Two-photon excitation endoscopy through a multimode optical fiber

The vast number of propagating solutions to the wave equation in multimode optical fibers represents a larger information capacity than provided by fiber bundles of the same diameter. Therefore, in the field of imaging, multimode fibers potentially allow the transmission of images with higher resolution. However, image transmission through multimode fibers is not direct as in the fiber bundle case, in which each of the fiber cores can relay a portion of the distal image. In multimode fiber transmission, a distribution of intensity is scrambled in time and space by the propagating modes, leading to a speckle-like pattern that does not resemble the initial distribution. Here, we demonstrate two-photon excitation imaging of fluorescent beads through a multimode optical fiber. We show that our method maintains the advantages of two-photon excitation microscopy compared to single-photon excitation such as reduced photo-bleaching, deeper penetration depth and sectioning capability. Our method is based on time-gated digital phase conjugation, which allows the generation of focused pulses on the other side of a multimode fiber. To acquire an image, the focused femtosecond pulse is scanned in a three-dimensional mesh, producing two-photon excitation on each spatial location of the sample. By collecting the fluorescence through the fiber, a 3D two-photon image is reconstructed.

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

Delivery of ultrashort spatially focused pulses through a multimode fiber for two photon endoscopic imaging

Salma Farahi, Edgar Emilio Morales Delgado, Christophe Moser, Demetri Psaltis

Due to their high number of supported modes, multimode optical fibers carry large amount of spatio-temporal information. However, propagation of a light pulse through a multimode optical fiber suffers from spatial distortions due to superposition of the various exited modes and from time broadening due to modal dispersion. Here, we present a method based on digital phase conjugation to selectively excite specific optical fiber modes in a multimode fiber that follow similar optical paths as they travel through the fiber. In this way, they can be made to interfere constructively at the fiber output to generate an ultrashort spatially focused pulse. The excitation of a limited number of modes limits modal dispersion, allowing the transmission of an ultrashort pulse. We also show that the short spatially focused pulse can be scanned digitally without movable elements. We experimentally demonstrate that the pulse at the output of the multimode fiber generate a two-photon signal. We show delivery of a 1550 nm pulse with 500 fs duration, spatially focused to a spot size of 7 micrometers, through a 30 cm long, 200 micrometers core multimode step-index fiber. We show how this technique is applied to endoscopic two-photon imaging.

Spie-Int Soc Optical Engineering2015

Multimode fiber optical imaging using wavefront control

Nicolino Stasio

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.

EPFL2017

Multimode fiber optical imaging using wavefront control

Nicolino Stasio

EPFL2017