We show that the modulation of the phases of the laser beams of ultra-short pulses leads to modulation of the two photon fluorescence intensity. The phase modulation technique when used in multi-photon microscopy can improve the signal to noise ratio. The technique can also be used in multiplexing the signals in the frequency domain in multi-focal raster scanning microscopy. As the technique avoids the use of array detectors as well as elaborate spatiotemporal multiplexing schemes it provides a convenient means to multi-focal scanning in axial direction. We show examples of such uses. Similar methodology can be used in other non-linear scanning microscopies, such as second or third harmonic generation microscopy.
Many optical approaches have been used to examine the composition and structure of gemstones, both recently and throughout history. The nonlinear optical behavior of different gemstones has not been investigated, and the higher order terms to the refractive index represent an unused tool for qualifying and examining a stone. We have used a multiphoton microscope to examine the nonlinear optical properties of 36 different gemstones and demonstrate that it is a useful tool for imaging them three-dimensionally up to the millimeter scale below the sample surface. The polarization dependence of sec…
Super-resolved far-field microscopy has emerged as a powerful tool for investigating the structure of objects with resolution well below the diffraction limit of light. Nearly all super-resolution imaging techniques reported to date rely on real energy states of probe molecules to circumvent the diffraction limit, preventing super-resolved imaging of contrast mechanisms that occur via virtual energy states such as harmonic generation (HG). Here we report a super-resolution technique based on SPatIal Frequency modulated Imaging (SPIFI) that permits super-resolved nonlinear microscopy with any c…
An integrated microscope that combines different optical techniques for simultaneous imaging is demonstrated. The microscope enables spectral-domain optical coherence microscopy based on optical backscatter, and multi-photon microscopy for the detection of two-photon fluorescence and second harmonic generation signals. The unique configuration of this integrated microscope allows for the simultaneous acquisition of both anatomical (structural) and functional imaging information with particular emphasis for applications in the fields of tissue engineering and cell biology. In addition, the cont…
Multiphoton microscopy (MPM) has gained enormous popularity over the years for its capacity to provide high resolution images from deep within scattering samples1. However, MPM is generally based on single-point laser-focus scanning, which is intrinsically slow. While imaging speeds as fast as video rate have become routine for 2D planar imaging, such speeds have so far been unattainable for 3D volumetric imaging without severely compromising microscope performance. We demonstrate here 3D volumetric (multiplane) imaging at the same speed as 2D planar (single plane) imaging, with minimal compro…
Compressive sensing can overcome the Nyquist criterion and record images with a fraction of the usual number of measurements required. However, conventional measurement bases are susceptible to diffraction and scattering, prevalent in high-resolution microscopy. Here, we explore the random Morlet basis as an optimal set for compressive multiphoton imaging, based on its ability to minimise the space-frequency uncertainty. We implement this approach for the newly developed method of wide-field multiphoton microscopy with single-pixel detection (TRAFIX), which allows imaging through turbid media …
Two-Photon Microscopy has become an invaluable tool for biological and medical research, providing high sensitivity, molecular specificity, inherent three-dimensional sub-cellular resolution and deep tissue penetration. In terms of imaging speeds, however, mechanical scanners still limit the acquisition rates to typically 10-100 frames per second. Here we present a high-speed non-linear microscope achieving kilohertz frame rates by employing pulse-modulated, rapidly wavelength-swept lasers and inertia-free beam steering through angular dispersion. In combination with a high bandwidth, single-p…
Label-Free Multiphoton Microscopy is a very powerful optical microscopy that can be applied to study samples with no need for exogenous fluorescent probes, keeping the main benefits of a Multiphoton approach, like longer penetration depths and intrinsic optical sectioning, while opening the possibility of serial examinations with different kinds of techniques. Among the many variations of Label-Free MPM, Higher Harmonic Generation (HHG) is one of the most intriguing due to its generally low photo-toxicity, which enables the examination of specimens particularly susceptible to photo-damages. HH…
We report second- and third-harmonic generation in monolayer MoS$_\mathrm{2}$ as a tool for imaging and accurately characterizing the material's nonlinear optical properties under 1560 nm excitation. Using a surface nonlinear optics treatment, we derive expressions relating experimental measurements to second- and third-order nonlinear sheet susceptibility magnitudes, obtaining values of $|χ_s^{(2)}|=2.0\times10^{-20}$ m$^2$ V$^{-1}$ and for the first time for monolayer MoS$_\mathrm{2}$, $|χ_s^{(3)}|=1.7\times10^{-28}$ m$^3$ V$^{-2}$. These sheet susceptibilities correspond to effective bulk n…
Structured illumination in Single Molecule Localization Microscopy provides new information on the position of molecules and thus improves the localization precision compared to standard localization methods. Here, we used a time-shifted sinusoidal excitation pattern to modulate the fluorescence signal of the molecules whose position information is carried by the phase and recovered by synchronous demodulation. We designed two flexible fast demodulation systems located upstream of the camera, allowing us to overcome the limiting camera acquisition frequency and thus to maximize the collection …
Multiphoton microscopy has enabled us to image cellular dynamics in vivo. However, the excitation wavelength for imaging with commercially available lasers is mostly limited between 650-1040 nm. Here we develop a femtosecond fiber laser system that produces $\sim$150 fs pulses at 1.8 $μ$m. Our system starts from an erbium-doped silica fiber laser, and its wavelength is converted to 1.8 $μ$m using a Raman shift fiber. The 1.8 $μ$m pulses are amplified with a two-stage Tm:ZBLAN fiber amplifier. The final pulse energy is $\sim$1 $μ$J, sufficient for in vivo imaging.
Second-order nonlinear optical interactions, including second harmonic generation (SHG) and sum-frequency generation (SFG), can reveal a wealth of information about chemical, electronic, and vibrational dynamics at the nanoscale. Here, we demonstrate a powerful and flexible new approach, called phase-modulated degenerate parametric amplification (DPA). The technique, which allows for facile retrieval of both the amplitude and phase of the second-order nonlinear optical response, has many advantages over conventional or heterodyne-detected SHG, including the flexibility to detect the signal at …
Scanning Microwave Impedance Microscopy (MIM) measurement of photoconductivity with 50 nm resolution is demonstrated using a modulated optical source. The use of a modulated source allows for measurement of photoconductivity in a single scan without a reference region on the sample, as well as removing most topographical artifacts and enhancing signal to noise as compared with unmodulated measurement. A broadband light source with tunable monochrometer is then used to measure energy resolved photoconductivity with the same methodology. Finally, a pulsed optical source is used to measure local …
Confocal and multiphoton microscopy are effective techniques to obtain high-contrast images of 2-D sections within bulk tissue. However, scattering limits their application to depths only up to ~1 millimeter. Multimode fibers make excellent ultrathin endoscopes that can penetrate deep inside the tissue with minimal damage. Here, we present Multiview Scattering Scanning Imaging Confocal (MUSSIC) Microscopy that enables high signal-to-noise ratio (SNR) imaging through a multimode fiber, hence combining the optical sectioning and resolution gain of confocal microscopy with the minimally invasive …
We present a new two-snapshot structured light illumination (SLI) reconstruction algorithm for fast image acquisition. The new algorithm, which only requires two mutually π phase-shifted raw structured images, is implemented on a custom-built temporal focusing fluorescence microscope (TFFM) to enhance its axial resolution via a digital micromirror device (DMD). First, the orientation of the modulated sinusoidal fringe patterns is automatically identified via spatial frequency vector detection. Subsequently, the modulated in-focal-plane images are obtained via rotation and subtraction. Lastly, …
Learned, uncertainty-driven adaptive acquisition for photon-efficient scanning microscopy
Fu Foundation School of Engineering and Applied Science, Columbia University in the City of New York（纽约市哥伦比亚大学傅氏工程与应用科学学院）；University of California, Berkeley（加利福尼亚大学伯克利分校）；Department of Biological Engineering, Massachusetts Institute of Technology（麻省理工学院生物工程系）
Femtosecond point-projection microscopy (fs-PPM) is an electron microscopy technique that possesses a combination of high spatio-temporal resolution and sensitivity to local electric fields. This allows it to visualize ultrafast charge carrier dynamics in complex nanomaterials. We benchmark the capability of the fs-PPM technique by imaging the ultrafast dynamics of charge carriers produced by multiphoton ionization of silver nanowires. The space-charge driven motion of photoelectrons is followed on \mbox{sub-100}\,nm length scales, while the dynamics are captured on 30-100 fs time scales. The …
Nonlinear optical microscopy allows rapid high-resolution microscopy with image contrast generated from intrinsic properties of the sample. Established modalities such as multiphoton excited fluorescence and second/third-harmonic generation can be combined with other nonlinear techniques, such as coherent Raman spectroscopy which typically allow chemical imaging of a single resonant vibrational mode of a sample. Here, we utilize a single ultrafast laser source to obtain broadband coherent Raman spectra on a microscope, together with other nonlinear microscopy approaches on the same instrument.…
We present a laser scanning reflection-matrix microscopy combining the scanning of laser focus and the wide-field mapping of the electric field of the backscattered waves for eliminating higher-order aberrations even in the presence of strong multiple light scattering noise. Unlike conventional confocal laser scanning microscopy, we record the amplitude and phase maps of reflected waves from the sample not only at the confocal pinhole, but also at other non-confocal points. These additional measurements lead us to constructing a time-resolved reflection matrix, with which the sample-induced ab…
An experiment demonstrating single-pixel single-arm complementary compressive microscopic ghost imaging based on a digital micromirror device (DMD) has been performed. To solve the difficulty of projecting speckles or modulated light patterns onto tiny biological objects, we instead focus the microscopic image onto the DMD. With this system, we have successfully obtained a magnified image of micron-sized objects illuminated by the microscope's own incandescent lamp. The image quality of our scheme is more than an order of magnitude better than that obtained by conventional compressed sensing w…
Two-photon light-sheet live imaging at kilohertz frame rate using birefringence-based pulse splitting
Laboratoire d’Optique et Biosciences, CNRS, INSERM, École Polytechnique, Institut Polytechnique de Paris（光学与生物科学实验室，法国国家科学研究中心，法国国家健康与医学研究院，巴黎综合理工学院，巴黎理工学院）；ICube Research Institute, CNRS, University of Strasbourg（ICube研究所，法国国家科学研究中心，斯特拉斯堡大学）；Sorbonne Université, CNRS, Laboratoire Jean Perrin（索邦大学，法国国家科学研究中心，让·佩兰实验室）
Modulating the electric potential on a conducting electrode is presented to generate an optical contrast for scattering microscopy that is sensitive to both surface charge and local topography. We dub this method Electric-Double-Layer-Modulation microscopy. We numerically compute the change in the local ion concentration that is the origin of this optical contrast for three experimentally relevant geometries: nanosphere, nanowire, and nanohole. In absence of plasmonic effects and physical absorption, the observable optical contrast is proportional to the derivative of the ion concentration wit…
Here we report nonlinear focal modulation microscopy (NFOMM) to achieve super-resolution imaging. Abandoning the previous persistence on minimizing the size of Gaussian emission pattern by directly narrowing (e.g. Minimizing the detection pinhole in Airyscan, Zeiss) or by indirectly peeling its outer profiles (e.g., Depleting the outer emission region in STED, stimulated emission microscopy) in pointwise scanning scenarios, we stick to a more general basis------ maximizing the system frequency shifting ability. In NFOMM, we implement a nonlinear focal modulation by applying phase modulations w…
High-resolution optical microscopy suffers from a low contrast in scattering media where a multiply scattered wave obscures a ballistic wave used for image formation. To extend the imaging depth, various gating operations - confocal, coherence, and polarization gating - have been devised to filter out the multiply scattered wave. However, these gating methods are imperfect as they all act on the detection plane located outside a scattering medium. Here, we present a new gating scheme, called 'space' gating, that rejects the multiply scattered wave directly at the object plane inside a scatteri…
We experimentally investigated the contrast mechanism of infrared photoinduced force microscopy (PiFM) for recording vibrational resonances. Extensive experiments have demonstrated that spectroscopic contrast in PiFM is mediated by opto-mechanical damping of the cantilever oscillation as the optical wavelength is scanned through optical resonance. To our knowledge, this is the first time opto-mechanical damping has been observed in the AFM. We hypothesize that this damping force is a consequence of the dissipative interaction between the sample and the vibrating tip; the modulated light source…
Two-Photon Microscopy (TPM) can provide three-dimensional morphological and functional contrast in vivo. Through proper staining, TPM can be utilized to create virtual, H&E equivalent images and thus can improve throughput in histology-based applications. We previously reported on a new light source for TPM that employs a compact and robust fiber-amplified, directly modulated laser. This laser is pulse-to-pulse wavelength switchable between 1064 nm, 1122 nm, and 1186 nm with an adjustable pulse duration from 50ps to 5ns and arbitrary repetition rates up to 1MHz at kW-peak powers. Despite the l…
We report a novel lensless on-chip microscopy platform based on near-field blind ptychographic modulation. In this platform, we place a thin diffuser in between the object and the image sensor for light wave modulation. By blindly scanning the unknown diffuser to different x-y positions, we acquire a sequence of modulated intensity images for quantitative object recovery. Different from previous ptychographic implementations, we employ a unit magnification configuration with a Fresnel number of ~50,000, which is orders of magnitude higher than previous ptychographic setups. The unit magnificat…
