Gfp filter set

TIRF microscopy was combined with an AFM-based SCFS (CellHesion200) mounted on an inverted microscope (Observer.Z1, Zeiss, Germany) with a × 100/1.45 a Plan-FLUOR objective (Zeiss). TIRF illumination was achieved by coupling a beam emitted by a solid-state laser (Sapphire 488 LP, 50 mW, Coherent) into a single mode fibre (coupler: HPUC-2-488-4.5AS-11, fibre: QPMJ-A3A, 3S-488-3.5/125-SAS-4, OZ Optics) connected to a slider TIRF condenser (Laser TIRF, Zeiss). An optimized GFP filter set (Chroma Technology Corp.) and 10% laser power using quantum dots (Crystalplex, USA) was used for TIRF. Images were recorded using a camera (Evolve, Photometrics) and imaging software (Axiovision, Zeiss). The experimental setup for TIRF combined SCFS using paxillin-GFP, lifeact-mCherry expressing pKO-αVβ1, pKO-αV and pKO-β1 fibroblasts was as described above except for an extended contact time of 500 s. TIRF images were acquired at initial 5 s and thereafter at 20 s intervals for the fibroblast attached to the cantilever, brought in contact with FNIII7-10.

All images were collected on an Axio Observer Z1 microscope (Carl Zeiss Microscopy, LLC), which is equipped with a halogen lamp for bright-field mode, and a 120 W Metal Halide lamp (Lumen Dynamics Group, Inc., model: X-Cite 120PC Q) for fluorescence excitation. The microscope is fully automated, including a linear-encoded x-y translation stage (Ludl Electronics Products, Ltd., model: 96S108-LE), filter wheel, shutters, and is equipped with a CoolSNAP HQ camera (Photometrics, 6.45 µm pixels, 1392×1040 resolution). A 63× glycerol-immersion, phase-contrast objective (N.A. 1.3) was used to collect both phase contrast and fluorescence images. A GFP filter-set (Chroma Technology Corp., set 49002) with the excitation band centered at 470 nm (full-width of 40 nm) and emission band centered at 525 nm (full-bandwidth of 50 nm) was used to image Venus-expressing cells with an exposure time of 75 ms. The computer-controllable ONIX microfluidic system (EMD Millipore, model: EV-262) was used to trap cells, provide them with a continuous flow of fresh media, and to change media during the experiment.
All hardware control and image-processing is performed on a PC running Windows 7 with 4 GB of RAM, and a dual core, 32-bit Intel i5 processor.

Cells were incubated in the dark in Tyrode’s solution supplemented with 10 μg/ml IB4 conjugated to Alexa Fluor^® 488 dye (Invitrogen Life Technologies) for 10–12 min. After washout fluorescent images were captured during the first 15 min of each experiment (before prolonged calcium imaging) using a standard GFP filter set (Chroma Technology, Bellows Falls, VT). Cells demonstrating bright fluorescence and halo around the neuronal plasma were considered as IB4−positive (IB4+), whereas those having intensity below 20% of maximum for selected field of view were considered as IB4−negative (IB4−).