We recently developed a novel optical method for observing submicron intracellular structures in living cells which is
called confocal light absorption and scattering spectroscopic (CLASS) microscopy. It combines confocal microscopy, a
well-established high-resolution microscopic technique, with light scattering spectroscopy (LSS). CLASS microscopy
requires no exogenous labels and is capable of imaging and continuously monitoring individual viable cells, enabling the
observation of cell and organelle functioning at scales on the order of 100 nm with 10 nm accuracy. To demonstrate the
ability of the CLASS microscope to monitor unstained living cells on submicrometer scale we studied human bronchial
epithelial cells undergoing apoptosis. Fluorescence microscopy of living cells requires application of molecular markers
which can affect normal cell functioning. CLASS microscopy is not affected by this avoiding potential interference of
fluorescence molecular markers with cell processes. In addition, it provides not only size information but also
information about the biochemical and physical properties of the cell. CLASS microscopy can provide unique
capabilities for the study of cell interactions with the environment, cell reproduction and growth and other functions of
viable cells, which are inaccessible by other techniques.
We recently developed a new microscopic optical technique capable of noninvasive analysis of cell structure and cell
dynamics on the submicron scale . It combines confocal microscopy, a well-established high-resolution microscopic
technique, with light scattering spectroscopy (LSS) and is called confocal light absorption and scattering spectroscopic
(CLASS) microscopy. CLASS microscopy requires no exogenous labels and is capable of imaging and continuously
monitoring individual viable cells, enabling the observation of cell and organelle functioning at scales on the order of
To test the ability of CLASS microscopy to monitor cellular dynamics in vivo we performed experiments with human
bronchial epithelial cells treated with DHA and undergoing apoptosis. The treated and untreated cells show not only
clear differences in organelle spatial distribution but time sequencing experiments on a single cell show disappearance of
certain types of organelles and change of the nuclear shape and density with the progression of apoptosis.
In summary, CLASS microscopy provides an insight into metabolic processes within the cell and opens doors for the
noninvasive real-time assessment of cellular dynamics. Noninvasive monitoring of cellular dynamics with CLASS
microscopy can be used for a real-time dosimetry in a wide variety of medical and environmental applications that have
no immediate observable outcome, such as photodynamic therapy, drug screening, and monitoring of toxins.
Introduction: Light Scattering Spectroscopy has been a recently developed as a non-invasive technique capable of sizing the cellular organelles. With this technique, we monitor the heat-induced sub-cellular structural transformations in a human RPE cell culture.
Material and Methods: A single layer of human RPE cells (ATCC) was grown on a glass slide. Cells are illuminated with light from a fiber-coupled broadband tungsten lamp. The backscattered (180 degree) light spectra are measured with an optical multichannel analyzer (OMA). Spectra are measured during heating of the sample.
Results: We reconstructed the size distribution of sub-micron organelles in the RPE cells and observed temperature-related changes in the scattering density of the organelles in the 200-300nm range (which might be peroxisomes, microsomes or lysosomes). The sizes of the organelles did not vary with temperature, so the change in scattering is most probably due to the change in the refractive indexes. As opposed to strong spectral variation with temperature, the total intensity of the backscattered light did not significantly change in the temperature range of 32-49 °C.
Conclusion: We demonstrate that Light Scattering Spectroscopy is a powerful tool for monitoring the temperature-induced sub-cellular transformations. This technique providing an insight into the temperature-induced cellular processes and can play an important role in quantitative assessment of the laser-induced thermal effects during retinal laser treatments, such as Transpupillary Thermal Therapy (TTT), photocoagulation, and Photodynamic Therapy (PDT).