It has been shown that exposure to green (λ=550 nm, I=16 W/m2) fluorescent excitation light can induce force relaxation in living cells. Minimizing illumination time is critical to reducing light-induced force relaxation. We utilized a microscopic setup equipped with a mechanical shutter to evaluate the effect of short (<1s) exposure of living fibroblast cells to green light for one hour following the initial exposure. Cells were plated on polyacrylamide hydrogels embedded with dark red fluorescent nano particles which serve as fiducial markers. Motion of the nano particles was used to quantify change in cell force. Using a Zeiss Apotome Fluorescent Microscope with a Zeiss Axioobserver Z1 built-in reflected light shutter (<60 ms frequency), we exposed cells to green (λ=550 nm, I=16 W/m2) fluorescent excitation light for t=500 ms. Following this initial exposure, an image of the beads under each cell  was captured every 5 minutes for one hour with dark red fluorescent excitation light (λ=650 nm, I=8.6 W/m2), which does not induce force changes. A widefield fluorescent metal halide lamp (X-Cite® Series 120Q) was used in conjunction with Semrock Brightline FP (green) and Cy5 (red) filters to illuminate the sample. The video shows the one-hour time lapse sped up 3000x (0.1s = 5 min in real time).  The bead motion in the video illustrates that cells exposed to green light for only 500 ms do not exhibit widespread relaxation following exposure. The inward-outward-inward bead fluctuations show that localized cell forces continue to oscillate long after exposure to green fluorescent excitation light.  Reducing exposure time to 500 ms may eradicate the widespread, light-induced force relaxation commonly observed in cells following initial illumination.

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This image, taken with the LSM 710 confocal microscope, shows two embryoid bodies (EBs), which are spherical aggregates of pluripotent cells that can be differentiated into the three germ layers. The EBs in this image are composed of mouse embryonic stem cells that have been differentiated into motor neurons (green) and glia (red); nuclei are stained in blue. Over time, the cells extend neurites across the underlying collagen substrate to form synapses with each other. This research is supported by an NSF-funded STC, Emergent Behaviors of Integrated Cellular Systems (www.ebics.net).

This is an image of a neuromuscular junction in a rat larynx, imaged with the LSM 710 confocal system and using two photon second harmonic generation imaging microscopy. The nerve terminal (red) attaches to the motor endplate (green) together with the muscle (grey SHG image) imaged with a GaAsP BiG detector without any label. This image shows how the neuromuscular junction interacts with the muscles of the vocal fold to cause contractions after receiving signals from the brain. By imaging the neuromuscular junctions with a variety of instruments including superresolution techniques, this project aimed to study the effects of age on neuromuscular junctions.