Light-based in-vivo brain imaging relies on light transport over large distances of highly scattering tissues. Scattering gradually reduces imaging contrast and resolution, making it difficult to reach structures at greater depths even with the use of multiphoton techniques. To reach deeper, minimally invasive endo-microscopy techniques have been established. These most commonly exploit graded-index rod lenses and enable a variety of modalities in head-fixed and freely moving animals. A recently proposed alternative is the use of holographic control of light transport through multimode optical fibres promising much less traumatic application and superior imaging performance. We present a 110 μm thin laser-scanning endo-microscope based on this prospect, enabling in-vivo volumetric imaging throughout the whole depth of the mouse brain. The instrument is equipped with multi-wavelength detection and three-dimensional random access options, and it performs at lateral resolution below 1 μm. We showcase various modes of its application through the observations of fluorescently labelled neurones, their processes and blood vessels. Finally, we demonstrate how to exploit the instrument to monitor calcium signalling of neurones and to measure blood flow velocity in individual vessels at high speeds.
Multimode fiber-based endoscopes have recently emerged as a tool for
minimally invasive endoscopy in tissue, at depths well beyond the
reach of multiphoton imaging. Here, we demonstrate label-free
second-harmonic generation (SHG) microscopy through such a fiber
endoscope. We simultaneously fully control the excitation polarization
state and the spatial distribution of the light at the fiber tip, and
we use this to implement polarization-resolved SHG imaging, which
allows imaging and identification of structural proteins such as
collagen and myosin. We image mouse tail tendon and heart tissue,
employing the endoscope at depths up to 1 mm, demonstrating that we
can differentiate these structural proteins. This method has the
potential for enabling instant and in
situ diagnosis of tumors and fibrotic conditions in sensitive
tissue with minimal damage.
Microendoscopes based on optical fibres have recently come to the fore as promising candidates allowing in-vivo observations of otherwise inaccessible biological structures in animal models. Despite being still in its infancy, imaging can now be performed at the tip of a single multimode fibre, by relying on powerful holographic methods for light control. Fibre based endoscopy is commonly performed en face, resulting in possible damage of the specimen owing to the direct contact between the distal end of the probe and target. On this ground, we designed an all-fibre probe with an engineered termination that reduces compression and damage to the tissue under investigation upon probe insertion. The geometry of the termination brings the field of view to a plane parallel to the fibre’s longitudinal direction, conveying the probe with off-axis imaging capabilities. We show that its focusing ability also benefits from a higher numerical aperture, resulting in imaging with increased spatial resolution. The effect of probe insertion was investigated inside a tissue phantom comprising fluorescent particles suspended in agarose gel, and a comparison was established between the novel side-view probe and the standard en face fibre probe. This new concept paves the way to significantly less invasive deep-tissue imaging.
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