Super-resolution computational saturated absorption microscopy.

Imaging beyond the diffraction limit barrier has attracted wide attention due to the ability to resolve previously hidden image features. Of the various super-resolution microscopy techniques available, a particularly simple method called saturated excitation microscopy (SAX) requires only simple modification of a laser scanning microscope: The illumination beam power is sinusoidally modulated and driven into saturation. SAX images are extracted from the harmonics of the modulation frequency and exhibit improved spatial resolution. Unfortunately, this elegant strategy is hindered by the incursion of shot noise that prevents high-resolution imaging in many realistic scenarios. Here, we demonstrate a technique for super-resolution imaging that we call computational saturated absorption (CSA) in which a joint deconvolution is applied to a set of images with diversity in spatial frequency support among the point spread functions (PSFs) used in the image formation with saturated laser scanning fluorescence microscopy. CSA microscopy allows access to the high spatial frequency diversity in a set of saturated effective PSFs, while avoiding image degradation from shot noise.

Nearly degenerate two-color impulsive coherent Raman hyperspectral imaging.

Impulsive stimulated Raman scattering (ISRS) is a robust technique for studying low frequency (<300 cm-1) Raman vibrational modes, but ISRS has faced difficulty in translation to an imaging modality. A primary challenge is the separation of the pump and probe pulses. Here we introduce and demonstrate a simple strategy for ISRS spectroscopy and hyperspectral imaging that uses complementary steep edge spectral filters to separate the probe beam detection from the pump and enables simple ISRS microscopy with a single-color ultrafast laser source. ISRS spectra are obtained that span from the fingerprint region down to <50 cm-1 vibrational modes. Hyperspectral imaging and polarization-dependent Raman spectra are also demonstrated.

Line-scan compressive Raman imaging with spatiospectral encoding.

We report a line-scanning imaging modality of compressive Raman technology with a single-pixel detector. The spatial information along the illumination line is encoded onto one axis of a digital micromirror device, while spectral coding masks are applied along the orthogonal direction. We demonstrate imaging and classification of three different chemical species.

Fluorescent coherent diffractive imaging with accelerating light sheets.

Fluorescence microscopy is a powerful method for producing high fidelity images with high spatial resolution, particularly in the biological sciences. We recently introduced coherent holographic image reconstruction by phase transfer (CHIRPT), a single-pixel imaging method that significantly improves the depth of field in fluorescence microscopy and enables holographic refocusing of fluorescent light. Here we demonstrate that by installing a confocal slit conjugate to the illuminating light sheets used in CHIRPT, out-of-focus light is rejected, thus improving lateral spatial resolution and rejecting noise from out-of-focus fluorescent light. Confocal CHIRPT is demonstrated and fully modeled. Finally, we explore the use of beam shaping and point-spread-function engineering to enable holographic single-lens light-sheet microscopy with single-pixel detection.

Compressive Raman imaging with spatial frequency modulated illumination.

We report a line scanning imaging modality of compressive Raman technology with spatial frequency modulated illumination using a single pixel detector. We demonstrate the imaging and classification of three different chemical species at line scan rates of 40 Hz.

Single pixel quantitative phase imaging with spatial frequency projections.

We introduce a new single pixel imaging technique that automatically co-registers quantitative phase and incoherent image modalities through the simultaneous acquisition of identical object spatial frequency information. The technique consists of using a time varying groove density diffraction grating to produce a reference and scan beam. The interference between the beams produce time varying spatial frequencies in the sample. The collected light on a single pixel detector produces a time trace that allows easy recovery of coherent and incoherent contrast mechanisms. We derive theory for the quantitative phase and show excellent agreement with experimental data and numeric model. Additionally, we derive a general theory of single pixel quantitative phase theory that can be applied broadly to general methods that use a sequence of modulated light patterns for single pixel phase imaging.

Superresolved multiphoton microscopy with spatial frequency-modulated imaging.

Superresolved 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 superresolution imaging techniques reported to date rely on real energy states of fluorescent molecules to circumvent the diffraction limit, preventing superresolved imaging with contrast mechanisms that occur via virtual energy states, including harmonic generation (HG). We report a superresolution technique based on spatial frequency-modulated imaging (SPIFI) that permits superresolved nonlinear microscopy with any contrast mechanism and with single-pixel detection. We show multimodal superresolved images with two-photon excited fluorescence (TPEF) and second-harmonic generation (SHG) from biological and inorganic media. Multiphoton SPIFI (MP-SPIFI) provides spatial resolution up to 2η below the diffraction limit, where η is the highest power of the nonlinear intensity response. MP-SPIFI can be used to provide enhanced resolution in optically thin media and may provide a solution for superresolved imaging deep in scattering media.

Three-dimensional single-pixel imaging of incoherent light with spatiotemporally modulated illumination.

We derive analytic expressions for the three-dimensional coherent transfer function (CTF) and coherent spread function (CSF) for coherent holographic image reconstruction by phase transfer (CHIRPT) microscopy with monochromatic and broadband illumination sources. The 3D CSF and CTF were used to simulate CHIRPT images, and the results show excellent agreement with experimental data. Finally, we show that the formalism presented here for computing the CSF/CTF pair in CHIRPT microscopy can be readily extended to other forms of single-pixel imaging, such as spatial-frequency-modulated imaging.

Simultaneous spatial frequency modulation imaging and micromachining with a femtosecond laser.

A Ti:Al2O3 chirped-pulse amplification system is used to simultaneously image and machine. By combining simultaneous spatial and temporal focusing (SSTF) with spatial frequency modulation for imaging (SPIFI), we are able to decouple the imaging and cutting beams to attain a resolution and a field-of-view that is independent of the cutting beam, while maintaining single-element detection. This setup allows for real-time feedback with the potential for simultaneous nonlinear imaging and imaging through scattering media. The novel SSTF machining platform uses refractive optics that, in general, are prohibitive for energetic, amplified pulses that might otherwise compromise the integrity of the focus as a result of nonlinear effects.

General theoretical treatment of spectral modulation light-labeling spectroscopy.

We theoretically derive the analytic relationship between experimental parameters and the measured incident (or illumination) optical power spectrum for a new form of spectroscopy, entitled light labeling spectroscopy. The light labeling signals are shown to arise from the interference between fields diffracted from a grating with time varying ruling density. A Gaussian model is used to illustrate the bounds of the method for recovering power spectra without artificial spectral apodization. Finally, several example systems are tabulated to give numerical insight into the possible system performances across a range of wavelength regions.

Submillisecond second harmonic holographic imaging of biological specimens in three dimensions.

Optical microscopy has played a critical role for discovery in biomedical sciences since Hooke's introduction of the compound microscope. Recent years have witnessed explosive growth in optical microscopy tools and techniques. Information in microscopy is garnered through contrast mechanisms, usually absorption, scattering, or phase shifts introduced by spatial structure in the sample. The emergence of nonlinear optical contrast mechanisms reveals new information from biological specimens. However, the intensity dependence of nonlinear interactions leads to weak signals, preventing the observation of high-speed dynamics in the 3D context of biological samples. Here, we show that for second harmonic generation imaging, we can increase the 3D volume imaging speed from sub-Hertz speeds to rates in excess of 1,500 volumes imaged per second. This transformational capability is possible by exploiting coherent scattering of second harmonic light from an entire specimen volume, enabling new observational capabilities in biological systems.

Third harmonic generation microscopy of a mouse retina.

PURPOSE: To demonstrate lipid-specific imaging of the retina through the use of third harmonic generation (THG), a multiphoton microscopic technique in which tissue contrast is generated from optical inhomogeneities. METHODS: A custom fiber laser and multiphoton microscope was constructed and optimized for simultaneous two-photon autofluorescence (TPAF) and THG retinal imaging. Imaging was performed using fixed-frozen sections of mouse eyes without the use of exogenous fluorescent dyes. In parallel experiments, a fluorescent nuclear stain was used to verify the location of the retinal cell nuclei. RESULTS: Simultaneous THG and TPAF images revealed all retinal layers with subcellular resolution. In BALB/c strains, the THG signal stems from the lipidic organelles of the cellular and nuclear membranes. In the C57BL/6 strain, the THG signal from the RPE cells originates from the pigmented granules. CONCLUSIONS: THG microscopy can be used to image structures of the mouse retina using contrast inherent to the tissue and without the use of a fluorescent dye or exogenously expressed recombinant protein.

Two-dimensional single-pixel imaging by cascaded orthogonal line spatial modulation.

Two-dimensional (2D) images are taken using a single-pixel detector by temporally multiplexing spatial frequency projections from orthogonal, time varying spatial line modulation gratings. Unique temporal frequencies are applied to each point in 2D space, applying a continuous spread of frequencies to one dimension, and an offset frequency applied to each line in the orthogonal dimension. The object contrast information can then be recovered from the electronic spectrum of the single pixel, and through simple processing be reformed into a spatial image.

Plane wave analysis of coherent holographic image reconstruction by phase transfer (CHIRPT).

Fluorescent imaging plays a critical role in a myriad of scientific endeavors, particularly in the biological sciences. Three-dimensional imaging of fluorescent intensity often requires serial data acquisition, that is, voxel-by-voxel collection of fluorescent light emitted throughout the specimen with a nonimaging single-element detector. While nonimaging fluorescence detection offers some measure of scattering robustness, the rate at which dynamic specimens can be imaged is severely limited. Other fluorescent imaging techniques utilize imaging detection to enhance collection rates. A notable example is light-sheet fluorescence microscopy, also known as selective-plane illumination microscopy, which illuminates a large region within the specimen and collects emitted fluorescent light at an angle either perpendicular or oblique to the illumination light sheet. Unfortunately, scattering of the emitted fluorescent light can cause blurring of the collected images in highly turbid biological media. We recently introduced an imaging technique called coherent holographic image reconstruction by phase transfer (CHIRPT) that combines light-sheet-like illumination with nonimaging fluorescent light detection. By combining the speed of light-sheet illumination with the scattering robustness of nonimaging detection, CHIRPT is poised to have a dramatic impact on biological imaging, particularly for in vivo preparations. Here we present the mathematical formalism for CHIRPT imaging under spatially coherent illumination and present experimental data that verifies the theoretical model.

Three-photon excitation source at 1250 nm generated in a dual zero dispersion wavelength nonlinear fiber.

We demonstrate 1250 nm pulses generated in dual-zero dispersion photonic crystal fiber capable of three-photon excitation fluorescence microscopy. The total power conversion efficiency from the 28 fs seed pulse centered at 1075 nm to pulses at 1250 nm, including coupling losses from the nonlinear fiber, is 35%, with up to 67% power conversion efficiency of the fiber coupled light. Frequency-resolved optical gating measurements characterize 1250 nm pulses at 0.6 nJ and 2 nJ, illustrating the change in nonlinear spectral phase accumulation with pulse energy even for nonlinear fiber lengths < 50 mm. The 0.6 nJ pulse has a 26 fs duration and is the shortest nonlinear fiber derived 1250 nm pulse yet reported (to the best of our knowledge). The short pulse durations and energies make these pulses a viable route to producing light at 1250 nm for multiphoton microscopy, which we we demonstrate here, via a three-photon excitation fluorescence microscope.

Nonlinear fiber amplifier with tunable transform limited pulse duration from a few 100 to sub-100-fs at watt-level powers.

We report a fiber amplifier system with an output transform limited pulse duration that is broadly tunable from 400 to 60 fs. We produce <100 fs pulses with >200 kW of peak power by compensating a significant amount of third-order dispersion. The spectral noise characteristics are also investigated to insure highly stable supercontinuum generation.

Overcoming temporal polarization instabilities from the latent birefringence in all-normal dispersion, wave-breaking-extended nonlinear fiber supercontinuum generation.

The intrinsic weak birefringence in all-normal dispersion highly nonlinear fiber, particularly ultra-high-numerical-aperture fiber, generates supercontinuum with long term polarization instabilities, even for seed pulses launched along the perceived slow axis of the fiber. Highly co/anti-correlated fluctuations in energy between regions of power spectral density mask the extent of the spectral noise in total integrated power measurements. The instability exhibits a seed pulse power threshold above which the output polarization state of the supercontinuum seeds from noise. Eliminating this instability through the utilization of nonlinear fiber with a large designed birefringence, encourages the exploration of compression schemes and seed sources. Here, we include an analysis of the difficulties for seeding supercontinuum with the highly attractive ANDi-type lasers. Lastly, we introduce an intuitive approach for understanding supercontinuum development and evolution. By modifying the traditional characteristic dispersion and nonlinear lengths to track pulse properties within the nonlinear fiber, we find simple, descriptive handles for supercontinuum evolution.

Two-dimensional spatial-frequency-modulated imaging through parallel acquisition of line images.

This Letter demonstrates a two-dimensional imaging technique that uses a line scan camera to resolve one spatial dimension and temporal modulation to resolve the perpendicular dimension. A temporal intensity modulation, which increases linearly in frequency along one direction is applied to an illumination beam. The modulated light distribution is imaged onto an object then onto a line scan camera oriented perpendicularly to the direction of the modulation sweep. A line diffuser is placed shortly before the line scan camera and diffuses light along the direction of modulation so that each pixel collects all modulation frequencies.

Hilbert reconstruction of phase-shifted second-harmonic holographic images.

New techniques are presented that make phase-shifting holography viable for second-harmonic generation (SHG) holography with weak object fields. We developed an intrinsic phase shift calibration of SHG holograms, an algorithm that extracts the reference and object intensity directly from a set of phase-shifted holographic data, and a more robust phase-shifting holography reconstruction algorithm based on π-shifted hologram pairs that permits self-calibration of the phase shift and recovery of the complex field through a Hilbert transform.

Theory of diffraction effects in spatial frequency-modulated imaging.

An analytic theory describing the effects of diffraction and aberrations on single-pixel imaging performed by temporally modulating illumination light is presented. This method encodes spatial information using sinusoidal temporal modulations that are chirped in frequency across the extent of an illumination line focus. With some approximations, a point spread function relationship as a function of defocus or other aberrations is found for both spatially coherent and incoherent cases. The theory is validated through experiments and simulations, including measurement of the transverse and longitudinal optical transfer function, and confirmation of insensitivity to aberrations and significant optical scattering after encoding of spatial information through temporal modulation.