Imaging with third harmonic generation (THG) provides a powerful label-free approach to probing biological systems, particularly looking at structures formed by lipids. We present the first demonstration of THG widefield holographic microscopy. Holographic microscopy preserves the amplitude and phase of the measured field, which enables us to construct a synthetic spatial aperture with expanded field of view for complex valued THG images. Additionally, we implement a matrix approach postprocessing algorithm to reconstruct aberration-free THG holographic images by estimating and correcting for phase aberrations and scattering distortions.
SignificanceMultiphoton 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.AimWe 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.ApproachSpatial 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.ResultsThe 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.ConclusionsImplementing SPIFI in-line in two domains enables full-path dispersion compensation optimization through to the sample for enhanced resolution multiphoton microscopy.
Using the structured illumination, single pixel detection imaging technique SPatIal Frequency modulation Imaging (SPIFI), we demonstrate a cascaded Wavelength Domain and Spatial Domain (WD-SD-SPIFI) system enabling real-time, in-line, second order dispersion compensation optimization for multiphoton imaging. Enhanced resolution is demonstrated by imaging a sub-diffractive 140 nm fluorescent nanodiamond with Two Photon Excitation Fluorescence (2PEF) to measure the Point Spread Function (PSF). With a 1034 nm pulsed laser through a Numerical Aperture (NA) of 0.5, a PSF Full Width at Half Max (FWHM) of 780 nm was measured with minimal post processing analysis that only requires Fast Fourier Transforms (FFTs).
Non-invasive imaging with high resolution deep within biological materials without the use of harmful ionizing radiation is of great interest in the field of medical imaging. Second harmonic generation is an excellent mechanism to circumvent this issue by providing outstanding contrast and optical sectioning. In general, these signals are weak and prone to scattering which introduce great challenges when imaging deep within turbid media. We will discuss recently demonstrated Epi-SHG holography, which can detect very weak backscattered SHG signals and enables the application of recently developed techniques which utilize the phase information to allow diffraction limited imaging within deep tissue.
Raman spectra provide a rich and powerful label-free probe of biological systems. While the narrow spectral features make Raman spectroscopy extremely attractive, the weak strength of Raman scattering makes it difficult to deeply in many situations. Low frequency Raman scattering spectroscopy provides the potential for probing macromolecular and local material properties. Until recently, the acquisition of such low frequency Raman spectra has been limited by technical challenges. We will discuss newly developed tools that allow for high-speed time-domain Raman spectroscopy of low and fingerprint region Raman spectra. Imaging performance and robustness of the signal to scattering will also be discussed.
Raman microscopy is a valuable approach to label-free chemical imaging. Recent advances have enabled the development of rapid methods for hyperspectral microscopy using impulsive stimulated Raman scattering (ISRS) that focus on low-frequency Raman modes of materials. The persistent challenge with Raman spectroscopic methods is low signal due to the relatively weak Raman scattering. ISRS microscopy allows for high quality hyperspectral imaging of low and fingerprint Raman vibrational frequencies but is normally subject to poor performance in the presence of optical scattering. We will present methods that solve this issue and allow for high-quality imaging when strong optical scattering is present.
In recent years, new super resolution imaging methods based on the anti-bunching properties of photons emitted by single quantum emitters have emerged. Thus far, these methods have been extremely limited in speed as they rely on very low repetition lasers to match the speed of cameras or use high-speed photon counting at individual points scanned across the surface of the object. Here, we study the use of spatio-temporally modulated illumination light to acquire photon counts from an extended region of the object. Thus, we combine high speed photon detection with extended illumination to enhance the imaging speed of anti-bunching super resolution microscopy.
Raman microscopy is a powerful mode of label free nonlinear optical microscopy that is hampered by weak cross-sections, leading to slow imaging. We will discuss two advances in coherent Raman microscopy: 1) high speed coherent anti-Stokes Raman scattering microscopy imaging based on spatial frequency modulation imagining (SPIFI) where a structured line focus is used to image is used to image specimens with a single pixel detector. 2) Doppler Raman microscopy that exploits the extremely low timing jitter of modelocked lasers for ultrasensitive Raman spectroscopy and microscopy.
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.
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.
Doppler Raman (DR) spectroscopy is a coherent Raman technique that combines impulsive Raman excitation with novel frequency shift detection to enable high-sensitivity Raman spectroscopy in the biological fingerprint region (500cm-1-1500cm-1) and the low frequency regime from 10cm-1 – 500cm-1. Using DR, we demonstrate nonresonant Raman spectroscopy on a suite of biologically significant targets involved in cell respiration including cytochrome c, adenosine triphosphate (ATP), flavin adenine dinucleotide (FAD), and nicotinamide adenine dinucleotide (NADH). High-sensitivity detection of low-to-medium frequency Raman vibrational modes may provide a tool to monitor states of cell respiration along with large molecular structural changes such as protein conformational dynamics.
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.
The majority of optical super-resolution imaging methods have been developed for thin or transparent biological samples, where the effects of scattering are minimized. Moreover, most of these techniques are based on the manipulation of fluorescent probes, or other molecular real-energy states. Multiphoton spatial frequency modulated imaging (MP-SPIFI) provides a pathway for super-resolving fine structures through multiple scattering lengths by making use of a modulated line focus and nonlinear excitation, and is applicable to both fluorescence and harmonic generation imaging. The technique works by projecting a set of 1D spatial frequencies onto the object, and utilizing the multiphoton interaction to drive harmonics of the spatial frequencies. The result is that an n-photon interaction yields frequency support of nearly 2n beyond the lens NA in the lateral x-dimension, along the line focus. However, the axial resolution of the object is limited in the conventional way by how tightly the line is focused. Here, we improve axial resolution by employing a limited-angle diffraction tomography, where the illumination is rotated in the x-z plane relative to the sample. The set of angular measurements are coherently combined in spatial-frequency space. Using a priori information about the location of each measurement in this kx-kz space greatly enhances the signal-to-noise ratio of the reconstructed object. We expect the method to be a useful way to improve resolution in deep-tissue imaging, or with any sample that exhibits strong scattering.
While fluorescence microscopy has revolutionized a broad range of biological studies, one of several challenges that remain is the need to increase image acquisition rates to image in dynamic specimens. Spatial-frequency-projection techniques, such as CHIRPT and SPIFI, utilize spatiotemporally structured illumination patterns to enable rapid acquisition of multidimensional images with a single-pixel detector. CHIRPT in particular shows promise for enhancing image acquisition rates because it encodes the spatial phase difference between two interfering illumination beams into temporal modulations of the fluorescent light emitted from the specimen. Consequently, the complex-valued, 1D image measured with CHIRPT can be digitally propagated to recover a 2D image of the fluorophore distribution in the specimen. Moreover, the depth-of-field (DOF) in CHIRPT with planar illumination approaches 100x the conventional limit and thus allows for large volumes of the specimen to be imaged simultaneously. Unfortunately, all configurations of CHIRPT reported to date require the use of focused light sheets to form 2D and 3D images – thereby restricting the effective DOF provided by CHIRPT to the conventional limit. In this work, we show that the addition of a confocal slit to the CHIRPT microscope allows one to control the effective DOF with linearly-excited fluorescence. We present experimental data and a complete optical theory that describes the experimental results. Confocal CHIRPT may enable rapid imaging by dramatically reducing the number of axial translations required to form a complete 3D image, particularly when coupled with remote focusing of the confocal filter.
Optical microscopy and spectroscopy are widely used in multiple research areas relating to biology. Label-free spectroscopy and imaging are valuable tools that permit interrogation of biological samples without the need for exogenous labels, allowing for investigation of unperturbed biological systems. We demonstrate a coherent Raman technique called Doppler Raman (DR) spectroscopy which combines impulsive excitation with a novel frequency shift detection scheme for rapid, high sensitivity detection of low to medium frequency vibrational modes from 10-1800cm-1. Briefly, the DR spectroscope is a pump-probe system where the pump beam generates a time-varying index of refraction proportional to the Raman response of the sample. The time-delayed probe beam undergoes a frequency shift in the sample due to the time-varying index of refraction that is resolved using a novel high-sensitivity detection scheme. Other coherent Raman techniques such as Stimulated Raman Scattering (SRS) and Coherent Anti-Stokes Raman Spectroscopy (CARS) have been used to provide sensitive, label-free contrast for an array of biological targets, but their ability to detect low frequency vibrational modes is limited. Biologically significant targets like cytochrome c (740-760cm-1), DNA (782, 788, 1095cm-1), hydroxyapatite, and numerous pharmaceutical drugs exhibit rich Raman spectra across a range of low frequency modes below the well-known “fingerprint region”. Additionally, many proteins like hemoglobin, insulin, and bovine serum albumin have breathing modes below 50cm-1. Sensitive detection of low-frequency Raman vibrational modes unlocks a suite of potential biological and chemical dynamics like protein conformational changes and protein super complex formation.
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.
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.
Superresoltuion (SR) microscopy is a valuable tool for biological studies. While the ability to resolve features to 20 nm and below is now routine in transparent specimens, such as cell cultures and cleared specimens, many areas of biological study have not been probed with SR imaging. Further, SR microscopy has thus far been limited primarily to contrast mechanisms that rely on real energy states of a target molecule, with fluorescence being the dominant modality. We recently demonstrated that spatial-frequency modulated imaging (SPIFI) enables superresolved imaging for both multiphoton fluorescence and nonlinear coherent scattering with single-pixel detection. The technique operates by projecting a set of spatial frequencies in one dimension along a spatiotemporally modulated line-focus that illuminates the specimen. Harmonics of the spatial frequencies projected onto the specimen encode spatial information beyond the diffraction limit of the illumination light. This additional information scales with the order of the nonlinearity, but is limited to the single dimension in which the grating sequence is projected. Consequently, 2D images collected with SR SPIFI are diffraction limited in the dimension perpendicular to the line focus. In this work, we extend our technique to a two-dimensional resolution enhancement with an inverse-domain lateral computed tomography. By enabling 2D SR SPIFI while maintaining single-pixel detection, we anticipate more widespread use of this method for imaging in turbid media.
Spatial Frequency Modulated Imaging (SPIFI) with single element detection has previously been demonstrated with a time varying amplitude spatial frequency. This has been shown in a variety of modalities (linear, TPEF, SHG) and also with variations on the base design to provide additional dimensions of information. SPIFI is also capable of providing enhanced resolution images. However, the signal-to-noise is a limiting factor in the quality of the resolution enhancement. We present a microscope design which uses a nematic spatial light modulator to provide a time varying amplitude from an amplitude or phase grating. Twophoton excitation fluorescence images of 10-µm fluorescent polystyrene beads are presented using a phase grating. Additionally, the microscope can provide spatial gratings in polarization which provide an alternative means of imaging in third harmonic generation (THG). THG images are provided using an amplitude and polarization-grating modulation pattern.
MultiPhoton SPatIal Frequency modulated Imaging (MP-SPIFI) has recently demonstrated the ability to simultaneously obtain super-resolved images in both coherent and incoherent scattering processes — namely, second harmonic generation and two-photon fluorescence, respectively.1 In our previous analysis, we considered image formation produced by the zero and first diffracted orders from the SPIFI modulator. However, the modulator is a binary amplitude mask, and therefore produces multiple diffracted orders. In this work, we extend our analysis to image formation in the presence of higher diffracted orders. We find that tuning the mask duty cycle offers a measure of control over the shape of super-resolved point spread functions in an MP-SPIFI microscope.
Coherent holographic image reconstruction by phase transfer (CHIRPT) is an imaging method that permits digital holographic propagation of fluorescent light. The image formation process in CHIRPT is based on illuminating the specimen with a precisely controlled spatio-temporally varying intensity pattern. This pattern is formed by focusing a spatially coherent illumination beam to a line focus on a spinning modulation mask, and image relaying the mask plane to the focal plane of an objective lens. Deviations from the designed spatio-temporal illumination pattern due to imperfect mounting of the circular modulation mask onto the rotation motor induce aberrations in the recovered image. Here we show that these aberrations can be measured and removed non-iteratively by measuring the disk aberration phase externally. We also demonstrate measurement and correction of systematic optical aberrations in the CHIRPT microscope.
We present a laser scanning microscope capable of producing multiple focal volumes. These volumes can be displaced
vertically, to acquire simultaneous images from multiple planes, or superimposed at the same depth but with different
polarization states. We call this last implementation, differential multiphoton laser scanning microscopy (dMPLSM). To
our knowledge, this constitutes the first report of a multifocal microscope with this capacity. The microscope is able to
take images in different modalities, two-photon excited fluorescence, second, and third harmonic generation. In this
work, we demonstrate several capabilities of our microscope: simultaneous acquisition of two and six images from two
focal planes separated by several microns, and a pair of simultaneous images taken at the same focal plane but with two
different polarizations. Some potential applications include following microorganism motion, studies of phase matching
in microscopic environments, studies of blood flow, etc. The microscope is based on a pulsed ultrafast laser. The pulses
are split, manipulated and recombined in an interlaced pattern in order to generate a sequence of pulses with different
divergences, and possibly different polarizations. This pulse train is sent to the objective and focused at different depths.
The signal is recorded using a photoncounting photomultiplier tube. Images from different foci are separated using time
demultiplexing based on a low cost field programmable gate array. The use of a single element detector, instead of a
multi-element (CCD camera), allows for imaging of scattering media. The use of photon counting leads to lower signal
to noise ratio in the images.
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