INTRODUCTION
PRINCIPLES
The biomembrane force probe
1 Biomembrane force probe instrumentation. A Picture of a home-made BFP. It is built based on an inverted microscope. B Schematic of the necessary parts required to build a BFP. Optional parts are indicated. The experimental chamber and the micromanipulators are positioned on the mechanical stage |
2 Setup of the mechanical stage and the experimental chamber. A The mechanical stage. Three micromanipulators are installed in the mechanical stage, controlling the probe, target and helper micropipettes respectively. Piezoes are mounted on the target micromanipulator which can provide movement control in nanometer resolution. The chamber holder is placed on the stage at the position right above the objective lens. B Schematics of the chamber holder. The experimental chamber was formed by the facing edges of the opposite metal plates of the holder, and the coverslips glued on the top and the bottom of the metal plates, leaving the other two sides open to allow the insertion of the micropipettes. In the experiment, the experimental buffer, the RBC, beads, and the cell or droplet samples are injected into the chamber, and the two open sides of the chamber are sealed with mineral oil. The mineral oil can prevent the experimental buffer from evaporation. If temperature control is required in the experiment, a revised version of the chamber holder (right) can be manufactured |
3 The micropipettes system for target and probe aspiration. A Image of the whole view field in the BFP experiment. The probe micropipette first aspirates a swollen biotinylated RBC, then an SA-bead was attached at the opposite side of the RBC by the helper micropipette. A cell or liquid droplet is aspirated by the target micropipette whose movement is controlled by a Piezo actuator. B Measureing the spring constant of the RBC. A camera images the whole view field including the micropipette, the RBC and the bead. The radii of the probe micropipette tip (Rp), the RBC (R0) and the binding interface of the RBC/SA-bead (Rc) can then be measured. Together with the pressure differences Δp detected by the pressure sensor, the spring constant kp of the RBC system could be calculated |
4 Measuring RBC deformation in BFP. A–C In BFP, a high speed camera images a thin line at the boundary between RBC and the SA-bead (cyan in Panel A, purple in Panel B, and yellow in Panel C). The intensity profiles of the images (insets) are captured and analyzed in the unit of the pixel of the camera. The profile of no force condition (A) serves as a reference for RBC conformation, and that for pushing (B) or pulling (C) can be compared with the reference. D Gaussian distribution fitting of the intensity profile of each image indicates the deformation of the RBC in pixel (ΔX1 and ΔX2), which can be converted to distance change (Δd) by multiplying the resolution of each pixel |
Elasticity measurement with BFP
5 Measuring the Young’s modulus of LLPS droplets. A A droplet is aspirated by the target micropipette. An experimental cycle of the droplet approaching, pressing and retracting from the bead on the probe. B A typical force curve generated from the experimental cycle shown in Panel A, the approaching phase is shown in red. C In the pressing process (red), the force and time of starting and ending points can be used to calculate the Young’s modulus |
MATERIAL, EQUIPMENT AND SOFTWARE
Buffers, reagents and other supplies
Equipments
Softwares
EXPERIMENTAL PROCEDURE
Force probe (human RBCs) preparation
LLPS droplet preparation
Micropipettes preparation
Preparation of the experimental chamber
Micromanipulation and equipment setting
Data acquisition
6 Young’s modulus of LLPS droplets measured with BFP. A Representative force curves of two LLPS droplet samples measured with BFP. The Young’s modulus of the droplets can be calculated by using the starting and ending points indicated on the curves. For each sample, hundreds of such measurements will be performed. B, C Histogram of the Young’s modulus of the softer Sample 1 (B) and the harder Sample 2 (C), Gaussian distribution fitting (solid red curve) gives the optimal Young’s modulus of the samples, ~0.5 kPa and ~4.8 kPa respectively |