Aberration free synthetic aperture second harmonic generation holography.

Second harmonic generation (SHG) microscopy is a valuable tool for optical microscopy. SHG microscopy is normally performed as a point scanning imaging method, which lacks phase information and is limited in spatial resolution by the spatial frequency support of the illumination optics. In addition, aberrations in the illumination are difficult to remove. We propose and demonstrate SHG holographic synthetic aperture holographic imaging in both the forward (transmission) and backward (epi) imaging geometries. By taking a set of holograms with varying incident angle plane wave illumination, the spatial frequency support is increased and the input and output pupil phase aberrations are estimated and corrected - producing diffraction limited SHG imaging that combines the spatial frequency support of the input and output optics. The phase correction algorithm is computationally efficient and robust and can be applied to any set of measured field imaging data.

Cascaded domain multiphoton spatial frequency modulation imaging.

Significance: Multiphoton microscopy is a powerful imaging tool for biomedical applications. A variety of techniques and respective benefits exist for multiphoton microscopy, but an enhanced resolution is especially desired. Additionally multiphoton microscopy requires ultrafast pulses for excitation, so optimization of the pulse duration at the sample is critical for strong signals. Aim: We aim to perform enhanced resolution imaging that is robust to scattering using a structured illumination technique while also providing a rapid and easily repeatable means to optimize group delay dispersion (GDD) compensation through to the sample. Approach: Spatial frequency modulation imaging (SPIFI) is used in two domains: the spatial domain (SD) and the wavelength domain (WD). The WD-SPIFI system is an in-line tool enabling GDD optimization that considers all material through to the sample. The SD-SPIFI system follows and enables enhanced resolution imaging. Results: The WD-SPIFI dispersion optimization performance is confirmed with independent pulse characterization, enabling rapid optimization of pulses for imaging with the SD-SPIFI system. The SD-SPIFI system demonstrates enhanced resolution imaging without the use of photon counting enabled by signal to noise improvements due to the WD-SPIFI system. Conclusions: Implementing SPIFI in-line in two domains enables full-path dispersion compensation optimization through to the sample for enhanced resolution multiphoton microscopy.

Design and analysis of polygonal mirror-based scan engines for improved spatial frequency modulation imaging.

Spatial frequency modulation imaging (SPIFI) is a structured illumination single pixel imaging technique that is most often achieved via a rotating modulation disk. This implementation produces line images with exposure times on the order of tens of milliseconds. Here, we present a new architecture for SPIFI using a polygonal scan mirror with the following advances: (1) reducing SPIFI line image exposure times by 2 orders of magnitude, (2) facet-to-facet measurement and correction for polygonal scan design, and (3) a new anamorphic magnification scheme that improves resolution for long working distance optics.

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.

Single-shot spatial frequency modulation for imaging.

Spatial frequency modulation for imaging (SPIFI) has traditionally employed a time-varying spatial modulation of the excitation beam. Here, for the first time to our knowledge, we introduce single-shot SPIFI, where the spatial frequency modulation is imposed across the entire spatial bandwidth of the optical system simultaneously enabling single-shot operation.

Fisher information and the Cramér-Rao lower bound in single-pixel localization microscopy with spatiotemporally modulated illumination.

Single-pixel imaging, the concept that an image can be captured via a single-pixel detector, is a cost-effective yet powerful technique to reduce data acquisition duration without sacrificing image resolution when properly structured illumination patterns are introduced. Normally, the image reconstruction process is subject to the diffraction limit. Here, we study the possibility of exploiting the information contained in the illumination patterns to enable a form of single-pixel localization microscopy (SPLM) for super-resolution. This concept is inspired by coherent holographic image reconstruction by phase transfer (CHIRPT) microscopy. CHIRPT microscopy is a single-pixel imaging technique that uses structured illumination that is spatiotemporally modulated (STM) so that a unique temporal modulation pattern is imparted to each point within a large illumination volume. The fluorescent light emitted by molecules contains the same temporal modulations as the illumination patterns at the locations of the molecules. By recording a portion of the total emitted fluorescent power, the signal may be numerically processed to form an image. Unique temporal modulation patterns that excite fluorescent probes at each point can also be used to localize individual molecules by matching their particular temporal light emission patterns to the measured temporal signal. This paper evaluates the feasibility of SPLM with STM illuminations used in and inspired by CHIRPT microscopy via the information content its data carry about the emitter location(s). More specifically, we provide the mathematical formalism of Fisher information (FI) and the Cramér-Rao lower bound (CRLB) associated with the location parameters of the emitter(s). The FI and CRLB are then numerically evaluated under different experimental assumptions to assess the effects of experimental parameters on localization precision. Last, we compare the single-pixel CRLB to that from camera-based single-molecule localization microscopy in the localization of a single emitter. We show that SPLM has several distinguishing characteristics that provide certain advantages, such as relatively constant CRLB over a very large illumination volume and improved CRLB for 3D localization due to the information coupling introduced by simultaneous modulations of the transverse axes.

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.

Two-dimensional random access multiphoton spatial frequency modulated imaging.

Spatial frequency modulated imaging (SPIFI) enables the use of an extended excitation source for linear and nonlinear imaging with single element detection. To date, SPIFI has only been used with fixed excitation source geometries. Here, we explore the potential for the SPIFI method when a spatial light modulator (SLM) is used to program the excitation source, opening the door to a more versatile, random access imaging environment. In addition, an in-line, quantitative pulse compensation and measurement scheme is demonstrated using a new technique, spectral phase and amplitude retrieval and compensation (SPARC). This enables full characterization of the light exposure conditions at the focal plane of the random access imaging system, an important metric for optimizing, and reporting imaging conditions within specimens.

Interferometric spatial frequency modulation imaging.

Interferometric spatial frequency modulation for imaging (I-SPIFI) is demonstrated for the first time, to our knowledge. Significantly, this imaging modality can be seamlessly combined with nonlinear SPIFI imaging and operates through single-element detection, making it compatible for use in scattering specimens. Imaging dynamic processes with submicrometer axial resolution through long working distance optics is shown, and high contrast images compared to traditional wide-field microscopy images. Finally, enhanced lateral resolution is achieved in I-SPIFI. To our knowledge, this is the first single platform that enables multimodal linear and nonlinear imaging, with enhanced resolution, all of which can be performed simultaneously.

Fabrication and characterization of modulation masks for multimodal spatial frequency modulated microscopy.

Spatial frequency modulated imaging (SPIFI) is a powerful imaging method that when used in conjunction with multiphoton contrast mechanisms has the potential to improve the spatial and temporal scales that can be explored within a single nonlinear optical microscope platform. Here we demonstrate, for the first time to our knowledge, that it is possible to fabricate inexpensive masks using femtosecond laser micromachining that can be readily deployed within the multiphoton microscope architecture to transform the system from a traditional point-scanning system to SPIFI and gain the inherent advantages that follow.

High-resolution mosaic imaging with multifocal, multiphoton photon-counting microscopy.

High-resolution mosaic imaging is performed for the first time to our knowledge with a multifocal, multiphoton, photon-counting imaging system. We present a novel design consisting of a home-built femtosecond Yb-doped KGdWO(4) laser with an optical multiplexer, which is coupled with a commercial Olympus IX-71 microscope frame. Photon counting is performed using single-element detectors and an inexpensive electronic demultiplexer and counters.