp Spectroscopy

This protocol presents a complete experimental workflow for studying RNA-protein interactions using optical tweezers. Several possible experimental setups are outlined including the combination of optical tweezers with confocal microscopy.

Optical Tweezers to Study RNA-Protein Interactions in Translation Regulation

Formation of actomyosin bundles in vitro and measuring myosin ensemble force generation using optical tweezers is presented and discussed.

Probing Myosin Ensemble Mechanics in Actin Filament Bundles Using Optical Tweezers

Here, we present a protocol for reconstituting microtubule bundles in vitro and directly quantifying the forces exerted within them using simultaneous optical trapping and total internal reflection fluorescence microscopy. This assay allows for nanoscale-level measurement of the forces and displacements generated by protein ensembles within active microtubule networks.

Directly Measuring Forces Within Reconstituted Active Microtubule Bundles

Visual, single-molecule biochemistry studied through microfluidic chambers is greatly facilitated using glass barrel, gas-tight syringes, stable connections of tubing to flow cells, and elimination of bubbles by placing switching valves between the syringes and tubing. The protocol describes dual optical traps that enable visualization of DNA transactions and intermolecular interactions.

Use of Dual Optical Tweezers and Microfluidics for Single-Molecule Studies

Here, we describe a high-speed magnetic tweezer setup that performs nanomechanical measurements on force-sensitive biomolecules at the maximum rate of 1.2 kHz. We introduce its application to DNA hairpins and SNARE complexes as model systems, but it will be also applicable to other molecules involved in mechanobiological events.

High-Speed Magnetic Tweezers for Nanomechanical Measurements on Force-Sensitive Elements

Learn to measure subcellular mechanics using optical tweezers on isolated embryonic zebrafish cells. This protocol enables direct force measurement of nuclear properties without disrupting the cell membrane or cytoskeleton.

Direct Force Measurements of Subcellular Mechanics in Confinement using Optical Tweezers

Explore More Videos