Holographic multimode fibre endoscopes have recently shown their promising potential as a minimally invasive imaging tool, particularly in the field of neurobiology. Currently, their resolution is limited by diffraction, constrained by the relatively low numerical aperture of the multimode fibres used (typically less than 0.4). Overcoming the diffraction-limit barrier would open new possibilities for detailed observation of dendritic spines and their motility in-vivo.
In this presentation we demonstrate pulsed STED microscopy delivered through a holographic multimode fibre endoscope. We show resolution improvements over 3-fold of the diffraction limit. Moreover, we discuss and showcase its applicability to bio-imaging.
SignificanceHair-thin multimode optical fiber-based holographic endoscopes have gained considerable interest in modern neuroscience for their ability to achieve cellular and even subcellular resolution during in-vivo deep brain imaging. However, the application of multimode fibers in freely moving animals presents a persistent challenge as it is difficult to maintain optimal imaging performance while the fiber undergoes deformations.AimWe propose a fiber solution for challenging in-vivo applications with the capability of deep brain high spatial resolution imaging and neuronal activity monitoring in anesthetized as well as awake behaving mice.ApproachWe used our previously developed M3CF multimode-multicore fiber to record fluorescently labeled neurons in anesthetized mice. Our M3CF exhibits a cascaded refractive index structure, enabling two distinct regimes of light transport that imitate either a multimode or a multicore fiber. The M3CF has been specifically designed for use in the initial phase of an in-vivo experiment, allowing for the navigation of the endoscope’s distal end toward the targeted brain structure. The multicore regime enables the transfer of light to and from each individual neuron within the field of view. For chronic experiments in awake behaving mice, it is crucial to allow for disconnecting the fiber and the animal between experiments. Therefore, we provide here an effective solution and establish a protocol for reconnection of two segments of M3CF with hexagonally arranged corelets.ResultsWe successfully utilized the M3CF to image neurons in anaesthetized transgenic mice expressing enhanced green fluorescent protein. Additionally, we compared imaging results obtained with the M3CF with larger numerical aperture (NA) fibers in fixed whole-brain tissue.ConclusionsThis study focuses on addressing challenges and providing insights into the use of multimode-multicore fibers as imaging solutions for in-vivo applications. We suggest that the upcoming version of the M3CF increases the overall NA between the two cladding layers to allow for access to high resolution spatial imaging. As the NA increases in the multimode regime, the fiber diameter and ring structure must be reduced to minimize the computational burden and invasiveness.
SignificanceOver more than 300 years, microscopic imaging keeps providing fundamental insights into the mechanisms of living organisms. Seeing microscopic structures beyond the reach of free-space light-based microscopy, however, requires dissection of the tissue—an intervention seriously disturbing its physiological functions. The hunt for low-invasiveness tools has led a growing community of physicists and engineers into the realm of complex media photonics. One of its activities represents exploiting multimode optical fibers (MMFs) as ultra-thin endoscopic probes. Employing wavefront shaping, these tools only recently facilitated the first peeks at cells and their sub-cellular compartments at the bottom of the mouse brain with the impact of micro-scale tissue damage.AimHere, we aim to highlight advances in MMF-based holographic endo-microscopy facilitating microscopic imaging throughout the whole depth of the mouse brain.ApproachWe summarize the important technical and methodological prerequisites for stabile high-resolution imaging in vivo.ResultsWe showcase images of the microscopic building blocks of brain tissue, including neurons, neuronal processes, vessels, intracellular calcium signaling, and red blood cell velocity in individual vessels.ConclusionsThis perspective article helps to understand the complexity behind the technology of holographic endo-microscopy, summarizes its recent advances and challenges, and stimulates the mind of the reader for further exploitation of this tool in the neuroscience research.
Endoscopes and similar instruments use bundles of optical fibers to relay each pixel of an image from facet to facet. But even one of these multi-mode fibers supports enough modes to relay a complete image, the problem being that modal dispersion results in the image being scrambled. However, by treating the fiber as a complex aberration and applying corrective beam shaping it is possible to relay an image along the fiber length. Here we show that by beam-shaping of a pulsed laser we can produce a scanning spot at the distil end of the fiber and by measuring the time dependent intensity of the back-scattered light we can achieve 3D imaging. We demonstrate imaging up to 3m from the fiber with a lateral resolution of 60x60 pixels and a millimetric depth resolution. Such minimally invasive endoscopic 3D imaging has applications in healthcare and remote inspection.
Neuroscience research related to functionality, connectivity and metabolism of neuronal circuits, individual neuronal cells and sub-cellular structures, nowadays, experiences a burgeoning need to develop techniques for the detailed investigation inside the complexity of the living matter. Particularly, high-resolution observations combined with an extended depth of penetration in tissue represents an ongoing challenge.
Holographic control of light propagation in complex media opens a promising way to overcome this technological barrier via exploiting multimode fibres as hair-thin, minimally-invasive endoscopes. This concept allows for more than one order of magnitude reduction of the instrument’s footprint and a significant enhancement of imaging resolution, compared with current minimally invasive endoscopes.
Here, we demonstrate a compact and high-speed system for fluorescent imaging at the tip of a fibre. The instrument’s performance reaches micron-scale resolution across a field of view 50 micrometres, yielding 7-kilopixel image information at a rate of 3.5 frames per second. The resolution limit is dictated only by the numerical aperture of the fibre probe, and the contrast/pureness of the focal points, utilised for raster-scanning regime, approach the theoretical limits for phase-only holographic wavefront shaping.
The achieved performance allowed for in-vivo observations of neuronal somata and processes, residing deep inside the visual cortex and hippocampus of an animal model with minimal damage to the tissue surrounding the fibre penetration area.
We believe that this demonstration represents an important step towards implementations of various advanced forms of imaging through multimode fibre based endoscopes to address numerous key challenges in neuroscience.
We have developed an all-solid, step-index multimode fibre based on compound "soft-glasses" yielding a very-high NA reaching 0.96 at 1064nm. By further extending the methods of holographic control of light propagation in multimode fibres, we were able to mitigate the adverse effect of mode-dependent loss affecting the new fibre type. This enabled harnessing the full available NA almost completely, and demonstrating high-resolution focussing with output NAs up to 0.91 through lensless fibres. Further, we show that the NA and pureness of such foci allow stable three-dimensional optical confinement of micrometre-sized dielectric objects. Being inherently holographic, this technique is capable of generating an arbitrary number of optical tweezers, as well as precisely repositioning them independently in all directions. The versatility of the new instrument is demonstrated by simultaneous and dynamic 3D manipulation of large assemblies of dielectric microparticles, as well as manipulation of micro-objects inside optically inaccessible environments such a turbid cavity through an opening as small as 0.1mm.
Moreover, the possibility of generating aberration-free foci with NA approaching 0.9 across the fibre core opens new perspectives for high-resolution holographic micro-endoscopy, paving the way for the delivery of advanced microscopy techniques through hair-thin fibre-optic probes.
Digital micro-mirror devices (DMDs) have recently emerged as practical spatial light modulators (SLMs) for applications in photonics, primarily due to their modulation rates, which exceed by several orders of magnitude those of the already well-established nematic liquid crystal (LC)-based SLMs. This, however, comes at the expense of limited modulation depth and diffraction efficiency. Here we compare the beam-shaping fidelity of both technologies when applied to light control in complex environments, including an aberrated optical system, a highly scattering layer and a multimode optical fibre. We show that, despite their binary amplitudeonly modulation, DMDs are capable of higher beam-shaping fidelity compared to LC-SLMs in all considered regimes.
Multimode fibers are a promising tool for high resolution, low-cost, minimally invasive endoscopic imaging. The fiber can be used both to illuminate the sample, which may be buried deep inside the tissue, and to collect the backreflected light. Except for the bare fiber, no other imaging optics have to be inserted, enabling a device with a very small diameter. However, light propagating through the fiber is scrambled before it hits the sample. This renders straightforward imaging impossible, but if this scrambling is known with high accuracy, for instance because the transmission matrix has been measured, the scrambling process can be compensated before the light enters the fiber. For step index multimode fibers, where the refractive index profile consists of a cylindrical core with a constant but higher refractive index than the cladding, it has been shown that the transmission matrix can be predicted for any fiber orientation. Graded index fibers (GIF), where the refractive index profile resembles a parabola, offer numerous advantages, most prominently they are much less sensitive to bending. We measured the transmission matrix of a large GIF and show that we can fully understand the transmission matrix in terms of guided fiber modes, and simultaneously acquire accurate knowledge of the refractive index profile. We also show that although the quality of a commercially available graded index fiber is not sufficient to perform the same analysis, imaging performance of a graded index fiber is much more resilient to bending than the imaging performance of a comparable step index fiber. This demonstrates the need for a graded-index fiber with a high quality refractive index profile.
Using spatial light modulators(SLM) to control light propagation through scattering media is a critical topic for various applications in biomedical imaging, optical micromanipulation, and fibre endoscopy.
Having limited switching rate, typically 10-100Hz, current liquid-crystal SLM can no longer meet the growing demands of high-speed imaging. A new way based on binary-amplitude holography implemented on digital micromirror devices(DMD) has been introduced recently, allowing to reach refreshing rates of 30kHz.
Here, we summarise the advantages and limitations in speed, efficiency, scattering noise, and pixel cross-talk for each device in ballistic and diffusive regimes, paving the way for high-speed imaging through multimode fibres.
Access to the requested content is limited to institutions that have purchased or subscribe to SPIE eBooks.
You are receiving this notice because your organization may not have SPIE eBooks access.*
*Shibboleth/Open Athens users─please
sign in
to access your institution's subscriptions.
To obtain this item, you may purchase the complete book in print or electronic format on
SPIE.org.
INSTITUTIONAL Select your institution to access the SPIE Digital Library.
PERSONAL Sign in with your SPIE account to access your personal subscriptions or to use specific features such as save to my library, sign up for alerts, save searches, etc.