Tirf microscope

The measurements were done on a commercial Olympus TIRF microscope (Olympus, USA), for the EGFR-EGFP excitation a 488 nm line of the argon ion laser was used. In the emission path a filter set for EGFP a HQ500/20 and 515 nm long pass filter split by a Q515lp dichroic mirror (Chroma Technology Corporation., Bellows Falls, VT-USA) was used. The signal was collected using an Olympus TIRF UIS2 PlanApo N 60× (NA = 1.45) oil-immersion objective and the fluorescence fluctuation was recorded by an EMCCD camera 512B Cascade (Photometrics, Tucson, AZ, USA). The frame rate was 20 ms and the image size 256×256 pixels with a pixel size of 139 nm. For both methods (iMSD and 2D-pCF) we needed to acquire at least 8192 frames which correspond to about 80 seconds of data acquisition. Cells were CHO-K1 cells transiently transfected with EGFR-EGFP plasmid (#32751) from Addgene (Cambridge,MA)[7 (link)] All date sets were detrended for bleaching using the exponential detrend function of the SimFCS Software. Bleaching affects the amplitude of the correlation function but much less the variance. However, in the specific data set used for the examples of this paper to total bleaching was about 10% from the initial to the final frame.

Murine osteoblast cells (OB-6) were cultured in α-MEM medium supplemented with 15% fetal bovine serum (FBS) and 1% penicillin/streptomycin. To assess the in vitro cytotoxicity of microparticles, they were first soaked in culture medium for 24 h and UV sterilized for 30 mins. The microparticles settled at the bottom of the media were then transferred into 24-well plate for cell seeding. The cells were seeded at a density of 10⁵ cells/ml to each well containing microparticles and without any microparticles (control). Medium was changed every 3 days and the cells were cultured up to 10 days for cytotoxicity assay. The viability of cells was indicated by the green fluorescence of calcein obtained by the enzymatic conversion of nonfluorescent cell-permeant acetomethoxy derivative of calcein and the dead cells were recognized simultaneously by the red fluorescence obtained by the binding of ethidium homodimer-1 to nucleic acids. The assay was performed on day 1, 3, 5, 7 and 10 according to the manufacturer’s protocol. The top surface of microparticle was imaged under fluorescence microscope (TIRF Microscope, Olympus) along with the bottom of well to determine the viable cells attached to the microparticle. ImageJ was used to determine the percent area of microparticles that was covered with cells on day 5 and 10.

Membrane TIRF was performed as described previously [3 (link),24 (link)]. Briefly, HKC11 cells were grown to 60% confluence in a dish with a collagen coated coverslip bottom (no. 1.5, MatTek, Ashland, MA). Cells were transfected with the indicated plasmids as described in FRET method. Samples were observed using an Olympus TIRF microscope equipped with a 60× 1.45 numerical aperture (NA) objective under the control of SlideBook software (version 4.2, Olympus, Center Valley, PA). Laser excitation was derived from a multiline argon ion laser run at the same current setting for all experiments. The power at the sample was controlled by a neutral density filter wheel. Excitation and emission wavelengths were selected using filter set for mCherry and GFP. The laser was aligned per the manufacturer’s instructions to achieve TIRF illumination. Images were taken using a Hamamatsu camera operating with 2 by 2 binning. Oxygen was provided by the ambient air which was supplemented by 5% CO₂ and warmed to 37°C in an environmental chamber surrounding the specimen.