David G. Winters

Spatially-chirped modulation imaging of absorbtion and fluorescent objects on single-element optical detector

Line imaging of fluorescent and absorptive objects with a single-pixel imaging technique that acquires one-dimensional cross-sections through a sample by imposing a spatially-varying amplitude modulation on the probing beam is demonstrated. The fluorophore concentration or absorber distribution of the sample is directly mapped to modulation frequency components of the spatially-integrated temporal signal. Time-domain signals are obtained from a single photodiode, with object spatial frequency correlation encoded in time-domain bursts in the electronic signal from the photodiode.

Subpicosecond fiber-based soliton-tuned mid-infrared source in the 9.7–14.9 μm wavelength region

We present a fiber-based mid-IR source of ultrafast laser pulses tunable in the 9.7-14.9 microm spectral region based on difference-frequency mixing between an Er:fiber laser and a first-order frequency-shifted soliton pulse. We have measured subpicosecond pulses at a repetition rate of 37 MHz, with a maximum power of 1.5 microW.

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.

Measurement of orientation and susceptibility ratios using a polarization-resolved second-harmonic generation holographic microscope

Three-dimensional second-harmonic fields, sample orientation, and susceptibility ratios of biological samples are measured using polarization-resolved second-harmonic generation (SHG) microscopy. The three-dimensional (3D) polarization is gathered by measurement of a series of holograms for which excitation and analyzer polarizations are systematically varied, and the 3D SHG field is recovered through numerical back propagation. Harmonophore orientation is resolved in 3D from a sub-set of polarization-resolved SHG holograms. We further expand on previous approaches for the determination of susceptibility ratios, adding the calculation of multiple ratio values to allow intrinsic verification.

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.

Submillisecond second harmonic holographic imaging of biological specimens in three dimensions

Significance Although nonlinear optical microscopy is a widely used technique, weak signal levels necessitate relatively slow 3D imaging rates. We demonstrate 3D second harmonic generation (SHG) with imaging speeds many orders of magnitude faster than previously described. A comparison of conventional laser scanning and holographic SHG imaging shows higher quality images are obtained with holography, and at higher speeds than in conventional experiments while using significantly reduced illumination intensity in the specimen. Rigorous new methods are introduced that allow robust quantification of image, noise, and thus signal-to-noise quality. This advance will open nonlinear optical imaging to the study of high-speed dynamics of biological specimens and tissues that display behavior current experimental methodologies fail to capture, such as neural circuit dynamics. 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.

Hyperspectral imaging via labeled excitation light and background-free absorption spectroscopy

We introduce a new method of hyperspectral imaging which encodes a varying temporal intensity modulation onto the excitation (or illumination) power spectrum. Functionally, we have moved the spectrometer from the back end of an experiment to the front, where intensity modulations uniquely label wavelengths within the excitation power spectrum at different frequencies, thereby creating a temporal light label which we can identify after subsequent light–matter interactions. To demonstrate this method, we acquire two-dimensional micrographs of background-free absorption spectra by capturing the intensity modulations transferred from the excitation spectrum into the emitted fluorescent intensity. Both the temporal light labeling method and the demonstrated excitation-labeled fluorescence application are readily adaptable to hyperspectral acquisition rates far beyond the frame rates of high-speed cameras.

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.

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.

Single-pixel fluorescent imaging with temporally labeled illumination patterns

Most coherent imaging methods, such as holography, cannot be directly applied to fluorescent light emission, because it is spatially incoherent. We introduce coherent holographic image reconstruction by phase transfer (CHIRPT), an imaging technique that transfers the spatial phase of coherent illumination light to temporal modulations of the emitted fluorescence intensity. The spatial phase information encoded in the temporal modulations is exploited to digitally propagate fluorescent light to render two-dimensional images from a temporal signal obtained from a single-pixel photodetector. The image formation mechanism of CHIRPT provides much larger imaging depths of field than conventional imaging, permitting data far away from the focal plane to be digitally refocused.