Multiphoton intravital microscopy in small animals: motion artefact challenges and technical solutions.

Since its invention 29 years ago, two-photon laser-scanning microscopy has evolved from a promising imaging technique, to an established widely available imaging modality used throughout the biomedical research community. The establishment of two-photon microscopy as the preferred method for imaging fluorescently labelled cells and structures in living animals can be attributed to the biophysical mechanism by which the generation of fluorescence is accomplished. The use of powerful lasers capable of delivering infrared light pulses within femtosecond intervals, facilitates the nonlinear excitation of fluorescent molecules only at the focal plane and determines by objective lens position. This offers numerous benefits for studies of biological samples at high spatial and temporal resolutions with limited photo-damage and superior tissue penetration. Indeed, these attributes have established two-photon microscopy as the ideal method for live-animal imaging in several areas of biology and have led to a whole new field of study dedicated to imaging biological phenomena in intact tissues and living organisms. However, despite its appealing features, two-photon intravital microscopy is inherently limited by tissue motion from heartbeat, respiratory cycles, peristalsis, muscle/vascular tone and physiological functions that change tissue geometry. Because these movements impede temporal and spatial resolution, they must be properly addressed to harness the full potential of two-photon intravital microscopy and enable accurate data analysis and interpretation. In addition, the sources and features of these motion artefacts are varied, sometimes unpredictable and unique to specific organs and multiple complex strategies have previously been devised to address them. This review will discuss these motion artefacts requirement and technical solutions for their correction and after intravital two-photon microscopy.

Partial depletion of the proinflammatory monocyte population is neuroprotective in the myenteric plexus but not in the basal ganglia in a MPTP mouse model of Parkinson's disease.

Parkinson's disease (PD) patients often suffer from gastrointestinal (GI) impairments that are associated with the alteration of dopaminergic (DAergic) neurons in the myenteric nervous system. Growing evidence suggests that inflammation originating from the gut may have a major impact in both the initiation and progression of PD. Here, we investigated the role of the innate immune response in neurodegeneration occurring in central nervous system (CNS) and enteric nervous system (ENS) in response to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a neurotoxin that produces Parkinsonism in both humans and animal models. We found a strong immune response in the gut of mice treated with MPTP, as demonstrated by the prominent presence of macrophages derived from CD115(+) CD11b(+) Ly6C(Hi) monocytes, known as M1 monocytes, and increased production of IL-1ß and IL-6. Partial depletion of proinflammatory M1 monocytes through intravenous injections of clodronate-encapsulated liposome protects against MPTP-induced reduction of tyrosine hydroxylase (TH) expression in the ENS. In contrast, loss of striatal TH expression in the CNS after MPTP intoxication occurs regardless of partial monocyte depletion. Examination of brain tissue revealed that microglial activation, comprising the majority of the immune response in the CNS after MPTP injections is unaffected by M1 depletion. In vitro experiments revealed that MPTP and MPP(+) act directly on monocytes to elicit a proinflammatory response that is, in part, dependent on the MyD88/NF-κB signaling pathway resulting in nitrite and proinflammatory cytokine production. Taken together, our results demonstrate a critical role for proinflammatory M1 monocytes/macrophages in DAergic alterations occurring in the GI, but not in the brain, in the MPTP model of PD.

Wide-field multiphoton imaging of cellular dynamics in thick tissue by temporal focusing and patterned illumination.

Wide-field temporal focusing is a novel technique that provides optical sectioning for imaging without the need for beam scanning. However, illuminating over large areas greatly reduces the photon density which limits the technique applicability to small regions, precluding functional imaging of cellular networks. Here we present a strategy that combines beam shaping and temporal focusing of amplified pulses (>1 µJ/pulse) for fast imaging of cells from the central nervous system in acute slices. Multiphoton video-rate imaging over total areas as wide as 4800 µm(2) with an optical sectioning under 10 µm at 800 nm is achieved with our setup, leading to imaging of calcium dynamics of multiple cells simultaneously in thick tissue.

p56lck stably associates with a 115 kDa substrate.

Using antibodies directed against p56lck, we have identified a 115 kDa protein (p115) that is specifically immunoprecipitated with p56lck from whole cell lysates. The p56lck/p115 complex is stable in the presence of nonionic detergents. p115 becomes phosphorylated on tyrosine residues in p56lck immune-complex kinase assays. Treatment of whole cells with 12-O-tetradecanoyl phorbol-13-acetate decreases the subsequent tyrosine phosphorylation of p115 in immune-complex kinase assays.