par Martin Oheim
Laboratory of Neurophysiology & New Microscopies
CNRS UMR 8154, INSERM U603
Université Paris Descartes
Résumé
Fluorescence microscopic imaging, at least with area detectors or upon confocal detection, establishes a point-to-point relationship between a voxel in object space and a group of pixels in image space. It thus relies on ballistic photons and all light, on the excitation or emission side that is deviated from its direct trajectory contributes to image formation as unwanted blur and background, eventually resulting in a loss of contrast and detectability.
One case where the contribution of scattered excitation light has been largely overlooked is total internal reflection fluorescence microscopy, based on evanescent-field excitation. I will show examples of how forward scattering in propagation direction of the evanescent wave compromises excitation light confinement and image contrast and discuss remedies to this phenomenon, based on non-linear evanescent-field excitation or a spinning TIRF geometry. A second example is drawn from our work on deep two-photon fluorescence microscopy. In this case, the situation is radically different, because the confinement of fluorescence excitation to a femtoliter volume prescribes the collection of even multiply scattered photons. Thus, design considerations for the collection optical path of two-photon fluorescence microscopes radically differ from, e.g., confocal microscopes in that they must optimize rather than counteract scattered-light collection.
Invitation : Anne Sentenac (SEMO)
ResearchGate
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