Single- and two-photon absorption are prominent additive manufacturing methods used in 3D light-based printing through polymerization. We propose the dual use of these methods simultaneously to speed up the printing process while maintaining high resolution. We show a blue light-sheet implementation to assist the polymerization by a femtosecond laser. The light-sheet speeds up the printing considerably by reducing the threshold as well as yielding good optical sectioning, and 2PP provides precise and complex structures in micro- and nanometer scales with high resolution. This method offers new possibilities for printing high-resolution 3D structures with a significant improvement in voxel printing speed.
Owing to its many outstanding properties, polydimethylsiloxane (PDMS) has been an important engineering elastomer. An integration of compact, low-loss, three-dimensional (3D) optical waveguides with fluidic functionalities will greatly enhance the capabilities of PDMS-based devices and enable heretofore unimaginable applications. Recently, we have demonstrated the fabrication of compact polymer waveguides in PDMS through multiphoton laser direct writing both with and without a photoinitiator. While the photoinitiator-free process enjoyed a high refractive index contrast and a very low optical loss, it was subject to excessive defects due to material damage during the high-intensity optical irradiation. Our process using a photoinitiator achieved a defect-free fabrication despite a lower refractive index contrast and a higher loss. We show that the defects are a result of uncontrolled optical beam collapse through self-focusing and discuss potential solutions to mitigate the defects.
Owing to its excellent elasticity, wide spectral range of transparency, and outstanding chemical and thermal stability, polydimethylsiloxane (PDMS) is an elastomer of great technological importance, being widely used in the fabrication of microfluidic and optofluidic devices in particular. Compact, low loss optical waveguides are crucial in such devices for a dense integration of optical functionalities. Compared with photolithographic methods, multiphoton laser direct writing through photopolymerization has shown great promise in the fabrication of three-dimensional (3D) optical waveguides in PDMS without the limit of planar structures. Here we demonstrate a multiphoton laser direct writing process that produces low-loss, ultra-compact waveguides in PDMS. The fabrication employs a self-initiated multiphoton polymerization of phenylacetylene infused in PDMS without additional photoinitiator. The elimination of the photoinitiator results in an excellent refractive index homogeneity and thus a low propagation loss due to scattering, as well as a greater biocompatibility by involving the fewest possible chemicals (monomer only) in the polymerization reaction. Our characterizations show that the waveguides fabricated as such are on average 1.3 µm wide with a refractive index contrast of 0.06 and a propagation loss of 0.03 dB/cm in the spectral band of 650-700 nm. Our technique will enable a broad range of applications spanning from wearable photonics to chip-scale optical interconnects.
High-resolution imaging in thick biological tissues beyond a few transport mean free path of photons remains a formidable challenge to modern science and technology. Recent advancements in optical tomography have enabled the reconstruction of the three-dimensional (3D) refractive index distribution in biological samples, which is crucial in the prediction of light propagation in that sample. Here we propose a new technique for imaging second harmonic radiating imaging probes (SHRIMPs) through a tissue using harmonic holography based on the extracted refractive index map. We analyze the spatiotemporal intensity distribution of light pulses using a new model that separate scattering by orders (times of deflections) and show that wide-field excitation and detection of the SHG in tissues beyond one cm thickness is possible with resonant nonlinear plasmonic nanocavities as the SHRIMP. We demonstrate with numerical simulations that the 3D image of SHRIMPs can be extracted by matching the measured second harmonic speckle pattern with that predicted for each point in the sample given the refractive index. We analyze the impact of measurement error in the refractive index on the SNR of the recovered image. We further discuss the influence of the frequency-domain memory effect.
Observing the fast dynamics of specific molecules or targets in three-dimensional (3D) space and time inside a crowded and complex environment, such as living cells or tissues, remain one of the grand open challenges in modern science. Harmonic holography tackle this challenge by combining the 3D imaging capability of holography with the ultrafast, coherent optical contrast offered by second-harmonic radiating imaging probes (SHRIMPs). Similar to fluorescence, the second-harmonic signal emitted from SHRIMPs provides a color contrast against the uninterested background scattering, which can be efficiently suppressed by an optical filter. We review the latest developments in SHRIMPs and harmonic holography and discuss their further applications in fluidics and biofluidics.
Compared with other imaging probes such as fluorescent dyes and quantum dots, second-harmonic radiating imaging
probes (SHRIMPs) provide a unique ultrafast, coherent optical contrast that is free of photobleaching and emission
intermittency. Using the second-harmonic signal emitted from SHRIMPs, harmonic holography achieves threedimensional
holographic imaging with a color contrast similar to fluorescence microscopy where the uninterested
background scattering is efficiently suppressed by an optical filter. The coherent contrast provided by SHRIMPs also
enables imaging through turbid media via digital phase conjugation. Here we review the developments and applications
of SHRIMPs and harmonic holography.
Intracochlear imaging is of great interest clinically because cochlea is the central organ of hearing. However, intracochlear imaging is technologically challenging due to the cochlea’s small size and encasement in bone. The state-of- the-art imaging techniques are not adequate for high resolution cellular imaging to establish diagnosis without destroying the cochlea. We report in situ imaging of intact mouse cochlea using endogenous two-photon excitation fluorescence (TPEF) as the contrast mechanism. TPEF eliminates the need for exogenous labeling and eradicating the staining-induced artifacts. We used a natural, membranous opening into the cochlea, the round window, as the optical access to reach the organ of Corti, requiring no additional slicing or opening. Our approach provides the maximum non-invasiveness in the imaging process. TPEF exhibits strong contrast allowing deep imaging of mouse cochlea with cellular and even subcellular resolution. Inner hair cell, outer hair cell and supporting cell are clearly identifiable in TPEF images. Distinct morphological differences are observed between healthy and noise-exposed cochleae, allowing detection of specific, noise-induced pathologic changes. The TPEF images taken through the round window are correlated with the whole mount sections, verifying their reliability. Compared with one-photon excitation fluorescence (OPEF) confocal microscope and wide-field transmission microscope images taken under the same magnification and resolution, TPEF images demonstrate clear advantages in terms of sharpness, signal to noise ratio and contrast. These capabilities provide a working foundation for microendoscopy-based clinical diagnostics of sensorineural hearing loss.
Sensorineural hearing loss is the most common type of hearing loss worldwide, yet the underlying cause is typically unknown because the inner ear cannot be biopsied today without destroying hearing, and intracochlear cells have not been imaged with resolution sufficient to establish diagnosis. Intracochlear imaging has been technologically challenging because of the cochlea's small size and encasement in bone. We report, for the first time, imaging of the mouse cochlea in situ without exogenous dyes, through a membranous round window, using a near-infrared femtosecond laser as the excitation and endogenous two-photon excitation fluorescence (TPEF) and second harmonic generation as the contrast mechanisms. We find that TPEF exhibits strong contrast allowing cellular, and even subcellular resolution, and detection of specific, noise-induced pathologic changes. Our results demonstrate that the round window provides a useful access to the cochlea through the middle ear, and they motivate future development of a new and efficient diagnostic tool based on two-photon micro-endoscopy.
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.
We report the hydrothermal synthesis of free-standing lithium niobate nanowires. We show that the versatile properties
of bulk lithium niobate such as nonlinear optical effects can be exploited at the nanoscale. We describe the fabrication of
polydimethylsiloxane (PDMS) microfluidics as well as indium tin oxide (ITO) electrodes with different design for
dedicated applications. The control of microfluidic channel dimensions and the corresponding particle concentration is
explored. Finally, the selection of fluidic conductivity for optimal dielectrophoretic trapping conditions is discussed.
We develop second-harmonic nanoparticles as the contrast agents for cell imaging. Second-harmonic nanoparticles show
promise as cell imaging probes due to their non-bleaching, non-blinking, and coherent signal. Nanoparticles of noncentrosymmetric
crystal structures have high second-harmonic generation (SHG) efficiency and provide high contrast in
a generally non-structured cell environment. Here, we use barium titanate (BaTiO3) nanoparticles with tetragonal crystal
structure as imaging probes. Cytotoxicity tests performed on BaTiO3 nanoparticles with mammalian cells did not result
in toxic effects. Specifically, we observed no change in the cell metabolism after 24 hours incubation of the cells with
high concentration of BaTiO3 nanoparticles. We demonstrate two methods of cell labeling with BaTiO3 nanoparticles for imaging. One is non-specific labeling via endocytosis of the cells, which results in a great number of the nanoparticles
randomly distributed inside the cells. The other is specific labeling via surface functionalization of the nanoparticles with
antibodies, which enables us to label specific cell membrane proteins with the nanoparticles. SHG imaging is compatible
to two-photon microscopy and the SHG signal from nanoparticles can be easily detected with a standard two-photon
confocal microscope. Our work provides the opportunity for long-term, three-dimensional cell tracking with secondharmonic
nanoparticles.
We have developed a luminescent marker using the second harmonic generation (SHG) from noncentrosymmetric
BaTiO3 nanocrystals. These nanoparticles are attractive due to their stable, non-saturating and coherent signal with a
femtosecond-scale response time and broad flexibility in the choice of excitation wavelength. In this paper, we report the
SHG response of BaTiO3 nanocrystals under a circularly polarized excitation. We observed a more uniform SHG signal
intensity from nanocrystals of different crystal orientations under a circularly polarized excitation. The threedimensional
(3D) SHG fields generated from the SHRIMPs were recorded by a harmonic holographic microscope. Submicron
resolution in both lateral and axial directions has been achieved. We show that the circularly polarized excitation
is useful for harmonic holographic microscopy.
Luminescent markers play a key role in imaging techniques for life science since they provide a contrast mechanism
between signal and background. We describe a new type of marker using second harmonic generation (SHG) from
noncentrosymmetric BaTiO3 nanocrystals. These nanoparticles are attractive due to their stable, non-saturating and
coherent signal with a femtosecond-scale response time and broad flexibility in the choice of excitation wavelength. In
this paper, we report the use of nanoparticles for cell imaging. We first stabilized the BaTiO3 nanoparticles in suspension
and characterized the optical properties. We also demonstrated the functionalization of BaTiO3 nanoparticles by
conjugating IgG antibody on the surface of the nanoparticles. These functionalized nanocrystals are capable of specific
labeling the antigens of interest.
Luminescent markers play a key role in imaging techniques for life sciences since they provide a contrast mechanism
between signal and background. We describe a new type of marker using second harmonic generation (SHG) from
noncentrosymmetric BaTiO3 nanocrystals. These nanoparticles are attractive due to their stable, non-saturating and
coherent signal with a femtosecond-scale response time and a broad flexibility in the choice of excitation wavelength.
We use the coherent SHG signal from BaTiO3 nanoparticles for three-dimensional (3D) imaging without scanning. We
built a harmonic holographic (H2) microscope which records digital holograms at the second harmonic frequency. Highresolution
3D distributions of these SHG markers in mammalian cells are successfully captured and interpreted by the H2
microscope.
Fluorescence microscopy has profoundly changed how cell and molecular biology is studied in almost
every aspect. However, the need of characterizing biological targets is largely unmet due to deficiencies
associated with the use of fluorescent agents. Dye bleaching, dye signal saturation, blinking, and tissue
autofluorescence can severely limit the signal-to-noise ratio (SNR). Given the photophysical properties are
fundamentally different to the fluorescent agents currently used in biomedical research, second harmonic
generating (SHG) nanoprobes can be suitable for biomedical imaging and can eliminate most of the
drawbacks encountered in classical fluorescence systems.
We describe a holographic technique capable of sampling dynamic events at 150 femtosecond time resolution. We apply the technique to the study of the nonlinear propagation of high energy pulses through gas and condensed media. The holograms are recorded as a digitized image from a CCD camera and reconstructed numerically to retrieve the refractive index change during the nonlinear optical process. We show dramatic differences in the pulse propagation characteristics depending on the strength of the nonlinear coefficient of the material and it's time response. Both positive and negative index changes have been measured in different media. The holographic technique allows us to distinguish the very fast positive index changes that are generally attributable to the Kerr nonlinearity from the negative index changes that result from free electrons generated by multiphoton ionization.
We have observed the filamentation of optical pulses in carbon disulfide(CS2) using femtosecond time-resolved optical polarigraphy(FTOP). A pump-probe setup is used to capture the propagation of a 150 femtosecond laser pulse in CS2. The probe pulse propagates in a direction perpendicular to the pump. The high intensity of the pump pulse causes a transient index change in the material through the Kerr effect. The induced birefringence is proportional to the intensity of the pump and can be captured by monitoring the polarization of the probe. The probe pulse is imaged on a CCD camera to recover the intensity profile of the pump pulse. We have used this technique to observe the spatial evolution of the pulse as a function of power and propagation distance. Initially, the pulse propagation causes a coarse redistribution of the intensity. The beam then breaks up into stable light filaments which propagate for several millimeters, and finally the beam profile becomes unstable to small fluctuations in the input power.
We present a holographic recording technique with 150 femtosecond time resolution. This technique allows us to capture either a single hologram with fine spatial resolution (4 micrometers), or a time-sequence of multiple holograms with reduced spatial resolution in a single-shot experiment, while preserving amplitude and phase information. The time resolution and the frame rate are limited only by the duration of the laser pulses. The holograms are recorded on a CCD camera and digitally reconstructed. We have used the technique to study the nonlinear propagation of high energy femtosecond pulses through liquids. We have observed dramatic differences in the pulse propagation characteristics depending on the strength of the nonlinear coefficient of the material and it's time response. The fine spatial resolution allows us to zoom in and visualize the spatial profile of the pulses breaking up into multiple filaments while the phase recovered from the holograms helps us identify the nonlinear index changes in the material. We have measured both positive and negative index changes. Very fast positive index changes are generally attributable to the Kerr nonlinearity. The negative index changes can be caused by electron plasma generated by multiphoton absorption.
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