Multimode fiber optical imaging using wavefront control

Optical microscopy enables us to observe at high resolution and with a large field of view objects otherwise invisible for the naked eye. This is why it is becoming a fundamental tool in biology and in diagnostics, where observation at cellular level can tell about the health status of a tissue. At visible wavelengths, light undergoes scattering when propagating through tissues, limiting the imaging depth to the first cellular layers of a biological sample. Endoscopic approaches allow the bypass of the scattering layers and move the accessible volume centimeters inside tissues, ideally keeping large field of view and high-resolution capabilities. In this thesis we explore the combination of wavefront control and ultrathin multimode optical fibers to develop a new family of endoscopes able to generate images with micrometric resolution being, at the same time, minimally invasive. We demonstrate that a variety of imaging techniques can be implemented in optical fiber-based endoscopy, maximizing the amount of information that can be collected through a few hundreds of micrometers-thick probe. Firstly, we describe digital phase conjugation (DPC), which is the utilized wavefront shaping technique to convert a multimode optical fiber in an ultrathin endoscope. The use of DPC in synergy with multimode optical fibers resulted in ultrathin probes able to perform fluorescence imaging, photoacoustic imaging, and multiphoton imaging. Moreover, we show that using saturation excitation of fluorescent samples we can beat the resolution limit imposed by the numerical aperture of the optical fiber and obtain optical sectioning. In the first part of the thesis we describe which steps have been performed towards the implementation of these techniques. In the second part, we exploit coherent fiber bundles as imaging probes. This kind of fiber is typically utilized in endoscopy, but their resolution is limited by the distance between the fiberâs cores. Here we show two techniques - one based on DPC and a second based on statistical properties of speckle patterns - to obtain images with a better resolution than the one imposed by the core-to-core spacing, both relying on a unique property of multicore fibers: the memory effect. We also accomplished complex pattern transmission through multicore fibers with high resolution. Overall, the presented work provides new insights in imaging through optical fibers based on wavefront shaping techniques, opening up pathways for addressing current issues in this research field.

Control of pulsed light propagation through multimode optical fibers

Edgar Emilio Morales Delgado

Visualization of organs and cells in the interior of living beings is a challenging task due to the light absorption and multiple light scattering occurring in biological tissue that prevents the di-rect transmission of images. A standard visualization approach is based on the use of endo-scopes which is accomplished through apertures in the body. Since their invention, the light transmission capability of optical fibers has enabled fiber optic based endoscopy with devices based on bundles of optical fibers that can directly relay images. Due to their small diameter, fiber optic endoscopes are standard in minimally invasive applications either with a white light illumination or as a fluorescent imaging device. The principle behind fluorescent endoscopy consists on the scanning of light intensity over a fluorescent sample, which is achieved by se-quential illumination of each one of the fiber bundles or by mechanical scanning of a single-mode double-cladding fiber. Collecting the fluorescent signal through the light guiding medium, an image can be reconstructed. The main drawback of fiber bundle endoscopes is the pixelated limited resolution given by the presence of thousands of cores that conform the bundle. In the double cladding fiber endoscopes, there is a limit to the miniaturization of the endoscope due to the requirement of mechanical elements at the tip of the fiber to scan the illumination over the sample. In this thesis, methods for focusing and digital scanning of optical light pulses through multimode optical fibers without any distal mechanical element are developed. Such techniques are based on spatial light modulation. Spatial phase modulation allows the control of light propagation in scrambling media, as in a multimode fiber, enabling the generation of intensity light foci or arbitrary intensity profiles. The high intensity of the transmitted pulses permits multi-photon imaging through multimode optical fibers, resulting in imaging probes of higher resolution than in the case of fiber bundles. We demonstrate in a working prototype that this approach provides all the advantages of multi-photon microscopy at the tip of an ultra-thin probe, such as a reduced photo bleaching of the sample, optical sectioning and enhanced image contrast. The developed methods can also be employed for material processing that requires high in-tensity light pulses. In specific, we demonstrate additive manufacturing, also known as 3D printing, at the tip of an ultra-thin needle. The working principle is based on two-photon polymerization, which is accomplished by scanning intense light pulses on a photosensitive material that hardens when exposed to light. All other additive manufacturing methods require very large nozzles or components in close proximity to the structure that they build, up to now. With an ultra-thin 3D printing probe we enable micro-fabrication through very small apertures, in places of difficult access or in the interior of living beings. We call it, endofabrication.

EPFL2017

Two-photon fluorescence imaging through multicore fiber with digital phase conjugation

Donald Benjaman Conkey, Christophe Moser, Demetri Psaltis, Nicolino Stasio

We present near diffraction limited two photon fluorescence (TPF) imaging through a lensless, multi-core fiber (MCF) endoscope utilizing digital phase conjugation. The ultra-small size of MCFs make them desirable tools for imaging deep into the body. TPF imaging enables optical sectioning and is widely used in brain and biological imaging and is a desired modality for fiber endoscopes. Previous implementations of TPF imaging through MCFs focus and scan the light from individual cores for image formation. In such systems the resolution is limited by the MCF core spacing, although a lens may be used to improve the resolution at the expense of the field of view. Other, more recent work has improved the resolution limitation using custom built MCFs for focusing and scanning of ultrafast pulses using wavefront shaping. Here we present digital phase conjugation for ultrafast pulse focusing through a MCF for an imaging resolution independent of the MCF core spacing. Furthermore, the phase conjugation technique does not require the use of a lens at the fiber end for focus formation and is compatible with commercially available MCFs with a large number of cores. Here, we present a 3000 core MCF endoscope and demonstrate ultrafast pulse focusing with sufficient focus spot contrast and power for TPF endoscope imaging. We construct TPF images by digital scanning of the phase conjugated focus on the target object and collection of the emitted fluorescence through the MCF. This work demonstrates the viability of digital conjugation combined with commercially available MCFs for higher resolution lensless, two photon endoscopy.

Spie-Int Soc Optical Engineering2016

Coherent imaging from bacteria to multicellular organisms

Séverine Coquoz

Structural and functional imaging of cells, tissues and organisms is crucial for understanding biomedical processes. Fluorescence microscopy is an established tool and has contributed to many discoveries in the life sciences. This technique provides molecular contrast but has limitations inherent to the fluorescent probes, namely phototoxicity and photobleaching. Phase microscopy provides a label-free alternative. Contrast is achieved by translating the phase variations induced by the sample into measurable intensity variations. While phase contrast and differential interference contrast microscopy offer a means to generate contrast without exogenous contrast agents, quantitative phase imaging (QPI) techniques have emerged, enabling the measurement of the phase delays. This measurement yields information on the thickness, refractive index and dry mass of the sample. QPI is thus a powerful tool and has proven its use in various biomedical applications. In the first part of this thesis, new concepts for extending the capabilities of QPI and applications exploiting them are presented. We implement piezo-based Fourier phase microscopy, exhibiting high spatial and temporal sensitivities and resolutions for label-free imaging of cell dynamics at various time scales. The phase is retrieved via phase-shifting intereferometry using a piezo-actuated module allowing very fast tunable phase modulation. Several examples of applications are described. Furthermore, we exploit the phase-to-mass equivalence for investigating the growth of uropathogenic E. coli and the effect of different antibiotics, thereby providing new insights into fundamental mechanisms of bacterial growth. Because QPI methods are limited to optically thin and weakly scattering objects, the second part of this thesis focuses on optical coherence microscopy (OCM). OCM is an interferometric technique providing 3D images of highly scattering biological samples with micrometric resolution and penetration depth of up to several hundreds of micrometers. Compared to optical coherence tomography, OCM uses high numerical aperture (NA) optics to achieve higher transverse resolution, leading to a decrease of the depth of field (DOF). Several methods have been developed to overcome this trade-off between resolution and DOF. Extended-focus OCM (xfOCM) provides a particularly interesting solution by illuminating the sample with a Bessel beam. Moreover, interferometric synthetic aperture microscopy (ISAM) achieves depth-independent resolution through an approximate solution to the inverse scattering problem. We develop extended ISAM (xISAM) to combine the benefits of both approaches, and demonstrate its potential on simulated and experimental data. We also propose a new application of visible OCM (visOCM), a xfOCM system using visible light and a high-NA objective to achieve 3D imaging with sub-micrometer spatial resolution. The capabilities of visOCM are well suited for 3D in vivo imaging of C. elegans, an extensively used model organism in biomedical research. We demonstrate that the anatomy of C. elegans can be visualized with high speed and high contrast down to the sub-cellular level. We further show that visOCM can be employed for imaging age-related morphological changes. Altogether, the methods developed in this thesis pave the way to many applications in the life sciences by featuring label-free imaging at various temporal and spatial scales. Several promising applications are pursued.

EPFL2017

Control of pulsed light propagation through multimode optical fibers

Edgar Emilio Morales Delgado

EPFL2017

Coherent imaging from bacteria to multicellular organisms

Séverine Coquoz

EPFL2017