title: High-resolution intravital microscopy.
abstract: Cellular communication constitutes a fundamental mechanism of life, for instance by permitting transfer of information through synapses in the nervous system and by leading to activation of cells during the course of immune responses. Monitoring cell-cell interactions within living adult organisms is crucial in order to draw conclusions on their behavior with respect to the fate of cells, tissues and organs. Until now, there is no technology available that enables dynamic imaging deep within the tissue of living adult organisms at sub-cellular resolution, i.e. detection at the level of few protein molecules. Here we present a novel approach called multi-beam striped-illumination which applies for the first time the principle and advantages of structured-illumination, spatial modulation of the excitation pattern, to laser-scanning-microscopy. We use this approach in two-photon-microscopy--the most adequate optical deep-tissue imaging-technique. As compared to standard two-photon-microscopy, it achieves significant contrast enhancement and up to 3-fold improved axial resolution (optical sectioning) while photobleaching, photodamage and acquisition speed are similar. Its imaging depth is comparable to multifocal two-photon-microscopy and only slightly less than in standard single-beam two-photon-microscopy. Precisely, our studies within mouse lymph nodes demonstrated 216% improved axial and 23% improved lateral resolutions at a depth of 80 µm below the surface. Thus, we are for the first time able to visualize the dynamic interactions between B cells and immune complex deposits on follicular dendritic cells within germinal centers (GCs) of live mice. These interactions play a decisive role in the process of clonal selection, leading to affinity maturation of the humoral immune response. This novel high-resolution intravital microscopy method has a huge potential for numerous applications in neurosciences, immunology, cancer research and developmental biology. Moreover, our striped-illumination approach is able to improve the resolution of any laser-scanning-microscope, including confocal microscopes, by simply choosing an appropriate detector.
authors.affiliation: LaVision Biotec GmbH, Bielefeld, Germany.
authors.forename: Volker
authors.initials: V
authors.lastname: Andresen
authors.forename: Karolin
authors.initials: K
authors.lastname: Pollok
authors.forename: Jan-Leo
authors.initials: JL
authors.lastname: Rinnenthal
authors.forename: Laura
authors.initials: L
authors.lastname: Oehme
authors.forename: Robert
authors.initials: R
authors.lastname: Günther
authors.forename: Heinrich
authors.initials: H
authors.lastname: Spiecker
authors.forename: Helena
authors.initials: H
authors.lastname: Radbruch
authors.forename: Jenny
authors.initials: J
authors.lastname: Gerhard
authors.forename: Anje
authors.initials: A
authors.lastname: Sporbert
authors.forename: Zoltan
authors.initials: Z
authors.lastname: Cseresnyes
authors.forename: Anja E
authors.initials: AE
authors.lastname: Hauser
authors.forename: Raluca
authors.initials: R
authors.lastname: Niesner
mesh_terms: D000818:Animals; D001402:B-Lymphocytes / Q000502:physiology*; D007107:Immune System / Q000502:physiology*; D051379:Mice; D018613:Microscopy, Confocal / Q000379:methods*; D038761:Photobleaching