Dynamic Cellular Measurements

In a recent case, cardiac myocytes from 1-2 day old neonatal Sprague Dawley (Harlan, Indianapolis, IN) rats were prepared at the University of Arizona and plated onto #1 round coverslips. After 2 weeks of incubation the cells were measured in a Bioptechs FCS3 perfusion chamber with an aluminized base plate and a 100μm gasket. Cells were kept at 37°C with an HBSS (Hank’s balanced salt solution) fluid bath. Figure 3 shows OT maps of an area of cells imaged at 40X (20X with 2X FOV lens) before and after pushing IPHC (isoproterenol hydrochloride – a beta adrenergic agonist).

The images below are the first in two time series of 200 datasets taken at 15 fps over a 13.3 sec time period. These images are the same 349 x 326 pixel subarea of 1050x1200 images. The optical volume of the subareas is determined by summing all the OT values and then scaling so that both traces have the same mean and same relative scaling. The actual physical volume is not obtainable from these data because the thickness of the cell culture and the index of refraction data are unknown.

A major advantage of this technique is that relative physical changes can easily be measured. The figure below shows the relative optical volume for these two time series before and after pushing IPHC. These cells spontaneously beat (or twitch) about once every 4 seconds when in the HBSS at 37°C. After the IPHC is pushed, the beating frequency increases by a factor of 8 to twice a second and the flexion is about 3 times stronger. The optical volume is getting smaller in this subarea when the cells beat because the cells are noticeably stretching out and expanding during the beats.

These measurements show how quantitative optical phase and optical volume measurements can be
used to study a group of cells over time.