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Annular Beam Shaping in Multiphoton Microscopy to Reduce Out-of-Focus Background

Artikel i vetenskaplig tidskrift
Författare Johan Borglin
Danni Wang
N. J. Durr
Dag Hanstorp
A. Ben-Yakar
Marica B Ericson
Publicerad i International Journal of Spectroscopy
ISSN 1687-9449
Publiceringsår 2017
Publicerad vid Institutionen för kemi och molekylärbiologi
Institutionen för fysik (GU)
Språk en
Länkar dx.doi.org/10.1155/2017/7560141
Ämnesord SPATIAL LIGHT-MODULATOR, FLUORESCENCE MICROSCOPY, HUMAN SKIN, IN-VIVO, EXCITATION, APERTURE
Ämneskategorier Spektroskopi, Atom- och molekylfysik och optik

Sammanfattning

Despite the inherent spatial confinement of multiphoton processes that arises from focusing through an objective, the maximum imaging depth in conventional multiphoton microscopy is ultimately limited by noise from out-of-focus fluorescence. This is particularly evident when imaging beyond shallow depths in highly scattering tissue as increased laser powers are necessary. The out-of-focus signal originates from multiphoton processes taking place primarily at shallow depths and deteriorates contrast and limits imaging depth. In this paper, annular laser beams are explored as a concept to reduce this background signal in multiphoton microscopy. The approach is theoretically verified by data from simulations and proof of principle is demonstrated on a custom-built experimental multiphoton microscopy platform. Annular laser beams were created by adopting wavefront control using a spatial light modulator and implemented for imaging tissue phantoms simulating turbid media and human skin ex vivo. The signal-to-background ratios were calculated and compared to images acquired with a traditional, filled-aperture Gaussian beam. Experiments in tissue phantom show an improvement in signal-to-background ratio of about 30% when using annular beam illumination in comparison to Gaussian illumination at specific depths. When laser power is not the limiting factor, this approach is expected to provide even greater benefits.

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