We thank Guillaume Troadec for helpful discussion, Zihao Wang for hardware consultation, and Sydney Manufacturing Hub, Gregg Suaning and Simon Ringer for support of our lab startup. This work was supported by Australian Research Council Discovery Project (DP200101970 - L.A.J.), NSW Cardiovascular Capacity Building Program (Early-Mid Career Researcher Grant - L.A.J.), Sydney Research Accelerator prize (SOAR - L.A.J.), Ramaciotti Foundations Health Investment Grant (2020HIG76 - L.A.J.), National Health and Medical Research Council Ideas Grant (APP2003904 - L.A.J.), and The University of Sydney Faculty of Engineering Startup Fund and Major Equipment Scheme (L.A.J.). Lining Arnold Ju is an Australian Research Council DECRA fellow (DE190100609).

1. Obtain Analyzable DFS Events
<ol>
	<li>Start the experiment in the software (e.g., LabVIEW VI) for the BFP control and parameter setting (Figure S1A).</li>
	<li>Observe the repetitive probe bead-target bead/cell touches in the software for BFP Monitor (Figure S1B).</li>
	<li>Test and achieve the adhesion frequency &#8804; 20% within the first 50 touches by tuning the impingement force and contact time, by which it ensures that &#8805; 89% of DFS adhesion event are mediated by single bonds12,13,14. 
	NOTE: For each Bead-Cell/Bead pair, we perform 200 repetitive touch cycles. To obtain publishable data quality, we usually perform n &#8805; 3 Bead-Bead or Bead-Cell pairs.
	<ol>
		<li>Save data, in the form of Force vs. Time, to the user directed folder by the end of each pair, prompted by the software for BFP control and parameter setting.</li>
	</ol>
	</li>
	<li>Collect the Force vs. Time raw data of Bond events, as exemplified in the Figure 2A, using the BFP acquisition platform (Figure S1C).
	<ol>
		<li>Open the BFP data analysis software. Click on the yellow folder icon and select the corresponding raw data file by double clicking on them.</li>
	</ol>
	</li>
	<li>Run the program, and then click the up and down button to switch between events. Use the outlier exclusion criteria (Figure S2) to screen out invalid events. Select the exporting data type as Force vs. Time format and click on the Export Plots Data button.</li>
</ol>
2. Convert the Force vs. Time Curve to the Force vs. Displacement Curve
<ol>
	<li>Export the data segment corresponding to the Retract stage to a spreadsheet (Figure 2A, square marquee), which is relevant to the spring constant analysis.</li>
	<li>Plot the Force vs. Time Curve using spreadsheet software. To obtain the Force vs. Displacment curve, convert the time values (Figure 2A, x-axis) to the total displacement values (&#916;xtot) by multiplying time values with piezo movement velocity (i.e., 4,000 nm/s by preset).</li>
	<li>Zero the first data point by subtracting the smallest displacement value from each acquired displacement value. This horizontal transformation does not affect the ascending slopes of the Retract stage nor the subsequent spring constant calculation.</li>
	<li>Notably, the BFP is considered as a serial spring system in which &#916;xtot (Table 1) sum deformations of the RBC,&#160;&#916;xRBC&#160;(Table 1), the molecular bond, &#916;xmol (Table 1), and the Target cell,&#160;&#916;xcell (Table 1), as the Eq. 2: 
	<img alt="Equation 2" src="/files/ftp_upload/62490/62490eq02.jpg" /> (Eq. 2)</li>
	<li>Plot the Force (F) vs. Displacement (&#916;xtot) curve as shown in the Figure 2B.</li>
</ol>
3. Spring Cnstant Analysis of Bead- Cell Mode
<ol>
	<li>In the Force vs. Displacement curve, two distinct slop can be identified, where each can represent the compressive phase and the tensile phase. Fit a regression line to each data group (Figure 2B), where the larger linear fit slope represents the total spring constant at compressive phase (Figure 2B, red), denoted as k1 (Table 1); and the smaller linear fit slope represents the total spring constant at tenslile phase (Figure 2B, blue), denotated as k2 (Table 1).</li>
	<li>For springs connected in series per step 2.2 description, express the reciprocal of the total spring constant, ktot (Table 1), as the sum of the spring constant inverses of RBC, kRBC&#160;(Table 1), the molecular bond, kmol&#160;(Table 1), and the Target cell, kcell (Table 1). During the compressive phase of the Bead-Cell mode, the molecular bond is not stretched, therefore kmol is not taken into consideration. The reciprocal of the ktot in this scenario (1/k1) is expressed as 
	<img alt="Equation 3" src="/files/ftp_upload/62490/62490eq03.jpg" /> (Eq. 3). 
	In the example data, kRBC&#160;is pre-determined (0.25 pN/nm by default). kcell can be derived from the Eq. 3 with the acquired k1 and kRBC (Figure 3B).</li>
	<li>During the tensile phase, adhesion is formed between the ligand-receptor pair. Express the reciprocal of the ktot in this scenario (1/k2) as 
	<img alt="Equation 4" src="/files/ftp_upload/62490/62490eq04.jpg" /> (Eq. 4) 
	where k2 (Table 1) represents the total spring constant during the tensile phase.</li>
	<li>Derive kmol from subtracting 1/k1 from 1/k2 (compare Eq. 3 vs. Eq. 4).</li>
</ol>
4. Spring Constant Analysis of Bead- Bead Mode
<ol>
	<li>Fit a regression line to the compressive phase data to obtain k1&#160;(similar to the Figure 2B, red). Of note, in the Bead-Bead mode, the Target cell is replaced by a glass bead coated with the receptor of interest (Figure 1C). Since bead deformation is negligible, the 1/kcell term can be removed from the Eq. 3 and Eq. 4 accordingly. The reciprocal ktot of the compressive phase (1/k1) can be expressed as: 
	<img alt="Equation 5" src="/files/ftp_upload/62490/62490eq05.jpg" /> (Eq. 5)</li>
	<li>Fit a regression line to the tensile phase data to obtain k2&#160;(similar to the Figure 2B, blue). The reciprocal ktot of the tensile phase (1/k2) can be expressed as: 
	<img alt="Equation 6" src="/files/ftp_upload/62490/62490eq06.jpg" /> (Eq. 6)</li>
	<li>Derive kmol from subtracting 1/k1 from 1/k2 (compare Eq. 5 vs. Eq. 6).</li>
</ol>

<ol>
	<li>Chen, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+Biomembrane+Force+Probe:+Concurrent+Quantitation+of+Receptor-ligand+Kinetics+and+Binding-induced+Intracellular+Signaling+on+a+Single+Cell.">Fluorescence Biomembrane Force Probe: Concurrent Quantitation of Receptor-ligand Kinetics and Binding-induced Intracellular Signaling on a Single Cell.</a> The Journal of Visualized Experiments. (102), e52975 (2015).</li><li>Su, Q. P., Ju, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biophysical+nanotools+for+single-molecule+dynamics.">Biophysical nanotools for single-molecule dynamics.</a> Biophysics Reviews. 10 (5), 1349-1357 (2018).</li><li>Ju, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+Force+Spectroscopy+Analysis+on+the+Redox+States+of+Protein+Disulphide+Bonds.">Dynamic Force Spectroscopy Analysis on the Redox States of Protein Disulphide Bonds.</a> Methods in Molecular Biology. 1967, 115-131 (2019).</li><li>An, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultra-stable+Biomembrane+Force+Probe+for+Accurately+Determining+Slow+Dissociation+Kinetics+of+PD-1+Blockade+Antibodies+on+Single+Living+Cells.">Ultra-stable Biomembrane Force Probe for Accurately Determining Slow Dissociation Kinetics of PD-1 Blockade Antibodies on Single Living Cells.</a> Nano Letters. 20 (7), 5133-5140 (2020).</li><li>Chen, Y., Ju, L., Rushdi, M., Ge, C., Zhu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Receptor-mediated+cell+mechanosensing.">Receptor-mediated cell mechanosensing.</a> Molecular Biology of the Cell. 28 (23), 3134-3155 (2017).</li><li>Ju, L., Chen, Y., Rushdi, M. N., Chen, W., Zhu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-Dimensional+Analysis+of+Cross-Junctional+Molecular+Interaction+by+Force+Probes.">Two-Dimensional Analysis of Cross-Junctional Molecular Interaction by Force Probes.</a> Methods in Molecular Biology. 1584, 231-258 (2017).</li><li>Evans, E., Ritchie, K., Merkel, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensitive+force+technique+to+probe+molecular+adhesion+and+structural+linkages+at+biological+interfaces.">Sensitive force technique to probe molecular adhesion and structural linkages at biological interfaces.</a> Biophysical Journal. 68 (6), 2580-2587 (1995).</li><li>Ju, L., Zhu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Benchmarks+of+Biomembrane+Force+Probe+Spring+Constant+Models.">Benchmarks of Biomembrane Force Probe Spring Constant Models.</a> Biophysical Journal. 113 (12), 2842-2845 (2017).</li><li>Evans, E., Ritchie, K., Merkel, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensitive+Force+Technique+to+Probe+Molecular+Adhesion+and+Structural+Linkages+at+Biological+Interfaces.">Sensitive Force Technique to Probe Molecular Adhesion and Structural Linkages at Biological Interfaces.</a> Biophysical Journal. 68, 2580 (1995).</li><li>Fiore, V. 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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+catch+of+a+Thy-1-alpha5beta1+syndecan-4+trimolecular+complex.">Dynamic catch of a Thy-1-alpha5beta1+syndecan-4 trimolecular complex.</a> Nature Communications. 5, 4886 (2014).</li><li>Passam, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechano-redox+control+of+integrin+de-adhesion.">Mechano-redox control of integrin de-adhesion.</a> Elife. 7, (2018).</li><li>Chen, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+integrin+alphaIIbbeta3+intermediate+affinity+state+mediates+biomechanical+platelet+aggregation.">An integrin alphaIIbbeta3 intermediate affinity state mediates biomechanical platelet aggregation.</a> Nature Materials. 18 (7), 760-769 (2019).</li><li>Chen, Y., Lee, H., Tong, H., Schwartz, M., Zhu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Force+regulated+conformational+change+of+integrin+&#945;V&#946;3.">Force regulated conformational change of integrin &#945;V&#946;3.</a> Matrix Biology. 60, 70-85 (2017).</li><li>Liu, B., Chen, W., Zhu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+force+spectroscopy+on+cells.">Molecular force spectroscopy on cells.</a> Annual Review of Physical Chemistry. 66, 427-451 (2015).</li><li>Piper, J. 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The authors declare that they have no competing interests to report regarding the present study.

In summary, we have provided a detailed data analysis protocol for preprocessing the DFS raw data and deriving molecular spring constants in the BFP Bead-Bead and Bead-Cell analysis modes. Biomechanical models and equations required for determining molecular and cellular spring constants are presented. Albeit different integrins are studied, the kmol measured by the Bead-Bead mode and the Bead-Cell mode possesses significant range differences (Figure 3A vs. Figure 3C). Of note, with the Bead-Bead mode, the receptor is covalently linked to the glass bead. In contrast, with the Bead-Cell mode, the surface receptor is adapted by the underlying plasma membrane and cytoskeletons, which most likely influence the measured kmol.
Data quality control is crucial to ensure the reproducibility. To this end, we have implemented the DFS data pre-screening and outlier exclusion criteria on the Force vs. Time plots. To demonstrate this, a representative dataset was selected, in which we categorized the DFS raw data into three levels of quality: Good (Figure S2A), Acceptable with noise (Figure S2B), and Poor unacceptable (Figure S2C). For beginners of using the BFP, we recommend the strict criteria to pre-screen the data with the Good quality (Figure S2A). Of note, based on the data pre-screening criteria, the regression fit line of the compressive phase should be steeper than that of the tensile phase, specifically k1 &#62; k2 (Figure S2C, vii). When measured k1 &#60; k2 (Figure S2C, vii), the derived kmol &#60; 0 is against the rationale per calculation in the step 4. Such events should then be considered as the invalid outliers and discarded.
To favor the BFP measurement on single-molecular level during data acquisition, multiple experimental configurations have been implemented according to previous study12. Firstly, protein coating density on beads is usually titrated down to a minimal level (e.g. 60 &#181;m-2) by strictly control the solution concentration, quantity of the protein and reaction conditions15. The average spatial distance between proteins on the bead is thereby estimated much larger than the linear dimensions of the protein, favoring our measurements on single-molecular level12,13,14. Secondly, we control the adhesion frequency for each ligand-receptor pair &#8804; 20%, under which molecular binding events will follow Poisson distribution predicting &#8805; 89% of events would be single-molecular binding14,15. To achieve so, impingement force and contact time are set accordingly and need to be consistent throughout the experiment12. Nevertheless, it is still possible that multiple bonds occur sequentially (Figure S2C, vi).&#160;In such cases, we will discard the events with signatures of multiple bonds. Last but not least, negative control experiments will be performed with beads coated with bovine serum albumin (Table of Materials) or SA alone to ensure the non-specific adhesion frequency is &#8804; 2%16,17.
Although BFP is powerful to investigate protein dynamics on living cell surface10,11,12, there are technical limitations. In BFP, only one ligand-receptor pair can be investigated at a time. It would be time consuming to obtain sufficient data with statistical significance. Besides, the experimental procedures are labor intensive with steep learning curves. Implementation wise, the current BFP system is susceptible to the environmental drifting and surrounding mechanical vibration. As a result, continuous manual adjustment is required to ensure the DFS data quality. To this end, one of our recent studies has introduced ultra-stable BFP feedback control algorithms to improve the stability of the BFP force-clamp DFS assays4. This technical advance enables measurements of stronger molecular interaction such as antigen-antibody binding with ultra-long bond lifetime (&#62;50 s). Nevertheless, we foresee future efforts will be made to automate and integrate the BFP data acquisition and DFS analysis into one computerized program, making the entire BFP operation and data analysis more user friendly and high-throughput.

As a live-cell DFS technique, BFP engineers a human red blood cell (RBC; Figure 1) into an ultrasensitive and tunable force transducer with a compatible spring constant range at 0.1-3 pN/nm1,2,3. To probe ligand-receptor interaction, BFP enables DFS measurements at ~1 pN (10-12 N), ~3 nm (10-9 m), and ~0.5 ms (10-3 s) in force, spatial, and temporal resolution4,5. Its experimental configuration consists of two opposing micropipettes, namely the Probe and the target. The Probe micropipette aspirates a RBC and a bead is glued at its apex via a biotin-streptavidin interaction. The bead is coated with the ligand of interest (Figure 1A). The Target micropipette aspirates either a cell or a bead bearing the receptor of interest, corresponding to the Bead-Cell (Figure 1B) and Bead-Bead (Figure 1C) modes, respectively5.
BFP construction, assembly and the DFS experimental protocols were described in detail previously1,6. Briefly, a BFP touch cycle consists of 5 stages: Approach, Impinge, Contact, Retract and Dissociate (Figure 1D). The horizontal RBC apex position is denoted as &#916;xRBC. At the beginning, the unstressed (zero-force) RBC deformation &#916;xRBC is 0 (Table 1). The Target is then driven by a piezotranslator to impinge on and retract from the Probe bead (Figure 1D). The RBC probe is first compressed by the Target with negative RBC deformation &#916;xRBC&#160;&#60; 0. In a Bond event, the Retract stage transitions from a compressive to a tensile phase with positive RBC deformation &#916;xRBC&#160;&#62; 0 (Figure 2C and D). According to Hooke&#39;s law, the BFP bearing force is able to be measured as F = kRBC&#160;&#215;&#160;&#916;xRBC, where kRBC (Table 1) is the RBC spring constant of the BFP. Upon bond rupture and the completion of one touch cycle, the probe bead returns to zero-force position with &#916;xRBC&#160;= 0 (Figure 1D).
To determine the kRBC, we measure and record the radii of the probe micropipette inner orifice (Rp), the RBC (R0) and the circular contact area (Rc) between the RBC and the probe bead (Figure 1A). Then kRBC is calculated according to the Evan&#39;s model (Eq. 1)7,8 using a LabVIEW program that acts as a virtual instrument (VI) to operate the BFP (Figure S1A)8,9.
<img alt="Equation 1" src="/files/ftp_upload/62490/62490eq01.jpg" /> (Eq. 1)
With a BFP established and DFS raw data obtained, hereby we present how to analyze the spring constant of ligand-receptor pair or cells. The DFS raw data on the interaction of the glycosylated protein Thy-1 and K562 cell bearing integrin &#945;5&#946;1 (Thy-1-&#945;5&#946;1; Figures 3A and 3B)10 and that of the fibrinogen and bead coated integrin &#945;IIb&#946;3 (FGN-&#945;IIb&#946;3; Figure 3C)11,12 have been used to demonstrate the Bead-Cell and Bead-Bead analysis modes, respectively.
BFP Experimental Preparation 
For details of BFP experimental preparation and instrumentation, please refer to the previously published protocols3. In brief, human RBC has been biotinylated using the Biotin-PEG3500-NHS in the carbon/bicarbonate buffer. Proteins of interest have been covalently coupled to the borosilicate glass beads using MAL-PEG3500-NHS in the phosphate buffer. To attach to the biotinylated RBC, the probe bead is also coated with streptavidin (SA) using the MAL-SA. Please see the Table of Materials and Table 2.
To assemble the BFP (Figure 1, left), the third micropipette termed &#39;Helper&#39; will be used to deliver the probe bead and glue it to the RBC&#39;s apex1,3. The covalent interaction between the SA coated probe bead and biotinylated RBC is much stronger than the ligand-receptor bond of interest. Thus, the Dissociate stage can be interpreted as the ligand-receptor bond rupture rather than the detachment of Probe bead from the RBC.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>3-Mercaptopropyltrimethoxysilane (MPTMS)</td>
			<td>Uct, Specialties, llc</td>
			<td>4420-74-0</td>
			<td>Glass bead functionalization</td>
		</tr>
		<tr>
			<td>Anhy. Sodium Phosphate Dibasic (Na2HPO4)</td>
			<td>Sigma-Aldrich</td>
			<td>S7907</td>
			<td>Phosphate buffer preparation</td>
		</tr>
		<tr>
			<td>BFP data acquisition VI</td>
			<td>LabVIEW</td>
			<td></td>
			<td>BFP control and parameter setting</td>
		</tr>
		<tr>
			<td>BFP data analysis VI</td>
			<td>LabVIEW</td>
			<td></td>
			<td>BFP raw data analysis</td>
		</tr>
		<tr>
			<td>Biotin-PEG3500-NHS</td>
			<td>JenKem</td>
			<td>A5026-1</td>
			<td>RBC biotinylation</td>
		</tr>
		<tr>
			<td>Borosilicate Glass beads</td>
			<td>Distrilab Particle Technology, Netherlands</td>
			<td>9002</td>
			<td>Glass bead functionalization</td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Sigma-Aldrich</td>
			<td>A0336</td>
			<td>Ligand functionalization</td>
		</tr>
		<tr>
			<td>Camera VI</td>
			<td>LabVIEW</td>
			<td></td>
			<td>BFP monitoring</td>
		</tr>
		<tr>
			<td>D-glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G7021</td>
			<td>Tyrode&#8217;s buffer preparation</td>
		</tr>
		<tr>
			<td>Hepes</td>
			<td>Sigma-Aldrich</td>
			<td>H3375</td>
			<td>Tyrode&#8217;s buffer preparation</td>
		</tr>
		<tr>
			<td>MAL-PEG3500-NHS</td>
			<td>JenKem</td>
			<td>A5002-1</td>
			<td>Glass bead functionalization</td>
		</tr>
		<tr>
			<td>Potassium Chloride (KCl)</td>
			<td>Sigma-Aldrich</td>
			<td>P9541</td>
			<td>Tyrode&#8217;s buffer preparation</td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate (NaHCO3)</td>
			<td>Sigma-Aldrich</td>
			<td>S5761</td>
			<td>Carbonate/bicarbonate buffer preparation; Tyrode&#8217;s buffer preparation</td>
		</tr>
		<tr>
			<td>Sodium Carbonate (Na2CO3)</td>
			<td>Sigma-Aldrich</td>
			<td>S2127</td>
			<td>Carbonate/bicarbonate buffer preparation</td>
		</tr>
		<tr>
			<td>Sodium Chloride (NaCl)</td>
			<td>Sigma-Aldrich</td>
			<td>S7653</td>
			<td>Tyrode&#8217;s buffer preparation</td>
		</tr>
		<tr>
			<td>Sodium Phosphate Monobasic Monohydrate (NaH2PO4&#8226;H2O)</td>
			<td>Sigma-Aldrich</td>
			<td>S9638</td>
			<td>Phosphate buffer preparation</td>
		</tr>
		<tr>
			<td>Streptavidin-Maleimide</td>
			<td>Sigma-Aldrich</td>
			<td>S9415</td>
			<td>Glass bead functionalization</td>
		</tr>
	</tbody>
</table>

molecular spring constant analysis by biomembrane force probe spectroscopy

A biomembrane force probe (BFP) has recently emerged as a native-cell-surface or in situ dynamic force spectroscopy (DFS) nanotool that can measure single-molecular binding kinetics, assess mechanical properties of ligand-receptor interactions, visualize protein dynamic conformational changes and more excitingly elucidate receptor mediated cell mechanosensing mechanisms. More recently, BFP has been used to measure the spring constant of molecular bonds. This protocol describes the step-by-step procedure to perform molecular spring constant DFS analysis. Specifically, two BFP operation modes are discussed, namely the Bead-Cell and Bead-Bead modes. This protocol focuses on deriving spring constants of the molecular bond and cell from DFS raw data.

In this work, we have demonstrated the protocol of the BFP spring constant analysis. For the Bead-Cell analysis mode, we analyzed the kmol&#160;of the molecular bond between the glycosylated protein Thy-1 coated on the Probe bead and the integrin &#945;5&#946;1 expressed on the Target K562 cell (Thy-1-integrin &#945;5&#946;1; Figure 3A)10. The kcell is also derived from the Bead-Cell mode (K562 Cell; Figure 3B). For the Bead-Bead mode, the molecular bond formed between fibrinogen and integrin &#945;IIb&#946;3 (FGN-integrin &#945;IIb&#946;3; Figure 3C)11,12 is used to demonstrate the Bead-Bead analysis mode.
For the Bead-Cell mode, we measured the spring constants of Thy-1-integrin &#945;5&#946;1 bond and K562 cell as kmol = 0.45 &#177; 0.28 pN/nm (Figure 3A) and kcell = 0.18&#160;&#177; 0.07 pN/nm (Figure 3B) from 27 pre-screened analyzable events. For the Bead-Bead mode, we measured the spring constants of FGN-integrin &#945;IIb&#946;3 bond as kmol = 0.53 &#177; 0.29 pN/nm (Figure 3C) from 33 pre-screened analyzable events.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Symbol</td>
			<td>Definition</td>
			<td>Symbol</td>
			<td>Definition</td>
		</tr>
		<tr>
			<td>&#916;xtot</td>
			<td>The total displacement of the piezo, which can also be interpreted as the total deformation of RBC, target cell and molecular bond.</td>
			<td>&#916;xRBC</td>
			<td>The RBC deformation, which can also be interpreted as the displacement of the Probe bead.</td>
		</tr>
		<tr>
			<td>ktot</td>
			<td>The total spring constant of the entire BFP serial spring system.</td>
			<td>kRBC</td>
			<td>The spring constant of the aspirated RBC by the Probe micropipette.</td>
		</tr>
		<tr>
			<td>kmol</td>
			<td>The spring constant of the BFP detected molecular bond</td>
			<td>kcell</td>
			<td>The spring constant of the Target cell.</td>
		</tr>
		<tr>
			<td>k1</td>
			<td>ktot of the compressive phase in the Retract stage.</td>
			<td>k2</td>
			<td>ktot of the tensile phase in the Retract stage.</td>
		</tr>
		<tr>
			<td>&#916;F1</td>
			<td>The increment of force sensed by the Probe bead in the compressive phase.</td>
			<td>&#916;F2</td>
			<td>The increment of force sensed by the Probe bead in the tensile phase.</td>
		</tr>
		<tr>
			<td>&#916;x1</td>
			<td>The increment of displacement in the compressive phase.</td>
			<td>&#916;x2</td>
			<td>The increment of displacement in the tensile phase.</td>
		</tr>
	</tbody>
</table>
Table 1. Symbol definitions for the BFP molecular spring constant analysis.&#160;Horizontal positions of all objects are defined as x, while &#916;x&#160;[nm]&#160;refers to the deformation relative to the original position. &#916;F [pN] refers to the force increment measured by BFP. k [pN/nm] refers to the spring constant. Subscripts 1 and 2 correspond to the compressive and tensile phases, respectively. The molecular spring constant is derived from the Force (F) vs. Displacement (&#916;xtot) curve.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62490/62490fig01.jpg" /> 
Figure 1: BFP configuration and DFS touch cycle. (A) The BFP system assembles two opposing micropipettes, namely the Probe (left) and the Target (right). The Probe micropipette aspirates a RBC (red) with a glass bead glued at its apex to serve as a force transducer. The Target micropipette aspirates a receptor-bearing cell (blue). The RBC spring constant (kRBC) is determined by the aspiration pressure (&#916;p)&#160;and the radii of aspirated RBC (R0), Probe micropipette (Rp) and circular contact area (Rc) between the RBC and the Probe bead. (B and C) Micrographs of the Bead-Cell (B) and Bead-Bead (C) BFP modes. Scale bars = 5 &#181;m. (D) A BFP touch cycle that consists of Approach, Impinge, Contact, Retract and Dissociate stages. The &#916;xRBC = 0 dash line indicates the BFP unstressed, or zero-force, position. The Retract stage includes compressive phase (&#916;xRBC &#60; 0, red), zero-force position (&#916;xRBC = 0, black) and tensile phase (&#916;xRBC &#62; 0, blue) in sequence. Black arrow indicates the position of Bond event. <a href="https://www.jove.com/files/ftp_upload/62490/62490fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62490/62490fig02.jpg" /> 
Figure 2: Derive molecular spring constants from DFS raw data. (A) Representative Force (F) vs. Time (t) curve&#160;of DFS raw data in one BFP touch cycle. (B)&#160;Representative converted Force (F) vs. Displacement (&#916;xtot) curve that depicts the Retract stage. k1 and k2 represent the fit slope of the consecutive compressive and tensile phases respectively. &#916;F1 and&#160;&#916;F2 represent the increments of force in the compressive phase and the tensile phase data, respectively, where &#916;x1 and&#160;&#916;x2 represent the increments of displacement in the compressive phase and tensile phase data, respectively. R2 values for spring constant during compressive stage (R12) and tensile phase (R22) are labelled on the graph to indicate good statistical fitness. (C and D) Illustrations of the Retract stage in the Bead-Cell (C) and Bead-Bead (D) experimental modes. kRBC represents the spring constant of RBC; kcell and kmol represent the spring constants of the Target cell and the molecular bond, respectively. During the tensile phase, adhesion is formed between the ligand-receptor pair, the RBC deflects in the same direction as piezo retracts beyond zero-force position (&#916;xRBC &#62; 0). <a href="https://www.jove.com/files/ftp_upload/62490/62490fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62490/62490fig03.jpg" /> 
Figure 3:&#160;Representative histograms of BFP measured spring constants. The event number (left y-axis) and frequency distribution (right y-axis) of measured spring constants for Thy-1-integrin &#945;5&#946;1 bond (A) and K562 Target cell (B) in the Bead-Cell mode and FGN-integrin &#945;IIb&#946;3 bond (C) in the Bead-Bead mode. Histograms are fit with Gaussian distribution curve (pink) and the statistical parameter, R2, is used to indicate the strength of fitness. <a href="https://www.jove.com/files/ftp_upload/62490/62490fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Figure S1: The homemade BFP interface. (A)&#160;BFP control and parameter setting interface. The parameters to determine RBC spring constant are entered from the panel of biophysical parameters. (B) BFP monitoring.&#160;Live BFP touch cycles will be observed from this camera view. (C) BFP DFS analysis interface where the Force (F) vs. Time (t) curves are offline reviewed and pre-processed for subsequent molecular spring constant analysis. <a href="https://www.jove.com/files/ftp_upload/62490/Fig S1.tif" target="_blank">Please click here to download this File.</a>
Figure S2. BFP analyzable data quality control and pre-screening criteria. (A) Good DFS events with high quality: (i) Bead-Cell rupture force event; (ii) Bead-Cell lifetime event; (iii) Bead-Bead rupture force event; (iv) Bead-Bead lifetime event. (B)&#160;Acceptable DFS events with some noise: (i) Data drifting but the zoom-in Retract stage remains valid; (ii)Slight data drifting after bond dissociation; (iii) Data kink at the zero-force regime; (iv) Holding force is small (&#60; 10 pN). (C)Poor-quality events that should be discarded: (i) No adhesion; (ii) Data oscillation; (iii) Data drifting all the time; (iv) Discontinuous data; (v) Compressive force too small (&#8776; 0 pN); (vi) Multiple bonds; (vii) Invalid data with derived kmol &#60; 0; (viii) Signal error. Zero force is indicated by the grey intermittent line. <a href="https://www.jove.com/files/ftp_upload/62490/Fig S2.tif" target="_blank">Please click here to download this File.</a>

Watch this Scientific Journal Video about Molecular Spring Constant Analysis by Biomembrane Force Probe Spectroscopy at JoVE.com

Molecular Spring Constant Analysis by Biomembrane Force Probe Spectroscopy

A biomembrane force probe (BFP) is an in situ dynamic force spectroscopy (DFS) technique. BFP can be used to measure the spring constant of molecular interactions on living cells. This protocol presents spring constant analysis for molecular bonds detected by BFP.

A biomembrane force probe (BFP) has recently emerged as a native-cell-surface or in situ dynamic force spectroscopy (DFS) nanotool that can measure ...

molecular-spring-constant-analysis-biomembrane-force-probe

Nanyang Technological University

Research

JoVE Journal

Bioengineering

2.9K Views.  The University of Sydney. A biomembrane force probe (BFP) is an in situ dynamic force spectroscopy (DFS) technique. BFP can be used to measure the spring constant of molecular interactions on living cells. This protocol presents spring constant analysis for molecular bonds detected by BFP. 

Video: Molecular Spring Constant Analysis by Biomembrane Force Probe Spectroscopy

Thermodynamics of Membrane Protein Folding Measured by Fluorescence Spectroscopy

Membrane protein folding is an emerging topic with both fundamental and health-related significance. The abundance of membrane proteins in cells underlies the need for comprehensive study of the folding of this ubiquitous family of proteins. Additionally, advances in our ability to characterize diseases associated with misfolded proteins have motivated significant experimental and theoretical efforts in the field of protein folding. Rapid progress in this important field is unfortunately hindered by the inherent challenges associated with membrane proteins and the complexity of the folding mechanism. Here, we outline an experimental procedure for measuring the thermodynamic property of the Gibbs free energy of unfolding in the absence of denaturant, &Delta;G&deg;H2O, for a representative integral membrane protein from E. coli. This protocol focuses on the application of fluorescence spectroscopy to determine equilibrium populations of folded and unfolded states as a function of denaturant concentration. Experimental considerations for the preparation of synthetic lipid vesicles as well as key steps in the data analysis procedure are highlighted. This technique is versatile and may be pursued with different types of denaturant, including temperature and pH, as well as in various folding environments of lipids and micelles. The current protocol is one that can be generalized to any membrane or soluble protein that meets the set of criteria discussed below.

This video article details the experimental procedure for obtaining the Gibbs free energy of membrane protein folding by tryptophan fluorescence.

Membrane protein folding is an emerging topic with both fundamental and health-related significance.  The abundance of membrane proteins in cells ...

Bacterial Immobilization for Imaging by Atomic Force Microscopy

AFM is a high-resolution (nm scale) imaging tool that mechanically probes a surface. It has the ability to image cells and biomolecules, in a liquid environment, without the need to chemically treat the sample. In order to accomplish this goal, the sample must sufficiently adhere to the mounting surface to prevent removal by forces exerted by the scanning AFM cantilever tip. In many instances, successful imaging depends on immobilization of the sample to the mounting surface. Optimally, immobilization should be minimally invasive to the sample such that metabolic processes and functional attributes are not compromised. By coating freshly cleaved mica surfaces with porcine (pig) gelatin, negatively charged bacteria can be immobilized on the surface and imaged in liquid by AFM. Immobilization of bacterial cells on gelatin-coated mica is most likely due to electrostatic interaction between the negatively charged bacteria and the positively charged gelatin. Several factors can interfere with bacterial immobilization, including chemical constituents of the liquid in which the bacteria are suspended, the incubation time of the bacteria on the gelatin coated mica, surface characteristics of the bacterial strain and the medium in which the bacteria are imaged. Overall, the use of gelatin-coated mica is found to be generally applicable for imaging microbial cells.

Live Gram-negative and Gram-positive bacteria can be immobilized on gelatin-coated mica and imaged in liquid using Atomic Force Microscopy (AFM).

AFM is a high-resolution (nm scale) imaging tool that mechanically probes a surface.  It has the ability to image cells and biomolecules, in a liquid ...

Stretching Short Sequences of DNA with Constant Force Axial Optical Tweezers

Single-molecule techniques for stretching DNA of contour lengths less than a kilobase are fraught with experimental difficulties. However, many interesting biological events such as histone binding and protein-mediated looping of DNA1,2, occur on this length scale. In recent years, the mechanical properties of DNA have been shown to play a significant role in fundamental cellular processes like the packaging of DNA into compact nucleosomes and chromatin fibers3,4. Clearly, it is then important to understand the mechanical properties of short stretches of DNA. In this paper, we provide a practical guide to a single-molecule optical tweezing technique that we have developed to study the mechanical behavior of DNA with contour lengths as short as a few hundred basepairs.
The major hurdle in stretching short segments of DNA is that conventional optical tweezers are generally designed to apply force in a direction lateral to the stage5,6, (see Fig. 1). In this geometry, the angle between the bead and the coverslip, to which the DNA is tethered, becomes very steep for submicron length DNA. The axial position must now be accounted for, which can be a challenge, and, since the extension drags the microsphere closer to the coverslip, steric effects are enhanced. Furthermore, as a result of the asymmetry of the microspheres, lateral extensions will generate varying levels of torque due to rotation of the microsphere within the optical trap since the direction of the reactive force changes during the extension.
Alternate methods for stretching submicron DNA run up against their own unique hurdles. For instance, a dual-beam optical trap is limited to stretching DNA of around a wavelength, at which point interference effects between the two traps and from light scattering between the microspheres begin to pose a significant problem. Replacing one of the traps with a micropipette would most likely suffer from similar challenges. While one could directly use the axial potential to stretch the DNA, an active feedback scheme would be needed to apply a constant force and the bandwidth of this will be quite limited, especially at low forces.
We circumvent these fundamental problems by directly pulling the DNA away from the coverslip by using a constant force axial optical tweezers7,8. This is achieved by trapping the bead in a linear region of the optical potential, where the optical force is constant-the strength of which can be tuned by adjusting the laser power. Trapping within the linear region also serves as an all optical force-clamp on the DNA that extends for nearly 350 nm in the axial direction. We simultaneously compensate for thermal and mechanical drift by finely adjusting the position of the stage so that a reference microsphere stuck to the coverslip remains at the same position and focus, allowing for a virtually limitless observation period.

We illustrate the use of a constant force axial optical tweezers to explore the mechanical properties of short DNA molecules. By stretching DNA axially, we minimize steric hindrances and artifacts arising in conventional lateral manipulation, allowing us to study DNA molecules as short as ~100 nm.

Single-molecule techniques for stretching DNA of contour lengths  less than a kilobase are fraught with experimental difficulties. However, many ...

Investigating Receptor-ligand Systems of the Cellulosome with AFM-based Single-molecule Force Spectroscopy

Cellulosomes are discrete multienzyme complexes used by a subset of anaerobic bacteria and fungi to digest lignocellulosic substrates. Assembly of the enzymes onto the noncatalytic scaffold protein is directed by interactions among a family of related receptor-ligand pairs comprising interacting cohesin and dockerin modules. The extremely strong binding between cohesin and dockerin modules results in dissociation constants in the low picomolar to nanomolar range, which may hamper accurate off-rate measurements with conventional bulk methods. Single-molecule force spectroscopy (SMFS) with the atomic force microscope measures the response of individual biomolecules to force, and in contrast to other single-molecule manipulation methods (i.e.&#160;optical tweezers), is optimal for studying high-affinity receptor-ligand interactions because of its ability to probe the high-force regime (&#62;120 pN). Here we present our complete protocol for studying cellulosomal protein assemblies at the single-molecule level. Using a protein topology derived from the native cellulosome, we worked with enzyme-dockerin and carbohydrate binding module-cohesin (CBM-cohesin) fusion proteins, each with an accessible free thiol group at an engineered cysteine residue. We present our site-specific surface immobilization protocol, along with our measurement and data analysis procedure for obtaining detailed binding parameters for the high-affinity complex. We demonstrate how to quantify single subdomain unfolding forces, complex rupture forces, kinetic off-rates, and potential widths of the binding well. The successful application of these methods in characterizing the cohesin-dockerin interaction responsible for assembly of multidomain cellulolytic complexes is further described.

Cellulosomes are multienzyme complexes designed for digesting cellulose. AFM-based SMFS was used to study the mechanical properties and folding configuration of cellulosome-associated protein assemblies. We present a complete workflow for protein immobilization, data acquisition, and data analysis to study the interactions of individual receptor-ligand complexes involved in cellulosome assembly.

Cellulosomes are discrete multienzyme complexes used by a subset of anaerobic bacteria and fungi to digest lignocellulosic substrates. Assembly of the ...

A Microfluidic Technique to Probe Cell Deformability

Here we detail the design, fabrication, and use of a microfluidic device to evaluate the deformability of a large number of individual cells in an efficient manner. Typically, data for ~102 cells can be acquired within a 1 hr experiment. An automated image analysis program enables efficient post-experiment analysis of image data, enabling processing to be complete within a few hours. Our device geometry is unique in that cells must deform through a series of micron-scale constrictions, thereby enabling the initial deformation and time-dependent relaxation of individual cells to be assayed. The applicability of this method to human promyelocytic leukemia (HL-60) cells is demonstrated. Driving cells to deform through micron-scale constrictions using pressure-driven flow, we observe that human promyelocytic (HL-60) cells momentarily occlude the first constriction for a median time of 9.3 msec before passaging more quickly through the subsequent constrictions with a median transit time of 4.0 msec per constriction. By contrast, all-trans retinoic acid-treated (neutrophil-type) HL-60 cells occlude the first constriction for only 4.3 msec before passaging through the subsequent constrictions with a median transit time of 3.3 msec. This method can provide insight into the viscoelastic nature of cells, and ultimately reveal the molecular origins of this behavior.

We demonstrate a microfluidics-based assay to measure the timescale for cells to transit through a sequence of micron-scale constrictions.

Here we detail the design, fabrication, and use of a microfluidic device to evaluate the deformability of a large number of individual cells in an ...

Formation of Biomembrane Microarrays with a Squeegee-based Assembly Method

Lipid bilayer membranes form the plasma membranes of cells and define the boundaries of subcellular organelles. In nature, these membranes are heterogeneous mixtures of many types of lipids, contain membrane-bound proteins and are decorated with carbohydrates. In some experiments, it is desirable to decouple the biophysical or biochemical properties of the lipid bilayer from those of the natural membrane. Such cases call for the use of model systems such as giant vesicles, liposomes or supported lipid bilayers (SLBs). Arrays of SLBs are particularly attractive for sensing applications and mimicking cell-cell interactions. Here we describe a new method for forming SLB arrays. Submicron-diameter SiO2 beads are first coated with lipid bilayers to form spherical SLBs (SSLBs). The beads are then deposited into an array of micro-fabricated submicron-diameter microwells. The preparation technique uses a "squeegee" to clean the substrate surface, while leaving behind SSLBs that have settled into microwells. This method requires no chemical modification of the microwell substrate, nor any particular targeting ligands on the SSLB. Microwells are occupied by single beads because the well diameter is tuned to be just larger than the bead diameter. Typically, more 75% of the wells are occupied, while the rest remain empty. In buffer SSLB arrays display long-term stability of greater than one week. Multiple types of SSLBs can be placed in a single array by serial deposition, and the arrays can be used for sensing, which we demonstrate by characterizing the interaction of cholera toxin with ganglioside GM1. We also show that phospholipid vesicles without the bead supports and biomembranes from cellular sources can be arrayed with the same method and cell-specific membrane lipids can be identified.

Supported lipid bilayers and natural membrane particles are convenient systems that can approximate the properties of cell membranes and be incorporated in a variety of analytical strategies. Here we demonstrate a method for preparing microarrays composed of supported lipid bilayer-coated SiO2 beads, phospholipid vesicles or natural membrane particles.

Lipid bilayer membranes form the plasma membranes of cells and define the boundaries of subcellular organelles. In nature, these membranes are ...

Surface Potential Measurement of Bacteria Using Kelvin Probe Force Microscopy

Surface potential is a commonly overlooked physical characteristic that plays a dominant role in the adhesion of microorganisms to substrate surfaces. Kelvin probe force microscopy (KPFM) is a module of atomic force microscopy (AFM) that measures the contact potential difference between surfaces at the nano-scale. The combination of KPFM with AFM allows for the simultaneous generation of surface potential and topographical maps of biological samples such as bacterial cells. Here, we employ KPFM to examine the effects of surface potential on microbial adhesion to medically relevant surfaces such as stainless steel and gold. Surface potential maps revealed differences in surface potential for microbial membranes on different material substrates. A step-height graph was generated to show the difference in surface potential at a boundary area between the substrate surface and microorganisms. Changes in cellular membrane surface potential have been linked with changes in cellular metabolism and motility. Therefore, KPFM represents a powerful tool that can be utilized to examine the changes of microbial membrane surface potential upon adhesion to various substrate surfaces. In this study, we demonstrate the procedure to characterize the surface potential of individual methicillin-resistant Staphylococcus aureus USA100 cells on stainless steel and gold using KPFM.

Here, we present a protocol explaining the use of Kelvin probe force microscopy as a tool for generating high resolution nano-scale surface potential maps. This tool was applied to assess the role of surface potential on the binding capacity of microorganisms to substrate surfaces.

Surface potential is a commonly overlooked physical characteristic that plays a dominant role in the adhesion of microorganisms to substrate surfaces. ...

Measurement of Maximum Isometric Force Generated by Permeabilized Skeletal Muscle Fibers

Analysis of the contractile properties of chemically skinned, or permeabilized, skeletal muscle fibers offers a powerful means by which to assess muscle function at the level of the single muscle cell. Single muscle fiber studies are useful in both basic science and clinical studies. For basic studies, single muscle fiber contractility measurements allow investigation of fundamental mechanisms of force production, and analysis of muscle function in the context of genetic manipulations. Clinically, single muscle fiber studies provide useful insight into the impact of injury and disease on muscle function, and may be used to guide the understanding of muscular pathologies. In this video article we outline the steps required to prepare and isolate an individual skeletal muscle fiber segment, attach it to force-measuring apparatus, activate it to produce maximum isometric force, and estimate its cross-sectional area for the purpose of normalizing the force produced.

Analysis of the contractile properties of chemically skinned, or permeabilized, skeletal muscle fibers offers a powerful means by which to assess muscle function at the level of the single muscle cell. In this article we outline a valid and reliable technique to prepare and test permeabilized skeletal muscle fibers in vitro.

Analysis of the contractile properties of chemically skinned, or permeabilized, skeletal muscle fibers offers a powerful means by which to assess muscle ...

Atomic Force Microscopy Imaging and Force Spectroscopy of Supported Lipid Bilayers

Atomic force microscopy (AFM) is a versatile, high-resolution imaging technique that allows visualization of biological membranes. It has sufficient magnification to examine membrane substructures and even individual molecules. AFM can act as a force probe to measure interactions and mechanical properties of membranes. Supported lipid bilayers are conventionally used as membrane models in AFM studies. In this protocol, we demonstrate how to prepare supported bilayers and characterize their structure and mechanical properties using AFM. These include bilayer thickness and breakthrough force. 
The information provided by AFM imaging and force spectroscopy help define mechanical and chemical properties of membranes. These properties play an important role in cellular processes such as maintaining cell hemostasis from environmental stress, bringing membrane proteins together, and stabilizing protein complexes.

We describe a protocol for preparation of supported lipid bilayers and its characterization using atomic force microscopy and force spectroscopy.

Atomic force microscopy (AFM) is a versatile, high-resolution imaging technique that allows visualization of biological membranes. It has sufficient ...

Fluorescence Biomembrane Force Probe: Concurrent Quantitation of Receptor-ligand Kinetics and Binding-induced Intracellular Signaling on a Single Cell

Membrane receptor-ligand interactions mediate many cellular functions. Binding kinetics and downstream signaling triggered by these molecular interactions are likely affected by the mechanical environment in which binding and signaling take place. A recent study demonstrated that mechanical force can regulate antigen recognition by and triggering of the T-cell receptor (TCR). This was made possible by a new technology we developed and termed fluorescence biomembrane force probe (fBFP), which combines single-molecule force spectroscopy with fluorescence microscopy. Using an ultra-soft human red blood cell as the sensitive force sensor, a high-speed camera and real-time imaging tracking techniques, the fBFP is of ~1 pN (10-12 N), ~3 nm and ~0.5 msec in force, spatial and temporal resolution. With the fBFP, one can precisely measure single receptor-ligand binding kinetics under force regulation and simultaneously image binding-triggered intracellular calcium signaling on a single live cell. This new technology can be used to study other membrane receptor-ligand interaction and signaling in other cells under mechanical regulation.

We describe a technique for concurrently measuring force-regulated single receptor-ligand binding kinetics and real-time imaging of calcium signaling in a single T lymphocyte.

Membrane receptor-ligand interactions mediate many cellular functions. Binding kinetics and downstream signaling triggered by these molecular interactions ...