Periscope for noninvasive two-photon imaging of murine retina in vivo

Stremplewski, Patrycjusz; Komar, Katarzyna; Palczewski, Krzysztof; Wojtkowski, Maciej; Palczewska, Grażyna

doi:10.1364/boe.6.003352

Cited by 22 publications

(19 citation statements)

References 29 publications

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“…Moreover, since neuroinflammatory processes are lasting days, weeks, or even months, whereas typical imaging time-windows in intravital microscopy are several hours, technologies allowing for repeated imaging in one and the same animal are requested. The potential of retinal two-photon imaging has been underlined by several recent publications (16, 18); however, to our knowledge, this is the first report demonstrating repeated time-lapse imaging of the retina, allowing us to monitor cellular dynamics, cellular interactions, and tissue function longitudinally in one mouse. Our approach reduces the number of animals needed for experiments and decreases inter-individual variance due to different responses to immunization across mice.…”

Section: Discussionmentioning

confidence: 91%

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Longitudinal Intravital Imaging of the Retina Reveals Long-term Dynamics of Immune Infiltration and Its Effects on the Glial Network in Experimental Autoimmune Uveoretinitis, without Evident Signs of Neuronal Dysfunction in the Ganglion Cell Layer

et al. 2016

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A hallmark of autoimmune retinal inflammation is the infiltration of the retina with cells of the innate and adaptive immune system, leading to detachment of the retinal layers and even to complete loss of the retinal photoreceptor layer. As the only optical system in the organism, the eye enables non-invasive longitudinal imaging studies of these local autoimmune processes and of their effects on the target tissue. Moreover, as a window to the central nervous system (CNS), the eye also reflects general neuroinflammatory processes taking place at various sites within the CNS. Histological studies in murine neuroinflammatory models, such as experimental autoimmune uveoretinitis (EAU) and experimental autoimmune encephalomyelitis, indicate that immune infiltration is initialized by effector CD4+ T cells, with the innate compartment (neutrophils, macrophages, and monocytes) contributing crucially to tissue degeneration that occurs at later phases of the disease. However, how the immune attack is orchestrated by various immune cell subsets in the retina and how the latter interact with the target tissue under in vivo conditions is still poorly understood. Our study addresses this gap with a novel approach for intravital two-photon microscopy, which enabled us to repeatedly track CD4+ T cells and LysM phagocytes during the entire course of EAU and to identify a specific radial infiltration pattern of these cells within the inflamed retina, starting from the optic nerve head. In contrast, highly motile CX3CR1+ cells display an opposite radial motility pattern, toward the optic nerve head. These inflammatory processes induce modifications of the microglial network toward an activated morphology, especially around the optic nerve head and main retinal blood vessels, but do not affect the neurons within the ganglion cell layer. Thanks to the new technology, non-invasive correlation of clinical scores of CNS-related pathologies with immune infiltrate behavior and subsequent tissue dysfunction is now possible. Hence, the new approach paves the way for deeper insights into the pathology of neuroinflammatory processes on a cellular basis, over the entire disease course.

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Section: Discussionmentioning

confidence: 91%

“…In studies using the technology, either adaptive optics (16) or a periscope-based setup (18) was used to visualize changes to the RPE in various mouse models. However, the complexity of the optical setup limited the imaging results to single static images.…”

Section: Introductionmentioning

confidence: 99%

Longitudinal Intravital Imaging of the Retina Reveals Long-term Dynamics of Immune Infiltration and Its Effects on the Glial Network in Experimental Autoimmune Uveoretinitis, without Evident Signs of Neuronal Dysfunction in the Ganglion Cell Layer

et al. 2016

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“…Such studies will be necessary to evaluate the full benefits of using a contact lens for imaging with beams of various sizes and at different retinal eccentricities, and help to inform the design of future mouse retinal imaging systems. Nevertheless, our results suggest that application of AO for mouse retinal imaging might be needed only if higher lateral resolution or precise axial sectioning than reported in this Letter is desired, or for efficient nonlinear (e.g., two-photon [16]) optical imaging. Based on these results, we also conclude that high lateral resolution mouse retinal SLO systems require an active axial focusing capability.…”

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confidence: 95%

Effect of scanning beam size on the lateral resolution of mouse retinal imaging with SLO

et al. 2015

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Scanning laser ophthalmoscopy (SLO) employs the eye’s optics as a microscope objective for retinal imaging in vivo. The mouse retina has become an increasingly important object for investigation of ocular disease and physiology with optogenetic probes. SLO imaging of the mouse eye, in principle, can achieve submicron lateral resolution thanks to a numerical aperture (NA) of ~0.5, about 2.5 times larger than that of the human eye. In the absence of adaptive optics, however, natural ocular aberrations limit the available optical resolution. The use of a contact lens, in principle, can correct many aberrations, permitting the use of a wider scanning beam and, thus, achieving greater resolution then would otherwise be possible. In this Letter, using an SLO equipped with a rigid contact lens, we report the effect of scanning beam size on the lateral resolution of mouse retinal imaging. Theory predicts that the maximum beam size full width at half-maximum (FWHM) that can be used without any deteriorating effects of aberrations is ~0.6 mm. However, increasing the beam size up to the diameter of the dilated pupil is predicted to improve lateral resolution, though not to the diffraction limit. To test these predictions, the dendrites of a retinal ganglion cell expressing YFP were imaged, and transverse scans were analyzed to quantify the SLO system resolution. The results confirmed that lateral resolution increases with the beam size as predicted. With a 1.3 mm scanning beam and no high-order aberration correction, the lateral resolution is ~1.15 μm, superior to that achievable by most human AO-SLO systems. Advantages of this approach include stabilization of the mouse eye and simplified optical design.

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“…Alternative approaches to coherence-gated wavefront sensing using a virtual Shack-Hartmann sensor have also been reported3637. The combination of coherence-gated detection with a lenslet-based Shack-Hartmann wavefront sensor was reported as well3839. A limitation of these methods is the added optical and electronics hardware complexity required for implementing the wavefront measurement.…”

Section: Discussionmentioning

confidence: 99%

Coherence-Gated Sensorless Adaptive Optics Multiphoton Retinal Imaging

Cua

Wahl

Zhao

et al. 2016

Sci Rep

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Multiphoton microscopy enables imaging deep into scattering tissues. The efficient generation of non-linear optical effects is related to both the pulse duration (typically on the order of femtoseconds) and the size of the focused spot. Aberrations introduced by refractive index inhomogeneity in the sample distort the wavefront and enlarge the focal spot, which reduces the multiphoton signal. Traditional approaches to adaptive optics wavefront correction are not effective in thick or multi-layered scattering media. In this report, we present sensorless adaptive optics (SAO) using low-coherence interferometric detection of the excitation light for depth-resolved aberration correction of two-photon excited fluorescence (TPEF) in biological tissue. We demonstrate coherence-gated SAO TPEF using a transmissive multi-actuator adaptive lens for in vivo imaging in a mouse retina. This configuration has significant potential for reducing the laser power required for adaptive optics multiphoton imaging, and for facilitating integration with existing systems.

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Periscope for noninvasive two-photon imaging of murine retina in vivo

Cited by 22 publications

References 29 publications

Longitudinal Intravital Imaging of the Retina Reveals Long-term Dynamics of Immune Infiltration and Its Effects on the Glial Network in Experimental Autoimmune Uveoretinitis, without Evident Signs of Neuronal Dysfunction in the Ganglion Cell Layer

Longitudinal Intravital Imaging of the Retina Reveals Long-term Dynamics of Immune Infiltration and Its Effects on the Glial Network in Experimental Autoimmune Uveoretinitis, without Evident Signs of Neuronal Dysfunction in the Ganglion Cell Layer

Effect of scanning beam size on the lateral resolution of mouse retinal imaging with SLO

Coherence-Gated Sensorless Adaptive Optics Multiphoton Retinal Imaging

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