Imaging of the interior of object with light has long been a challenge for optical imaging. Optical diffraction tomography (ODT) is able to obtain three-dimensional (3D) object information through object rotation. We will discuss harmonic optical tomography (HOT) that exploits a defocused illumination beam for nonlinear optical tomography. We will also discuss our demonstration of coherent ODT with incoherent light emission in a new optical tomography technique called fluorescent diffraction tomography (FDT) and the use of spatial frequency imaging for high speed nonlinear optical microscopy.
Raman microscopy has been widely developed for label free nonlinear optical microscopy of biological systems. Imaging speed in these systems is hampered by low Raman scattering cross sections and the requirement of scanning a focused laser beam through the sample in conventional Raman microscopy. The serial acquisition that is necessary in point scanning microscopy slows image acquisition and limits the dwell time are each image pixel. Here, we discuss two new imaging methods that are based on spatial frequency modulation imagining (SPIFI) [1-2], where a structured line focus is used to image is used to image specimens by collecting light on a single pixel detector. We discuss the use of SPIFI to improve the imaging speed of Spontaneous Raman scattering and coherent anti-Stokes Raman scattering microscopy. A detailed noise analysis highlighting the advantages and disadvantages of SPIFI as compared to conventional point scan imaging is presented.
In recent years, we demonstrated a new approach to super-resolution microscopy based on driving a nonlinear interaction with a Spatial Frequency Modulated Imaging (SPIFI). SPIFI is a line imaging technique that linearly sweeps all the frequencies supported by the band-pass of the objective lens. Here we introduce a new method of unrestricted super-resolution imaging based on driving saturated absorption in the specimen excitation. The saturated absorption drives harmonic distortions of the spatial frequencies used to illuminate the sample. These harmonics manifest themselves as temporal harmonic frequencies allowing for easy detection and separation of the super-resolution information in the far field.
Conventional CARS microscopy requires scanning a point focus through the specimen limits imaging speed. We present a spatial frequency projection imaging (SPIFI) method for CARS microscopy to spatially multiplex CARS microscopy. A spinning disk modulator is used to rapidly modulate the Stokes field with a rapidly swept spatially periodic transmission grating. SPIFI-CARS images are obtained by Fourier transforming the single pixel signal. Images of CARS and second harmonic generation from histological slices will be presented. The physics of image formation and the impact of multiplexing on SNR will be discussed. Prospects for scaling to high speed CARS imaging will be discussed.
Recently we have demonstrated that spatial frequency modulation imaging can use extended excitation sources in linear and nonlinear image modalities, is compatible with single element detection, and results in enhanced lateral resolution across the excitation beam. In this paper, we will present new methods where the SPIFI platform goes from one-dimensional to two-dimensional imaging while still exhibiting the enhanced resolution across the added dimension. Significantly, we present the physical mechanism responsible for the resolution enhancement for all imaging modalities, we provide computational models that support the physical model for the increased resolution, and finally, present experimental verification of the resolution enhancement.
We introduce a method for quantitative hyperspectral optical imaging in the spatial frequency domain (hs-SFDI) to image tissue absorption (μa) and reduced scattering (μs′) parameters over a broad spectral range. The hs-SFDI utilizes principles of spatial scanning of the spectrally dispersed output of a supercontinuum laser that is sinusoidally projected onto the tissue using a digital micromirror device. A scientific complementary metal–oxide–semiconductor camera is used for capturing images that are demodulated and analyzed using SFDI computational models. The hs-SFDI performance is validated using tissue-simulating phantoms over a range of μa and μs′ values. Quantitative hs-SFDI images are obtained from an ex-vivo beef sample to spatially resolve concentrations of oxy-, deoxy-, and met-hemoglobin, as well as water and fat fractions. Our results demonstrate that the hs-SFDI can quantitatively image tissue optical properties with 1000 spectral bins in the 580- to 950-nm range over a wide, scalable field of view. With an average accuracy of 6.7% and 12.3% in μa and μs′, respectively, compared to conventional methods, hs-SFDI offers a promising approach for quantitative hyperspectral tissue optical imaging.
Imaging with a single pixel confers many advantages for biological imaging, particularly in the case of tissues, where optical scattering obscured image signals for conventional imaging techniques. While laser scanning confocal and multiphoton imaging are powerful techniques that are routinely deployed for biological imaging, the signals must be acquired by scanning the focal spot sequentially through the entire region of interest. In recent years, we have introduced a new single pixel imaging method that speeds up imaging in tissues by spreading the conventional excitation spot to a spatial-temporally modulated line focus. In our method, the illumination beam is modulated with a spatial frequency that sweeps linearly in time, and is thus called spatial frequency projection imaging (SPIFI). SPIFI used with a nonlinear optical response also results in super-resolution imaging.
The challenge with SPIFI is that it is a one-dimensional imaging method, and consequentially, the spatial resolution enhancements afforded by nonlinear SPIFI imaging similarly only appear along the modulated spatial coordinate. Here, we introduce a new form of tomographic imaging that homogenized SPIFI imaging resolution along both coordinates of the object that is imaged. The method is a conjugate domain form of computed tomography (CT), that forms spatial frequency projections, parameterized by rotation angle, rather than spatial projections that are used in conventional CT. We develop theory and experimentally demonstrate Fourier coherent tomographic imaging of objects both with bright field (intensity transmission) and fluorescent emission modes. We demonstrate isotropic improvement in spatial resolution with this technique.
Single-pixel imaging is a developing family of techniques
which offer several advantages over conventional imaging with a segmented detector.
These include higher speed, improved availability and quality
of detectors at long wavelengths.
Examples include laser-scanning microscopy,
frequency-domain techniques, ghost imaging,
and methods employing an orthogonal mask sequence such as Hadamard masks.
We analyze this class of imaging techniques in terms of Frame theory,
which concerns sets of vectors that span a given vector space
but are not linearly independent as in the case of a basis.
The use of frames (rather than bases) allows for redundant measurements,
which can improve the signal-to-noise ratio (SNR) of the reconstructed image.
Current single-pixel techniques
admit an intuitive, physically-motivated reconstruction scheme,
but the reconstruction method is not always obvious.
The analysis provides a prescription
for reconstruction with any single-pixel imaging scheme.
For example, illumination with speckle-like patterns
which lack the statistical properties associated with speckle
does not allow accurate reconstruction with conventional methods,
but frame theory-inspired analysis allows
production of high-contrast, diffraction-limited images.
Even for schemes where reconstruction methods exist,
the theory can improve contrast, accuracy and resolution.
Frame theory-motivated reconstruction from simulated ghost imaging data
results in markedly improved contrast,
and resolution.
This analysis makes viable new single-pixel techniques
which lack intuitive reconstruction strategies,
and tuning of imaging properties such as noise for specific applications.
Through spatial frequency modulated imaging (SPIFI), multimodal, multiphoton microscopy (MPM) benefits from an extended excitation source without compromising the key performance characteristics afforded by point scanning MPM platforms. For example, the introduction of an in-house custom machined mask, which imparts a spatially distinct, temporal amplitude modulation to the extended excitation source, allows one and two-dimensional images to be captured with single element detection. This enables extended source imaging methods to retain a key feature of the point scanning systems; namely, the ability to image within scattering media, at depth.
Further, the range of contrast mechanisms for the extended source techniques presented here are not limited and readily extend to both linear and nonlinear imaging modalities. The SPIFI method developed here enables facile detection of such images with the added benefit of enhanced resolution. Notably, the resolution improvement holds across contrast mechanisms, and is independent of whether the contrast is generated through linear or nonlinear processes. Significantly, phase also comes into play as we present new SPIFI geometries that illustrate the role of phase in strategically controlling the source geometry and/or generating image contrast.
Optical microscopes are routinely employed for imaging live cell dynamics. Until recently, conventional optical microscopes lacked the ability to resolve spatial features significantly smaller than the wavelength of light. This kept the structure and dynamics of a vast array of biological processes hidden. Understanding the spatial organization and temporal dynamics of nanoscale molecular assemblies is critical to developing a comprehensive understanding of biology. In recent years, super-resolution (SR) microscopes have enabled routine live cell imaging at spatial resolutions <50nm. These new tools produced discoveries that challenged multiple paradigms of intracellular processes. Because optical scattering severely distorts SR methods, the SR imaging revolution has failed to be translated deep into scattering tissue. Yet it is well known that the behavior of cells in tissues and tumors deviates strongly from the behavior of 2D cell cultures. Here we present a new approach to optical SR imaging with spatial frequency modulated imaging that is, in principle, capable of providing unrestricted spatial resolution deep in live animal tissues. A broad illumination bandwidth homogenizes speckle that would otherwise be accumulated by the spatiotemporally structured illumination light, thereby preventing the speckle from distorting the image formation process. Further, scattering of the fluorescent light emitted from the object does not impact the quality of the measured image. We detail the principles of this SR imaging method and present both analytical and numerical calculations that test these concepts. Such discoveries will likely drive an improvement in our understanding of biology and disease.
Many single-pixel imaging techniques have been developed in recent years. Though the methods of image acquisition vary considerably, the methods share unifying features that make general analysis possible. Furthermore, the methods developed thus far are based on intuitive processes that enable simple and physically-motivated reconstruction algorithms, however, this approach may not leverage the full potential of single-pixel imaging. We present a general theoretical framework of single-pixel imaging based on frame theory, which enables general, mathematically rigorous analysis. We apply our theoretical framework to existing single-pixel imaging techniques, as well as provide a foundation for developing more-advanced methods of image acquisition and reconstruction. The proposed frame theoretic framework for single-pixel imaging results in improved noise robustness, decrease in acquisition time, and can take advantage of special properties of the specimen under study. By building on this framework, new methods of imaging with a single element detector can be developed to realize the full potential associated with single-pixel imaging.
Hyperspectral Imaging (HSI) is a growing field in tissue optics due to its ability to collect continuous spectral features of a sample without a contact probe. Spatial Frequency Domain Imaging (SFDI) is a non-contact wide-field spectral imaging technique that is used to quantitatively characterize tissue structure and chromophore concentration. In this study, we designed a Hyperspectral SFDI (H-SFDI) instrument which integrated a supercontinuum laser source to a wavelength tuning optical configuration and a sCMOS camera to extract spatial (Field of View: 2cm×2cm) and broadband spectral features (580nm-950nm). A preliminary experiment was also performed to integrate the hyperspectral projection unit to a compressed single pixel camera and Light Labeling (LiLa) technique.
We present a novel single-pixel imaging technique that simultaneously images fluorescence and quantitative phase of an object. To extract simultaneously co-registered fluorescence and phase images, the object is illuminated by a pair of spatially coherent monochromatic laser beams with a difference in illumination spatial frequency that is swept linearly in time. One of the beams is stationary – serving as a reference beam – and propagated along the optic axis. The other beam scans through the full range of transverse spatial frequencies supported by the illumination optic – sweeping the crossing angle of the two beams incident on the specimen as a function of time. The scanned beam also has a temporal modulation carrier frequency that allows the extraction of the products of interfering fields. To record a phase image, forward scattered light from a thin object is collected in the back Fourier plane of a collection optic. Placing a narrow slit in the back Fourier plane allows the complex spatial frequency spectrum of the object amplitude transmission to be recorded in time. At each time point, the spatial frequency value corresponding to the difference in transverse spatial frequency of the illumination beams is recorded. Simultaneously, the interference of the illumination beams in the object imparts a spatial frequency pattern on the fluorescent molecule excitation and the spatial frequency of the object’s fluorescent concentration is recorded at each time step. This single-pixel imaging method allows for simultaneous acquisition of the object phase and fluorescent images by collecting spatial frequency projections in time.
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