Structural and biological origins of light scattering in cells and tissue are still poorly understood. We demonstrate how this problem might be addressed through the use of transmission electron microscopy (TEM). For biological samples, TEM image intensity is proportional to mass-density, and thus proportional to refractive index (RI). By calculating the autocorrelation function (ACF) of TEM image intensity of a thin-section of cells, we essentially maintain the nanoscale ACF of the 3D cellular RI distribution, given that the RI distribution is statistically isotropic. Using this nanoscale 3D RI ACF, we can simulate light scattering through biological samples, and thus guiding many optical techniques to quantify specific structures. In this work, we chose to use Partial Wave Spectroscopy (PWS) microscopy as a one of the nanoscale-sensitive optical techniques. Hela cells were prepared using standard protocol to preserve nanoscale ultrastructure, and a 50-nm slice was sectioned for TEM imaging at 6 nm resolution. The ACF was calculated for chromatin, and the PWS mean sigma was calculated by summing over the power spectral density in the visible light frequency of a random medium generated to match the ACF. A 1-µm slice adjacent to the 50-nm slice was sectioned for PWS measurement to guarantee identical chromatin structure. For 33 cells, we compared the calculated PWS mean sigma from TEM and the value measured directly, and obtained a strong correlation of 0.69. This example indicates the great potential of using TEM measured RI distribution to better understand the quantification of cellular nanostructure by optical methods.
|Original language||English (US)|
|Title of host publication||Proc. SPIE - Biophysics, Biology and Biophotonics II|
|Subtitle of host publication||the Crossroads|
|Editors||Adam Wax, Vadim Backman|
|State||Published - May 1 2017|
Li, Y., Cherkezyan, L., Zhang, D., Almassalha, L., Roth, E., Chandler, J., Bleher, R., Subramanian, H., Dravid, V. P., & Backman, V. (2017). Nanoscale chromatin structure characterization for optical applications: a transmission electron microscopy study . In A. Wax, & V. Backman (Eds.), Proc. SPIE - Biophysics, Biology and Biophotonics II: the Crossroads (Vol. 10075)