We demonstrated a novel technique to improve the signal to noise ratio (SNR) of the frequency domain interferogram of a dual comb spectroscopy (DCS) setup, by about 5 dB, based on temporal shifting of the interfering pulse trains in the two channels. The experimental setup utilized a fiber based DCS architecture where electro-optic modulators (EOM) were used to generate two optical combs (or pulse trains). Due to this carefully adjusted periodic phase shifts, the interferogram now was time-multiplexed, or in other words included a larger number of peaks within a given time frame compared to when an unshifted case. There is nevertheless a tradeoff between the peak power and the bandwidth of the interferogram comb envelope in the spectral domain. The experimental results were also confirmed numerically and a relationship between the SNR improvement and the rate of phase shifting was established. These results open new possibilities in SNR improvement of EOM based multidimensional spectroscopic techniques and provide a powerful resource to execute sparse sampling and other complex techniques to maximize the amount of useful information in the interferogram data of a DCS setup.
The lensless endoscope represents the ultimate limit in miniaturization of imaging tools: an image can be transmitted through an optical fiber by numerical or physical inversion of the fiber's pre- measured transmission matrix. We present here a novel fiber-optic component, a "tapered multi-core fiber (MCF)", designed for integration into ultra-miniaturized endoscopes for minimally invasive two-photon point-scanning imaging. This new design addresses the power delivery issue that has faced MCF based lensless endoscopes. We achieve experimentally a factor 6.0 increase in two-photon signal yield while keeping the ability to point-scan by the memory effect. We report two-photon fluorescent imaging of cells and neurons with these improved MCF tapered fibers.
Complex touch panel displays development is requiring high performance glass cutting techniques. Femtosecond lasers, combined to Bessel beam generation based on reflective axicons already showed quality and efficiency improvements, while being able to handle high peak and average power.
We described here recent developments for high quality Bessel beam generation using a fully reflective system. This complex Bessel beam presents an intensity plateau along its propagation axis, being twice more homogeneous and having a five times sharper tail compared to a classical Bessel beam. This development paves the way to complex and selective multi-layer glass cutting.
The ability to combine incoherent sources with attractive performances enable hardware integration issues to be resolved using stable, good quality off-the-shelf components. Some new generation imaging systems can be found in the mid-infrared (MIR). The most portable laser technology at this range, our Quantum Cascade Laser source can provide light power of around 2 W, industrial grade.
With the Multi-plane Light Conversion technique and a modal approach, we present non-coherent beam combiner for QCL with optimal beam quality, demonstrating the state of the art in terms of M2.
Raman microscopy has been widely developed for label free nonlinear optical microscopy of biological systems. Imaging speed in these systems is hampered by low Raman scattering cross sections and the requirement of scanning a focused laser beam through the sample in conventional Raman microscopy. The serial acquisition that is necessary in point scanning microscopy slows image acquisition and limits the dwell time are each image pixel. Here, we discuss two new imaging methods that are based on spatial frequency modulation imagining (SPIFI) [1-2], where a structured line focus is used to image is used to image specimens by collecting light on a single pixel detector. We discuss the use of SPIFI to improve the imaging speed of Spontaneous Raman scattering and coherent anti-Stokes Raman scattering microscopy. A detailed noise analysis highlighting the advantages and disadvantages of SPIFI as compared to conventional point scan imaging is presented.
The lensless endoscope represents the ultimate limit in miniaturization of imaging tools: an image can be transmitted through a (multi-mode or multi-core) fiber by numerical or physical inversion of the fiber's pre- measured transmission matrix. However, the transmission matrix changes completely with only minute conformational changes of the fiber, which has so far limited lensless endoscopes to fibers that must be kept static. In this work we report a lensless endoscope which is exempt from the requirement of static fiber by designing and employing a custom-designed conformationally invariant fiber. We give experimental and theoretical validations and determine the parameter space over which the invariance is maintained.
We develop a high speed compressive Raman imaging technology using a programmable binary spectral filter and a single channel detector to perform fast Raman detection and concentration estimation of know species over millimeters field of view. The technology is x100 times faster than commercial CCD based systems and x10 times faster than the EMCCD based systems. We report fast imaging of breast micro-calcifications, SERS particles, pharmaceutical tablets and micro-plastics. We also present a novel fast line scan compressive Raman imaging technique using spatial frequency-modulated illumination (SPIFI) that enables to encode space into the frequency domain to acquire single shot line images. We demonstrate the imaging and classification of three different chemical species at line scan rates of 40 Hz.
Conventional CARS microscopy requires scanning a point focus through the specimen limits imaging speed. We present a spatial frequency projection imaging (SPIFI) method for CARS microscopy to spatially multiplex CARS microscopy. A spinning disk modulator is used to rapidly modulate the Stokes field with a rapidly swept spatially periodic transmission grating. SPIFI-CARS images are obtained by Fourier transforming the single pixel signal. Images of CARS and second harmonic generation from histological slices will be presented. The physics of image formation and the impact of multiplexing on SNR will be discussed. Prospects for scaling to high speed CARS imaging will be discussed.
Multicore fiber bundles are attractive candidates for lensless endscopes because of their ruggedness and the relative simplicity involved in their calibration and operation. Nevertheless, the measurement of the transmission matrix of the MCF is still an interferometric process, typically requiring high stability and possibly sequential measurements. A key challenge is to replace this with a much simpler and robust technique – phase retrieval. In this talk, we will examine the major challenge in using phase retrieval in conventional MCFs. This is related to the discrete and periodic nature of the auto-correlation of an ordered MCF, resulting in the stagnation of phase retrieval algorithms in one of a multitude of local minimums. We employ phase diversity, i.e. more the one complex illumination pattern with a known phase profile can help overcome this issue. In particular, we identify that spiral phase patterns are well-suited due to their generation of complementary speckle patterns, resulting in highly non-redundant information. We experimentally demonstrate that three intensity images are sufficient to retrieve the transmission matrix with very high accuracy and success rates. Furthermore, we will also present a novel disordered MCF, which facilitates phase retrieval with a single intensity image and a priori knowledge of the core positions. This is a simple and rapidly converging method which relies on the aperiodic arrangement and the sparsity of the cores. Both these computationally inexpensive techniques highlight the potential of phase retrieval as a tool for robust phase calibration of fiber bundles in lensless imaging.
A class of miniaturized imaged systems based on multicore fibers (MCFs) and wavefront shaping, also known as lensless endoscopes, have emerged as promising candidates for non-invasive imaging deep inside the tissue. At the current stage of their development, these systems already provide features like pixelation-free wide-field and point-scanning imaging and are compatible with the majority of nonlinear imaging techniques, offering diffraction-limited resolution over a field of view only limited by the single fiber numerical aperture.
Widely spaced single-mode cores in such an MCF are designed for very weak inter-core coupling, which ensures an infinite memory effect and provides good ultrashort pulse delivery framework for nonlinear imaging. In a true endoscopic setting, however, where the fiber geometry is subject to continuous deformation (at a rate from several Hz to several tens of Hz), results in inter-core phase and group delay dispersion (GDD) that changes as the fiber bends. This consequently degrades the PSF in terms of size and power in the focal spot, eventually rendering impossible to produce non-linear imaging.
We addressed this issue by implementing an active measurement and compensation loop at the proximal end of an MCF, allowing to correct in real time the GDD changes and to minimize the temporal dispersion of the delivered laser pulses. We evaluate this approach against a passive scheme where a static pre-compensation is used in a compliment with a specifically designed MCF, allowing to drastically increase the MCF bending robustness.
Non-linear optical microscopy proves to be an indispensable tool in natural sciences and becomes more and more attractive for clinical applications. Coherent Raman scattering, for instance, has the potential to become an in-vivo fast label-free histology tool as its chemical selectivity provides quantitative information on lipids and proteins locations and concentrations in tissues. Along with these techniques, second-harmonic generation of collagen and 2-photon excitation fluorescence broaden even more the non-linear imaging ability as collagen fibers represent an important role in human body construction. Whilst 2-photon excitation fluorescence allows to study auto-fluorescence (ex. NADH and NADHP molecules), and to excite a vast range of chromophores. However, absorption and scattering limit significantly the nonlinear imaging depth into tissues. As a solution, we offer a flexible, compact, and multimodal nonlinear endoscope (2.2 mm outer diameter, 35 mm rigid length) based on a resonantly piezo scanned hollow-core negative curvature double-clad fiber. The fiber design allows distortion-less, background-free delivery of femtosecond excitation pulses and the back-collection of nonlinear signals through the same fiber. The double-cladding of this fiber attends 10^5μm of silica collection surface which allows for a 4-fold collection improvement compared to previously used Kagomé hollow core fibers. Having a good control on the resonantly scanning fiber the endoscope can perform nonlinear imaging up to 8 frames per second over a field of view of 400μm. We demonstrate 2photon, SHG and CARS imaging in ex vivo gastric human tissue samples and in-vivo 2-photon fluorescence imaging of GFP-labeled neurons in mouse brain.
Lensless endoscopes have generated a great deal of interest in the development of minimally invasive probes for imaging in sensitive and hitherto inaccessible regions as found in deep brain imaging. Lifting the requirement for the opto-mechanical elements at the distal end of the fiber reduces the footprint of the endoscope down to the fundamental limit, the fiber itself. Our approach, using specially designed multicore fibers allows us to i) generate two-photon fluorescence contrast with femtosecond pulses, ii) image at high speeds with resonant scanners, iii) simple and non-interferometric calibration schemes and iii) exhibits a high resilience to spatio-temporal distortion of the focus due to fiber bending. In this contribution, we will discuss how novel designs of the MCFs can lift several of the instrumental complexity typically associated wavefront shaping and high speed imaging. We show that the use of sparse arrays of fiber cores can provide pixelation-free imaging with no artifacts when employed in the wavefront domain as opposed to conventional fiber bundles. Furthermore, we examine the unique properties of the MCFs which allow for fast and non-interferometric calibration schemes and can tolerate severe bending with an intact focus. The inclusion of a secondary cladding on the MCF allows us high sensitivity detection though the fiber (NA = 0.6) whilst preserving the advantages of a sparse MCF. The combination of these new developments brings us towards the application of these ultrathin probes in realistic imaging conditions.
The periodic arrangement of core positions in multi-core fiber bundles introduces ‘ghost’ artifacts to endoscopic images obtained through them, whether in wide-field imaging (based on either direct imaging or speckle correlations) or in confocal scanning microscopy using wavefront shaping. Here we introduce partially disordered multi-core bundles as a means to overcome these artifacts. The benefits of their use will be discussed in the context of multiphoton scanning microscopy utilizing a spatial light modulator in the proximal end, and in the more general case of widefield imaging. We also show that both numerically and experimentally that the presence of disorder also enables to apply phase retrieval methods to characterize the phase distortion introduced due to propagation in the bundle without the need of an interferometrically stabilized reference. Thus, in addition to overcoming the challenge of ghost artifacts, disordered multi-core fibers have the potential to overcome another challenge, movement-induced phase distortions, by enabling real-time characterization of this phase distortion in reflection mode only via the proximal end.
We take stock of the progress that has been made into developing ultrathin endoscopes assisted by wave front shaping. We focus our review on multicore fiber-based lensless endoscopes intended for multiphoton imaging applications. We put the work into perspective by comparing with alternative approaches and by outlining the challenges that lie ahead.
Recent advances in wavefront shaping techniques have enabled so-called lensless endoscopes using fiber probes. Unlocking the full potential of such endoscopes call for the capability of optically sectioned and/or label free imaging. Or in other words, imaging through fibers must retain the functionality of a nonlinear microscope. This is a key challenge due to the temporal broadening of ultrashort pulses in fibers owing to modal dispersion.
Here, we detail the first ever demonstration of two photon fluorescence imaging at the distal tip of a conventional graded index (GRIN) multimode fiber. GRIN fibers possess a high mode density, excellent throughput and limited temporal broadening. These features, in addition to its ready availability, make them attractive candidates for ultrathin endoscopes. In our approach, we apply the transmission matrix formalism and treat these fibers akin to highly scattering media. This lets us retrieve combinations of input modes that would generate intense focal spots throughout the field of view. Furthermore, we identify a regime where the modal dispersion in the fiber is minimal and two-photon excitation with femtosecond light pulses is possible. This allows us to perform two-photon imaging with ultrashort pulses in an epi-detection configuration analogous to conventional nonlinear microscopes. Finally, these concepts are validated by acquiring optically sectioned two photon fluorescence images of 3D samples with cellular resolution. We believe this first report of an ultrathin rigid endoscope of only 125 µm thickness would further accelerate the development of novel tools for demanding applications in biological imaging and opto-genetics.
The development of nonlinear fiber-endoscopes capable of imaging deeper in tissues and accessing internal organs represents a very attractive perspective for application of nonlinear optical microscopes to in-vivo research and diagnostics. The transmission of ultra-short laser pulses within a fiber is a critical issue in the development of such endoscopes. For instance, self-phase modulation (SPM), four-wave mixing (FWM) and Raman scattering occurring in conventional fibers severely affect transmitted pulses profiles in the time and frequency domains. Hollow-core (HC) fibers bring a solution to the problem, since propagation of the pulses in the air core limits nonlinear interactions. We employ here a novel double clad Kagomé-lattice HC fiber for the delivery of ultrafast pulses across a large spectral window (~400nm) with no pulse distortion. The epi-collection of the signal generated at the sample is efficiently performed with a specially designed outer multimode cladding. The fiber is incorporated in a prototype endoscope using a four-quartered piezo-electric tube to scan the laser beam on the sample. The low numerical aperture of the hollow-core (0.02) is efficiently increased by means of a dielectric microsphere attached to the fiber face. This results in tight focusing (~1 micron) of the beam at the HC fiber output. Resonant scanning of the fiber tip allows imaging over a field of 300 microns using low driving voltages. High-resolution images with different contrast mechanisms, such as SHG and TPEF, acquired with the prototype endoscope illustrate the potential of these fibers for nonlinear imaging in regions otherwise inaccessible to conventional optical microscopes.
We map out the group delays of all the 169 single-mode cores of a 30 cm long multi-core fiber using phaseshifting spectral interferometry. We then present and apply a new experimental concept suitable for partially compensating the measured inter-core group delay dispersion. The compensation scheme is based on two wave front shapers in a 4-f geometry and group delays are imposed in the Fourier plane between them. These results are of relevance for cases where one desires to perform imaging through optical fibers using femtosecond excitation as is the case in the two-photon lensless endoscopes which have recently been demonstrated.
Circumventing the limit imposed by diffraction is a major issue in the instrumental development to realize finer
resolutions in biological samples. With STED microscopy, we exploit the molecular transitions of the fluorescent
marker to image well below the Rayleigh criterion. Also in combination with STED, we propose to use an
alternative technique for optically sectioning fluorescent emitters close to the water-glass interface by selectively
filtering the supercritical emission at the pupil plane. We discuss the instrumental development of such a system
and its combination with other imaging techniques.
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