Concept and instrumentation of the dual biomembrane force probe (dBFP). (A) Schematic of a spatial crosstalk dual receptor system. Ligands 1 and 2 are presented by the respective Probes 1 and 2 to the cell at spatially separate locations. The signal initiated upon binding of Receptor 1 at one location travels a distance to activate Receptor 2 at another location. (B) Schematic of a temporal crosstalk dual receptor system. Ligands 1 and 2 are presented by the respective Probes 1 and 2 to the cell at temporally separate times. The signal initiated upon the binding of Receptor 1 upregulates Receptor 2, which remains upregulated after a period. (C–E) Images of the hardware system, consisting of two micropipettes (LP1, LP2) on the left side (C and D), the reaction chamber (C–E) and the other two micropipettes (RP1, RP2) on the right side (D and E).

Analyzing spatial crosstalk between TCR and LFA-1. (A–C) Dual ligand co-presentation setup and results. A T cell aspirated by LP1 was repetitively touched by a functionalized RBC (Probe 1) aspirated by RP1 (A). Probe 1 was coated with a mixture of pMHC and ICAM-1 to engage TCR and LFA-1 simultaneously (B). Adhesion frequency vs. contact duration curves of naïve OT1 CD8⁺ T cells tested against Probe 1 coated with ICAM-1 alone (purple), mutant OVA (OVA:H2-K^bα3A2) alone (blue), or a mixture of mutant OVA and ICAM-1 (C, red, n ≥ 3 cells for each point). (D–G) Dual ligand separate presentation setup and results. The setup is the same as in A, except the addition of a bead (Probe 2) aspirated by RP2 to make constant contact with the T cell at a distal site from the Probe 1 contact site (D). Binding to the ICAM-1-bearing Probe 1 reports the LFA-1 activity upregulated by TCR engagement with pMHC coated on Probe 2. (E). Representative spike trains of adhesion events between a T cell and an ICAM-1-bearing Probe 1 in 190 cycles. After the first 40 cycles of rare adhesions (blue spikes), Probe 2 coated with SA (left) or wild-type (WT) OVA (OVA:H2-K^b) (right) was moved to constantly contact with the T cell (green arrow), and Probe 1 continued to touch the T cell repeatedly for another 150 cycles of similarly rare (left) or much more frequent (right) adhesions (red spikes) (F). Time course of averaged adhesion frequency between T cell and ICAM-1-bearing Probe 1 calculated by binning the raw adhesion sequences with a 20-touch (72 s) window (G). The number of cells tested were: SA = 3 (gray), mutant OVA = 6 (purple), WT OVA = 7 (red), and WT OVA without Ca²⁺/Mg²⁺  = 3 (blue). Symbols *, $ and # indicate different groups of comparisons: WT OVA/MT OVA vs. SA, WT OVA vs. mutant OVA, and WT OVA vs. WT OVA without Ca²⁺/Mg²⁺, respectively. Single, double and triple symbols denote p < 0.05, 0.01 and 0.001, respectively, by Student t-test. Scale bars are 5 μm in all micrographs.

Analyzing temporal crosstalk between GPIb and GPIIb-IIIa. (A,B) dBFP setup for switching between interactions of the target cell with two immobilized ligands. Two beads functionalized with distinct proteins were attached to the apexes of two RBCs respectively aspirated by LP1 and LP2 to form Probes 1 and 2. A platelet held by RP1 was aligned against one probe at a time to sequentially engage two ligands, designated as the pre- (A) and post- (B) switch positions, respectively. Scale bars are 5 μm. (C–E) Schematics of the switching assay for studying temporal crosstalk within a platelet dual-receptor system (GPIb to GPIIb-IIIa). In the pre-switch position, the platelet was driven to repeatedly engage the first receptor GPIb with its ligand VWF-A1 coated on Probe 1 and exert a controlled pulling force on the A1–GPIb bonds (C, top; D). Intraplatelet Ca²⁺ signals triggered by GPIb mechanoreception were observed for 200 s (C, color gradient transition, middle). The subsequent upregulation of GPIIb-IIIa was probed by its ligand fibronectin (FN) on Probe 2 (C, bottom; E). (F–H) Concurrent measurement of platelet GPIb (receptor 1) triggered Ca²⁺ flux and bond kinetics. Top: Representative epi-fluorescence pseudo-colored images of intraplatelet Ca²⁺ in a platelet touched by BSA (non-adhesive control) (F) and VWF-A1 under ramped (1000 pN/s) (G) and clamped (25 pN) (H) forces at indicated times. Bottom: Representative time courses of normalized Ca²⁺ intensity (color matched curves and legends). The concurrent measurements of bond lifetime events (symbol) and the cumulative lifetime (purple curve) for the platelet exhibiting Ca²⁺ fluxes are overlaid. I. Pre- and Post-stimulation adhesion frequencies of platelet GPIIb-IIIa (receptor 2) binding to beads functionalized with FN. n ≥ 5 platelets were tested for each condition. *denotes p < 0.01 by Student t-test.

Analyzing temporal crosstalk between P₂Y receptors and CD62p. (A,B) Setup for switching between interactions of the target cell with a soluble and an immobilized ligand. It is similar to that in Fig. , B in both the pre- (A) and post- (B) switch positions except that Probe 1 is replaced by an open micropipette of large orifice (5–10 μm) as an agonist tank. Scale bars are 5 μm. (C–E) Schematics of the switch assay for investigating the upregulation of an activation marker (CD62p) by signaling via an agonist receptor (P₂Y). An aspired platelet was immersed into the tank micropipette filled with ADP (50 µM) in the pre-switch position (C, top), which activates the platelet through P2Y receptors (D). The Ca²⁺ flux inside the stimulated platelet was monitored for 600 s (C, middle), followed by the switching to examine the platelet activation marker CD62p by an anti-CD62p antibody coated on Probe 2 (E). (F) Representative normalized Ca²⁺ level vs. time. (G) Adhesion frequency (P _a) ratio between Post- vs Pre-stimulation for platelet CD62p binding to beads functionalized with the anti-CD62p antibody (AK4). P _a ratio for the “Pre-Stim” group equals 1. The results of stimulation by ADP tank immersion and immobilized VWF-A1 under a 25 pN clamped force (as set up in Fig. ) are compared. (H) FACS measurements of platelet CD62p expression between Post- vs Pre-stimulation by soluble ADP (50 µM) and VWF-A1 (100 µg/ml) (see Methods). Results depict the % of gated platelets positive for anti-CD62p antibody binding. Data were presented as the mean ± s.e.m. from n ≥ 3 platelets for dBFP and n = 3 experiments for FACS under each condition. ** and *** denote p < 0.01 and 0.001, respectively, by Student t-test.