Optical microscopy is an indispensable tool for researchers, allowing them to closely investigate different organisms, revealing new features and phenomena in biomedical research. Although very useful, conventional imaging techniques that rely only on ballistic, unaffected photons to form images inside inhomogeneous media, like biological tissue, are eventually limited up to the diffusion regime of optical propagation where scattering becomes dominant and no ballistic light can be detected.
Adaptive optics and nonlinear optimization methods that rely on so called guide stars have been employed to overcome this problem and image deeper inside biological tissue. These techniques attempt to recover the optimal wavefront that will enhance the image quality or that will render a focus spot inside the scattering biological tissue. In order to achieve that, they have to iterate through each correction mode (e.g. each pixel on a wavefront shaper) thus trading off measurement time with wavefront resolution. Here we present a new turbidity suppression approach, termed Focus Scanning Holographic Aberration Probing (F-SHARP or F♯) that allows us to directly measure the amplitude and phase of the scattered light distribution at the focal plane (scattered E-field PSF). Knowledge of the E-field enables rapid correction of both aberration and scattering with a high resolution. We demonstrate the power of F-SHARP by correcting for aberration and scattering and imaging neuronal structures through the larval zebrafish and mouse brain and through thinned mouse skull in vivo.
Endoscopy can be used to obtain high-resolution images at large depths in biological tissues. Usually endoscopic devices have a diameter ranging from 1 to few millimeters. Using digital phase conjugation, it is possible to adapt ultrathin multimode fibers to endoscopic purposes. Recently, we demonstrated that a 330 μm diameter, water-filled silica capillary waveguide can guide high frequency ultrasound waves through a 3 cm thick fat layer, allowing optical resolution photoacoustic imaging. Here we demonstrate that using digital phase conjugation, the same water-filled capillary waveguide (3 cm long) can be used as an endoscopic probe to obtain both fluorescence and optical resolution photoacoustic imaging, with no optical or acoustic elements at the tip of the waveguide. We study the consequences of using digital phase conjugation combined with a capillary waveguide and we conclude with possible future improvements of our endoscopic approach.
KEYWORDS: Multimode fibers, Optical fibers, Digital holography, Phase conjugation, Two photon imaging, Femtosecond phenomena, Dispersion, Graded index fibers, Two photon excitation microscopy, Digital imaging, Image transmission, Step index fibers
The large number of modes supported by multimode optical fibers potentially allows the transmission of larger amounts of information compared to single mode fibers. However, when pulsed light is transmitted through multimode fibers, the spatio-temporal profile of the incident beam is altered upon propagation, leading to a highly scrambled spatial profile and a broadened temporal duration due to modal and material dispersion. We present a digital phase conjugation method to counter-propagate through a multimode optical fiber only a group of modes of similar propagation constants which interfere constructively at a single location at the other side of the fiber, generating a spatially focused pulse. Since only modes with the same speed are excited, temporal broadening due to modal dispersion is suppressed, preserving the ultrashort duration of the propagating pulse. Using this technique, we experimentally demonstrate the transmission of focused pulses of 500 fs through a 30 cm length, 200 micrometer core step-index multimode fiber. Additionally, using a graded-index fiber, which allows the propagation of a larger number of modes of the same speed than a graded index fiber (hence a better focusing capability), we have been able to deliver and scan high-intensity focused femtosecond pulses. Moreover, based on the described principle, we demonstrate for the first time two-photon excitation imaging through a multimode optical fiber.
Confocal laser-scanning microscopy is a well-known optical imaging method where a pinhole is used in the illumination and detection pathways of a normal microscope, in order to selectively excite and detect a particular focal volume. The advantage of this method is a significant increase in contrast, due to the rejection of background contributions to the signal. Here, we propose to apply this method in the context of multimode fiber endoscopy. Due to modal scrambling, it is not possible to use a physical pinhole to filter light signals that have travel through multimode fibers. Instead, we use a transmission matrix approach to characterize the propagation of light through the fiber, and we apply the filtering operation in the digital domain.
Multimode optical fibers potentially allow the transmission of larger amounts of information than their single mode counterparts because of their high number of supported modes. However, propagation of a light pulse through a multimode fiber suffers from spatial distortions due to the superposition of the various exited modes and from time broadening due to modal dispersion.
We present a method based on digital phase conjugation to selectively excite in a multimode fiber specific optical fiber modes that follow similar optical paths as they travel through the fiber. The excited modes interfere constructively at the fiber output generating an ultrashort spatially focused pulse. The excitation of a limited number of modes following similar optical paths limits modal dispersion, allowing the transmission of the ultrashort pulse. We have experimentally demonstrated the delivery of a focused spot of pulse width equal to 500 fs through a 30 cm, 200 micrometer core step index multimode fiber. The results of this study show that two-photon imaging capability can be added to ultra-thin lensless endoscopy using commercial multimode fibers.
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.
Multimode optical fibers potentially allow the transmission of larger amounts of information than their single mode counterparts because of their high number of supported modes. However, propagation of a light pulse through a multimode fiber suffers from spatial distortions due to the superposition of the various exited modes and from time broadening due to modal dispersion. We present a method based on digital phase conjugation to selectively excite in a multimode fiber specific optical fiber modes that follow similar optical paths as they travel through the fiber. The excited modes interfere constructively at the fiber output generating an ultrashort spatially focused pulse. The excitation of a limited number of modes following similar optical paths limits modal dispersion, allowing the transmission of the ultrashort pulse. We have experimentally demonstrated the delivery of a focused spot of pulse width equal to 500 fs through a 30 cm, 200 micrometer core step-index multimode fiber. The results of this study show that two-photon imaging capability can be added to ultra-thin lensless endoscopy using commercial multimode fibers.
Microscopes are usually thought of comprising imaging elements such as objectives and eye-piece lenses. A different type of microscope, used for endoscopy, consists of waveguiding elements such as fiber bundles, where each fiber in the bundle transports the light corresponding to one pixel in the image. Recently a new type of microscope has emerged that exploits the large number of propagating modes in a single multimode fiber. We have successfully produced fluorescence images of neural cells with sub-micrometer resolution via a 200 micrometer core multimode fiber. The method for achieving imaging consists of using digital phase conjugation to reproduce a focal spot at the tip of the multimode fiber. The image is formed by scanning the focal spot digitally and collecting the fluorescence point by point.
We demonstrate a digital phase conjugation technique for generating a sharp focus point at the end of a multimode optical fiber. A sharp focus is experimentally obtained at the distal end of a 200μm core multimode fiber. By recording the digital holograms for different excitation conditions, the sharp focus can be digitally scanned over a 175μm diameter regular grid. The demonstrated technique is used for high resolution scanning lensless imaging based on multimode fibers, of stained biological samples that can enable diagnosis from the investigation of cellular phenotype.
Nanocrystals with second harmonic response is a new class of nonlinear optical nanoprobes with dramatically different
properties from fluorescent agents. Compared with two-photon fluorescence, second harmonic generation is an ultrafast,
lossless, and coherent process. In particular, the absence of photobleaching and emission intermittency in the optical
response of the second harmonic nanoparticles is likely to complement the fluorescent agents widely used today in many
imaging applications. Furthermore, the coherent emission from the second harmonic generation process provides unique
opportunities for the application of coherence domain techniques that are not available with fluorescent agents. We
review the application of the second harmonic nanocrystals in imaging applications, especially those pertaining to
biomedicine.
Kerstin Winkler, Lienhard Koerner, Peter Gussek, Istvan Balogh, Stefan Breitfelder, Johannes Schlichting, Jean-Pierre Dupraz, Jean-Paul Fabre, Jaap Panman, Ioannis Papadopoulos, Piero Zucchelli, Bart van de Vyver
Experiments such as CHORUS at CERN require the inspection of a large amount of nuclear emulsion plates exposed to particle beams. Rare events need to be found, measured and analyzed. Their features are stored as grains in microscopic dimensions in a 3D stack of plates. A new, fully automatic immersion microscope system was developed for this purpose. It features high resolution, small depth of focus, large working distance, large field of view and synchronization of illumination and detector. An additional requirement is given by variations in the refraction index and in the relative thickness of immersion oil and emulsion. The approach used here is an imaging system based on a various objective lens with extreme numerical aperture, large working distance and wide field, combined with a matched high-aperture Koehler illuminator. The light source is a mercury arc lamp, combined with a filter package for the g- line. It includes liquid crystal elements for synchronized shuttering and variable attenuation. The theoretical resolution is less than 1 micron in x, y, z within a volume of 0.5mm diameter times 1 mm scanning depth in all situations within a predefined index range. Three identical pieces of the system have been built. The identical pieces of the system have been built. The experimentally measured resolution confirms the expectations and is better than 1 micron in all three dimensions. This is the result of a complex process of system design and manufacturing, unifying optical, opto-mechanical and opto-electronical contributions. This process spans from the early stages of feasibility and manufacturing up to the test and adjustment procedures. The three prototypes are operational since the fall of 1998 in the frame of the CHORUS project. Practical experience and application results are presented.
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