User:Yeem/20.309/Mod 3 lab report

Fluorescence microscopy

White light characterization

We know that there are 600 lines per mm on the Ronchi ruling. Under 40x magnification, we counted 11 pixels from center of one line to the next. Therefore our calculated resolution was approximately 138 nm/pixel at 40x magnification. Our field of view was 640x480 pixels, or about 88um by 66um. Our actual observed resolution was such that the Ronchi ruling could not be resolved at 10x magnification.

Fluorescence characterization

Due to the brightness of our laser, we were forced to use a polarizer to reduce the intensity of the excitation light sufficiently to avoid saturating the detector. After taking our rhoadmine distribution, the 4um spheres were so bright that dividing out the rhodamine distribution had no effect on our processed image.

Actin imaging

Cytochalasin D is a potent inhibitor of actin polymerization. Cyto D binds to actin filaments, blocking the addition of actin monomers. Cells treated with cyto D are unable to rearrange their actin cytoskeletons, preventing movement and other functions. These cells eventually fall apart.

In our experiments, we observed less filamentous actin in cells treated with cyto D. Whereas the untreated images contain large amounts of wispy, elongated actin, the fibroblasts incubated with cyto D appeared contracted. Treated 3T3 cells were also noticeably fewer in number, suggesting that some had undergone apoptosis.

Microrheology

Methodology
We used mostly self authored code, except for generating our plots. These were performed using Heejin's code, albeit heavily modified.

Our tracking script read individual frames of movies, preprocessed them by applying a Gaussian filter, and returned trajectories of specified beads.

Calibration data for our microscope stability resulted in a mean squared displacement (MSD) of about 57 nm².

Results The movie for one of our cyto D treated cells was unusable due to apparent apoptosis of the cell over the course of recording. Another clearly showed shifting of beads within the cells visualized and returned an enormous MSD.