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.

Spectral phase and amplitude retrieval and compensation technique for measurement of pulses.

In this Letter, an in-line, compact, and efficient quantitative pulse compensation and measurement scheme is demonstrated. This simple system can be readily deployed in multiphoton imaging systems and advanced manufacturing where multiphoton processes are exploited.

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.

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.

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.

Broadband interferometric characterization of divergence and spatial chirp.

We demonstrate a spectral interferometric method to characterize lateral and angular spatial chirp to optimize intensity localization in spatio-temporally focused ultrafast beams. Interference between two spatially sheared beams in an interferometer will lead to straight fringes if the wavefronts are curved. To produce reference fringes, we delay one arm relative to another in order to measure fringe rotation in the spatially resolved spectral interferogram. With Fourier analysis, we can obtain frequency-resolved divergence. In another arrangement, we spatially flip one beam relative to the other, which allows the frequency-dependent beamlet direction (angular spatial chirp) to be measured. Blocking one beam shows the spatial variation of the beamlet position with frequency (i.e., the lateral spatial chirp).

Spatial-spectral characterization of focused spatially chirped broadband laser beams.

Proper alignment is critical to obtain the desired performance from focused spatially chirped beams, for example in simultaneous spatial and temporal focusing (SSTF). We present a simple technique for inspecting the beam paths and focusing conditions for the spectral components of a broadband beam. We spectrally resolve the light transmitted past a knife edge as it was scanned across the beam at several axial positions. The measurement yields information about spot size, M2, and the propagation paths of different frequency components. We also present calculations to illustrate the effects of defocus aberration on SSTF beams.

Measurement of energy contrast of amplified ultrashort pulses using cross-polarized wave generation and spectral interferometry.

We present a method using spectral interferometry (SI) to characterize a pulse in the presence of an incoherent background such as amplified spontaneous emission (ASE). The output of a regenerative amplifier is interfered with a copy of the pulse that has been converted using third-order cross-polarized wave generation (XPW). The ASE shows as a pedestal background in the interference pattern. The energy contrast between the short-pulse component and the ASE is retrieved. The spectra of the interacting beams are obtained through an improvement to the self-referenced spectral interferometry (SRSI) analysis.

Advances in multiphoton microscopy technology.

Multiphoton microscopy has enabled unprecedented dynamic exploration in living organisms. A significant challenge in biological research is the dynamic imaging of features deep within living organisms, which permits the real-time analysis of cellular structure and function. To make progress in our understanding of biological machinery, optical microscopes must be capable of rapid, targeted access deep within samples at high resolution. In this Review, we discuss the basic architecture of a multiphoton microscope capable of such analysis and summarize the state-of-the-art technologies for the quantitative imaging of biological phenomena.

Direct diode-pumped Kerr-lens mode-locked Ti:sapphire laser.

We describe a Ti:sapphire laser pumped directly with a pair of 1.2 W 445 nm laser diodes. With over 30 mW average power at 800 nm and a measured pulsewidth of 15 fs, Kerr-lens-modelocked pulses are available with dramatically decreased pump cost. We propose a simple model to explain the observed highly stable Kerr-lens modelocking in spite of the fact that both the mode-locked and continuous-wave modes are smaller than the pump mode in the crystal.

Intuitive analysis of space-time focusing with double-ABCD calculation.

We analyze the structure of space-time focusing of spatially-chirped pulses using a technique where each frequency component of the beam follows its own Gaussian beamlet that in turn travels as a ray through the system. The approach leads to analytic expressions for the axially-varying pulse duration, pulse-front tilt, and the longitudinal intensity profile. We find that an important contribution to the intensity localization obtained with spatial-chirp focusing arises from the evolution of the geometric phase of the beamlets.

Eliminating the scattering ambiguity in multifocal, multimodal, multiphoton imaging systems.

In this work we present how to entirely remove the scattering ambiguity present in existing multiphoton multifocal systems. This is achieved through the development and implementation of single-element detection systems that incorporate high-speed photon-counting electronics. These systems can be used to image entire volumes in the time it takes to perform a single transverse scan (four depths simultaneously at a rate of 30 Hz). In addition, this capability is further exploited to accomplish single-element detection of multiple modalities (two photon excited fluorescence and second harmonic generation) and to perform efficient image deconvolution. Finally, we demonstrate a new system that promises to significantly simplify this promising technology.

Differential Multiphoton Laser Scanning Microscopy.

Multifocal multiphoton microscopy (MMM) in the biological and medical sciences has become an important tool for obtaining high resolution images at video rates. While current implementations of MMM achieve very high frame rates, they are limited in their applicability to essentially those biological samples that exhibit little or no scattering. In this paper, we report on a method for MMM in which imaging detection is not necessary (single element point detection is implemented), and is therefore fully compatible for use in imaging through scattering media. Further, we demonstrate that this method leads to a new type of MMM wherein it is possible to simultaneously obtain multiple images and view differences in excitation parameters in a single shot.

Blind frequency-resolved optical-gating pulse characterization for quantitative differential multiphoton microscopy.

We use a unique multifocal multiphoton microscope to directly characterize the pulse in the focal plane of a high-NA objective using second-harmonic generation frequency-resolved optical gating (FROG). Because of the nature of the optical setup, femtosecond laser pulses of orthogonal polarization states are generated in the focal plane, each acquiring a different spectral dispersion. By applying an additional constraint on the phase extraction algorithm, we simultaneously extract both the gate and probe pulses from a single spectrogram with a FROG error of 0.016.

Optimizing the fluorescent yield in two-photon laser scanning microscopy with dispersion compensation.

A challenge for nonlinear imaging in living tissue is to maximize the total fluorescent yield from each fluorophore. We investigated the emission rates of three fluorophores-rhodamine B, a red fluorescent protein, and CdSe quantum dots-while manipulating the phase of the laser excitation pulse at the focus. In all cases a transform-limited pulse maximized the total yield to insure the highest signal-to-noise ratio. Further, we find evidence of fluorescence antibleaching in quantum dot samples.

Spatiotemporal dynamics of cross-polarized wave generation.

We use time-domain spatially and spectrally resolved interferometry to investigate cross-polarized wave (XPW) generation in barium fluoride. We find that the XPW pulse is square root of 3 smaller than the input in the spatiotemporal domain, regardless of input chirp. Additionally, we calculate a temporally dependent focal length resulting from the nonlinear interaction and discuss its implications.

Remote focusing for programmable multi-layer differential multiphoton microscopy.

We present the application of remote focusing to multiphoton laser scanning microscopy and utilize this technology to demonstrate simultaneous, programmable multi-layer imaging. Remote focusing is used to independently control the axial location of multiple focal planes that can be simultaneously imaged with single element detection. This facilitates volumetric multiphoton imaging in scattering specimens and can be practically scaled to a large number of focal planes. Further, it is demonstrated that the remote focusing control can be synchronized with the lateral scan directions, enabling imaging in orthogonal scan planes.

Characterization of coupled nonlinear spatiospectral phase following an ultrafast self-focusing interaction.

We use collinear spatially resolved spectral interferometery to characterize the nonlinear phase changes experienced by an intense ultrashort pulse propagating in glass. The measurement yields the spectrally dependent wavefront, allowing us to measure the spatial and chromatic aberrations of the nonlinearly induced lens. For these conditions, we find that while the shape of the spatial wavefront follows the beam profile as expected, the spectral dependence of the lensing power is determined by the self-phase modulation. The simultaneous measurement of the nonlinear spatiospectral phase demonstrates how the nonlinear spectral phase is coupled to self-focusing.

Advancing multifocal nonlinear microscopy: development and application of a novel multibeam Yb:KGd(WO4)2 oscillator.

We present a novel Yb:KGd(WO(4))(2) oscillator design that generates six beams of temporally delayed, 253 fs, 11 nJ pulses. This allows multifocal nonlinear microscopy to be performed without the need for complicated optical multiplexers. We demonstrate our design with twelve simultaneously acquired two-photon, second-harmonic and/or third-harmonic images generated from six laterally separated foci.

A modular approach to the analytic calculation of spectral phase for grisms and other refractive/diffractive structures.

Analytic expressions for spectral phase for optical systems are very important for the design of wide-bandwidth optical systems. We describe a general formalism for analytically calculating the spectral phase for arbitrary optical structure made up of nested pairs of plane-parallel interfaces that can be diffractive or refractive. Our primary application is the calculation of the spectral phase of a grism pair, which is then used to analyze the behavior of higher-order phase terms. The analytic expressions for the grism spectral phase provide insight into the tunability of the third-order phase of grisms as well as the fourth-order limits. Our exact and approximate expressions are compared with a raytracing model.