Integration winkelaufgelöster Streulicht-Messungen in die Mikroskopie lebender Zellen in zwei- und dreidimensionaler Kultur
Light scattering of small particles is characterized by its angular characteristics including specific oscillations, which reflect their size, shape and refractive index. This scattering behavior is now used for characterization of various types of cells or for measurement of morphological changes of cells undergoing necrosis or apoptosis. This work describes a conventional wide-field microscope which has been modified for backscattering experiments of living cells with high angular resolution, permitting simultaneous visual control of cells and their sub-structures. The method is based on focusing a laser beam at various positions of the aperture plane within the microscope objective lens, which results in a parallel beam hitting the sample under variable inclination. Backscattered light collected by the same objective lens is focused to an exit pupil, where an angle of 180° is selected by a pinhole, while the illumination angle can be varied from 190° - 257° with a precision of 0.5°. In comparison with control cells, 3T3 human fibroblasts cultivated as 2D monolayers show pronounced changes after initiation of apoptosis (by staurosporine) including shrinking and almost spherical shape. Concomitantly scattering intensity increases and exhibits pronounced oscillations, which are typical for Mie scattering. The scattering function and its angular oscillations are similar to spheres simulated with a refractive index n = 1.38 and 12.4 µm diameter (surrounded by water), in agreement with visual observation. When light scattering microscopy is applied to more realistic 3D multi-cellular spheroids, scattering profiles are more complex, but similar to those of 2D cell cultures. In first experiments for differentiation of tumorigenic and non-tumorigenic cells the scattering behavior of 3D spheroids of 3T3 fibroblasts changes upon infiltration of HeLa cervix carcinoma cells, thus indicating a potential of elastic light scattering experiments for tumor cell recognition.
Erstellung / Fertigstellung
Normierte SchlagwörterApoptosis [GND]
Light; Scattering [LCSH]
Optical measurements [LCSH]
Scattering, radiation [MeSH]