Abstract B11: Label-free hyperspectral microscopy detects alterations in nanoscale cellular structure with high temporal resolution
Authors: Chandler John E. , Almassalha Luay M. , Backman Vadim 
Published: March 2017

We have previously shown that analysis of the spectral variance of a reflected light microscope image allows direct study of cellular nanoscale organization through a technique called Partial Wave Spectroscopic (PWS) microscopy. This information is produced by measuring the spectral variations in light scattering to quantify the refractive index fluctuations, which reflect the distribution of macromolecular structures, within a biological sample with length scale sensitivity on the order of 20-200nm. Previously, this approach has been used for the early detection of tumors and the structure-function study of chromatin changes in cells. Here we present a state-of-the-art system utilizing this spectroscopic principle to develop a label-free, live-cell light microscopy imaging method capable of sensing temporal changes in the nanoscale structural organization of live cells called Live-cell PWS microscopy. This system is built into a commercial epi-fluorescence live-cell microscope and data is acquired using a hyperspectral camera (IMEC), which simultaneously collects 16 spectral bands in the visible spectrum with each exposure at rates up to ~100 frames per second. In addition, the design of this instrument allows simultaneous phase contrast and hyperspectral nanoscopic measurements. Phase contrast data can be acquired simultaneously with the hyperspectral data by providing phase contrast transmission illumination within one of the cameras 16 collection bands and using a corresponding notch filter for that band on the epi-illumination source. Calculating the spectral variance of each hyperspectral data cube yields images with label-free contrast of the cells where the intensity corresponds to the heterogeneity of nanoscale macromolecular organization within each diffraction limited voxel. Using this configuration, we can simultaneously acquire both phase contrast and the nanoscale sensitive hyperspectral videos of rapidly evolving structural changes in live cells without the need of any exogenous labels.

To demonstrate the unique capabilities of this system, we perform a range of experiments to induce changes in cellular ultrastructure at rapid time-scales. In this work, we will show the changes in the cellular nanoarchitecture upon (1) addition of a glucose to live HeLa cells, (2) during chemical fixation by paraformaldehyde, and (3) upon cell exposure to UV-radiation. In these experiments, we observed immediate (&lt;100ms) changes in nanoscale structural organization throughout the cell, with distinct structural changes differing between the cytoplasm and nucleus. For instance, incubation of glucose results in immediate transformation of the nuclear organization, causing a rapid decrease in nanoscale heterogeneity, which then quickly returned to pretreatment levels. Comparatively in the cytoplasm, addition of glucose results in significantly increased macromolecular mass-density heterogeneity upon treatment, which remains elevated even after the nucleus had recovered to pre-treatment levels. Likewise, we also recorded the rapid changes induced by chemical fixation with 4% paraformaldehyde solution in HeLa cells. In this case, we observe a gradual homogenization of the nanoscale structure as we lose clear delineation of the nucleus from the cytoplasm after fixation. This observation can be attributed to the randomized cross-linking process of the fixative yielding a more homogenous nanoscale structural organization than the non-random structures present prior to fixation in the cells. Finally, we explore the real-time effects of UV-light exposure on cellular behavior and the chromatin nanoarchitecture. These experiments demonstrate that rapidly evolving changes in the native unlabeled nanostructure of cells can be studied with high temporal resolution making this a unique tool for studying the nanoscale cellular structure-function relationship in real-time.

Citation Format: John E. Chandler, Luay M. Almassalha, Vadim Backman. Label-free hyperspectral microscopy detects alterations in nanoscale cellular structure with high temporal resolution. [abstract]. In: Proceedings of the AACR Special Conference on Engineering and Physical Sciences in Oncology; 2016 Jun 25-28; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2017;77(2 Suppl):Abstract nr B11.