Coolled pe 300

FITC-labeled BSM-Tz was covalently
grafted on PS as described above (PS/BSM¹), and the coated
surface was washed with PBS for two days at 4 °C before fluorescent
imaging. Scratches on the coating were made by a pipette tip to show
the difference between the PS substrate and mucin coating. Fluorescent
images of PS/BSM¹ were taken with a fluorescence microscope
(Nikon Eclipse Ti, equipped with CoolLED pE-300) with a 20× objective.
For PAAm, we first labeled this with Alexa Fluor 647 hydroxylamine
(ThermoFisher, A30632) via 5 mM EDC/NHS (in PBS of pH 7.4) coupling,
and the molar ratio between Alexa Fluor 647 hydroxylamine and acrylic
acid in PAAm is 1:100 to get enough carboxylic acid groups to remain
for grafting mucin. The labeled PAAm was washed with PBS for two days
at room temperature, and then FITC-labeled BSM-Tz was covalently grafted
on the labeled PAAm as described above (PAAm/BSM¹). The
cross section of PAAm/BSM¹ was imaged with a confocal microscope
(Zeiss LSM 900-Airy2) with a 20× objective.

Phase contrast images of macrophages cultured on the surfaces for
one day were taken with a fluorescence microscope (Nikon Eclipse Ti,
equipped with CoolLED pE-300). Images from the same positions of each
well were taken, and then the cell clusters were analyzed by a CellProfiler
(4.2.0) based on two independent cell experiments in triplicate for
each sample. To study the cell attachment after culturing on the surfaces
for one day, the cell medium was taken out, and then the well was
washed with 100 μL of PBS without mixing and taken out immediately.
The remaining cells on the surfaces were lysed with a lysis buffer,
and then the DNA amount was measured by a Qubit 1× dsDNA HS Assay
Kit (ThermoFisher, Q33230) under the provided instructions of Qubit
4. The DNA ratio between the washed surface and unwashed surface was
calculated, and it is applied as the ratio of attached cells.

Imaging was conducted using an inverted microscope (Nikon Ti-U), equipped with a water immersion objective (Nikon Plan APO VC, 60×, NA = 1.2) and LED EPI illumination (CoolLED pE-300), enabling rapid alternation between transmitted and EPI illumination modalities. The transmitted mode was employed to assess the integrity of GUV membranes, while fluorescence illumination served to quantify the cargo content within the GUVs. Optical tweezers (Tweez, Aresis d.o.o., Slovenia, Nd:YAG laser, λ = 1064 nm) with acousto-optic deflectors (AOD) for beam steering were integrated into the microscope. The laser power was measured by a power meter Coherent PM USB PS10.
Shockwaves were triggered by focusing the laser on optically opaque polystyrene microparticles (Dynabeads M-270 Streptavidin, ThermoFisher Scientific, nominal diameter d_p = 2.8 μm), which had a streptavidin coating to enable binding to biotin molecules in the GUV membrane. A high-speed camera (Photron SA-Z type 2100K-M-64GB) was used for evaluation of the microparticle response to laser illumination.

The SICM setup was comprised of an Axon MultiClamp 700B amplifier, MM-6 micropositioner (Sutter Instrument, Novato, CA) and a P-753 Linear actuator (Physik Instrumente, Irvine, CA) attached to the pipette holder to allow precise, three-dimensional movement of the micropipette (Fig. 1a). SICM software was used to control the positioning and topographical scanning capabilities of the SICM system (ICAPPIC, London, UK). An Eclipse Ti2 confocal microscope (Nikon Instruments Inc., Melville, NY) and broad-spectrum LED illumination system (pE-300 CoolLED, Andover, USA) were used for bright-field (BF) and immunofluorescence (IF) visualisation of mitochondria and myonuclei, in skeletal muscle fibres, to ensure efficient biopsy. The SICM system is used to automatically approach the skeletal muscle tissue, moving the micropipette just above the region of interest (Fig. 1b), which is then inserted into the skeletal muscle fibre through manual control (Fig. 1c). Following successful biopsy of mitochondria, the micropipette was retracted and the tip snapped into a 0.2-ml microfuge tubes containing lysis buffer (Fig. 1d) as described in the “Laser capture microdissection (LCM) and tissue lysis” section.