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Figure 2 | Particle and Fibre Toxicology

Figure 2

From: Darkfield-Confocal Microscopy detection of nanoscale particle internalization by human lung cells

Figure 2

Detection of fluorescent polystyrene spheres by co-localized confocal and darkfield microscopy. A. Fluorescent polystyrene beads of the indicated sizes were illuminated with 488 nm laser light. Emitted particle fluorescence was detected using the confocal microscope in the green channel while scattered incident light collected by the darkfield condenser was simultaneously observed via the transmitted light detector. For the 140 nm spheres, insets in the lower left-hand corner show an enlarged area for clarity. B. Three dimensional colocalization analysis of a 500 nm fluorescent polystyrene sphere imaged by DF-CLSM. The sphere shown was excited by 488 nm laser light while simultaneous monitoring for fluorescence and scattered light occurred via the green and transmitted light channels. Each set of images shows the XY, XZ, and YZ orientation for the combined, green (CLSM), and transmission (DF) channels, respectively. The large crosshairs represent the same point in space across all the axial views. C. Variability in spatial localization of DF images obtained with multiple wavelengths of light. Shown are 10x pseudo-colored images of the same 27 nm TiO2 particle illuminated by 404 (Blue), 488 (Green), and 561 (Red) laser light. Each set of images depicts the X, Y, and Z orientation for the transmission (DF) channel. For the combined view, areas of overlap in the 488 and 561 excitations are observed in yellow. The arrows represent the midpoint of the same particle illuminated at each wavelength, showing a small x,y lateral distortion between the 488 and 561 nm excitation lines, while both the 488 and 561 have a much larger axial (z) dispersions from the 404 excitation line. Note the large separation between the blue exciation and the visible laser exciation lines. 60x Plan Fluor, Magnification 600X + 1, 5, or 10X zoom as designated.

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