3D X-Ray Imaging of Continuous Objects beyond the Depth of Focus Limit.

X-ray ptychography is becoming the standard method for sub-30 nm imaging of thick extended samples. Available algorithms and computing power have traditionally restricted sample reconstruction to 2D slices. We build on recent progress in optimization algorithms and high performance computing to solve the ptychographic phase retrieval problem directly in 3D. Our approach addresses samples that do not fit entirely within the depth of focus of the imaging system. Such samples pose additional challenges because of internal diffraction effects within the sample. We demonstrate our approach on a computational sample modeled with 17 million complex variables.

Adaptive optics via self-interference digital holography for non-scanning three-dimensional imaging in biological samples.

Three-dimensional imaging in biological samples usually suffers from performance degradation caused by optical inhomogeneities. Here we proposed an approach to adaptive optics in fluorescence microscopy where the aberrations are measured by self-interference holographic recording and then corrected by a post-processing optimization procedure. In our approach, only one complex-value hologram is sufficient to measure and then correct the aberrations, which results in fast acquisition speed, lower exposure time, and the ability to image in three-dimensions without the need to scan the sample or any other element in the system. We show proof-of-principle experiments on a tissue phantom containing fluorescence particles. Furthermore, we present three-dimensional reconstructions of actin-labeled MCF7 breast cancer cells, showing improved resolution after the correction of aberrations. Both experiments demonstrate the validity of our method and show the great potential of non-scanning adaptive three-dimensional microscopy in imaging biological samples with improved resolution and signal-to-noise ratio.

3D + time blood flow mapping using SPIM-microPIV in the developing zebrafish heart.

We present SPIM-µPIV as a flow imaging system, capable of measuring in vivo flow information with 3D micron-scale resolution. Our system was validated using a phantom experiment consisting of a flow of beads in a 50 µm diameter FEP tube. Then, with the help of optical gating techniques, we obtained 3D + time flow fields throughout the full heartbeat in a ∼3 day old zebrafish larva using fluorescent red blood cells as tracer particles. From this we were able to recover 3D flow fields at 31 separate phases in the heartbeat. From our measurements of this specimen, we found the net pumped blood volume through the atrium to be 0.239 nL per beat. SPIM-µPIV enables high quality in vivo measurements of flow fields that will be valuable for studies of heart function and fluid-structure interaction in a range of small-animal models.

Interferometric temporal focusing microscopy using three-photon excitation fluorescence.

Super-resolution microscopy has become a powerful tool for biological research. However, its spatial resolution and imaging depth are limited, largely due to background light. Interferometric temporal focusing (ITF) microscopy, which combines structured illumination microscopy and three-photon excitation fluorescence microscopy, can overcome these limitations. Here, we demonstrate ITF microscopy using three-photon excitation fluorescence, which has a spatial resolution of 106 nm at an imaging depth of 100 µm with an excitation wavelength of 1060 nm.

Image restoration approach to address reduced modulation contrast in structured illumination microscopy.

The performance of structured illumination microscopy (SIM) is hampered in many biological applications due to the inability to modulate the light when imaging deep into the sample. This is in part because sample-induced aberration reduces the modulation contrast of the structured pattern. In this paper, we present an image restoration approach suitable for processing raw incoherent-grid-projection SIM data with a low fringe contrast. Restoration results from simulated and experimental ApoTome SIM data show results with improved signal-to-noise ratio (SNR) and optical sectioning compared to the results obtained from existing methods, such as 2D demodulation and 3D SIM deconvolution. Our proposed method provides satisfactory results (quantified by the achieved SNR and normalized mean square error) even when the modulation contrast of the illumination pattern is as low as 7%.

FIMic: design for ultimate 3D-integral microscopy of in-vivo biological samples.

In this work, Fourier integral microscope (FIMic), an ultimate design of 3D-integral microscopy, is presented. By placing a multiplexing microlens array at the aperture stop of the microscope objective of the host microscope, FIMic shows extended depth of field and enhanced lateral resolution in comparison with regular integral microscopy. As FIMic directly produces a set of orthographic views of the 3D-micrometer-sized sample, it is suitable for real-time imaging. Following regular integral-imaging reconstruction algorithms, a 2.75-fold enhanced depth of field and [Formula: see text]-time better spatial resolution in comparison with conventional integral microscopy is reported. Our claims are supported by theoretical analysis and experimental images of a resolution test target, cotton fibers, and in-vivo 3D-imaging of biological specimens.

Comparison of four methods of surface roughness assessment of corneal stromal bed after lamellar cutting.

Corneal lamellar cutting with a blade or femtosecond laser (FSL) is commonly used during refractive surgery and corneal grafts. Surface roughness of the cutting plane influences postoperative visual acuity but is difficult to assess reliably. For the first time, we compared chromatic confocal microscopy (CCM) with scanning electron microscopy, atomic force microscopy (AFM) and focus-variation microscopy (FVM) to characterize surfaces of variable roughness after FSL cutting. The small area allowed by AFM hinders conclusive roughness analysis, especially with irregular cuts. FVM does not always differentiate between smooth and rough surfaces. Finally, CCM allows analysis of large surfaces and differentiates between surface states.

Fast varifocal two-photon microendoscope for imaging neuronal activity in the deep brain.

Fluorescence microendoscopy is becoming a promising approach for deep brain imaging, but the current technology for visualizing neurons on a single focal plane limits the experimental efficiency and the pursuit of three-dimensional functional neural circuit architectures. Here we present a novel fast varifocal two-photon microendoscope system equipped with a gradient refractive index (GRIN) lens and an electrically tunable lens (ETL). This microendoscope enables quasi-simultaneous imaging of the neuronal network activity of deep brain areas at multiple focal planes separated by 85-120 µm at a fast scan rate of 7.5-15 frames per second per plane, as demonstrated in calcium imaging of the mouse dorsal CA1 hippocampus and amygdala in vivo.

Multifocus structured illumination microscopy for fast volumetric super-resolution imaging.

We here report for the first time the synergistic implementation of structured illumination microscopy (SIM) and multifocus microscopy (MFM). This imaging modality is designed to alleviate the problem of insufficient volumetric acquisition speed in super-resolution biological imaging. SIM is a wide-field super-resolution technique that allows imaging with visible light beyond the classical diffraction limit. Employing multifocus diffractive optics we obtain simultaneous wide-field 3D imaging capability in the SIM acquisition sequence, improving volumetric acquisition speed by an order of magnitude. Imaging performance is demonstrated on biological specimens.

sideSPIM - selective plane illumination based on a conventional inverted microscope.

Previously described selective plane illumination microscopy techniques typically offset ease of use and sample handling for maximum imaging performance or vice versa. Also, to reduce cost and complexity while maximizing flexibility, it is highly desirable to implement light sheet microscopy such that it can be added to a standard research microscope instead of setting up a dedicated system. We devised a new approach termed sideSPIM that provides uncompromised imaging performance and easy sample handling while, at the same time, offering new applications of plane illumination towards fluidics and high throughput 3D imaging of multiple specimen. Based on an inverted epifluorescence microscope, all of the previous functionality is maintained and modifications to the existing system are kept to a minimum. At the same time, our implementation is able to take full advantage of the speed of the employed sCMOS camera and piezo stage to record data at rates of up to 5 stacks/s. Additionally, sample handling is compatible with established methods and switching magnification to change the field of view from single cells to whole organisms does not require labor intensive adjustments of the system.

Fitting-free algorithm for efficient quantification of collagen fiber alignment in SHG imaging applications.

Collagen fiber alignment derived from second harmonic generation (SHG) microscopy images can be important for disease diagnostics. Image processing algorithms are needed to robustly quantify the alignment in images with high sensitivity and reliability. Fourier transform (FT) magnitude, 2D power spectrum, and image autocorrelation have previously been used to extract fiber information from images by assuming a certain mathematical model (e.g. Gaussian distribution of the fiber-related parameters) and fitting. The fitting process is slow and fails to converge when the data is not Gaussian. Herein we present an efficient constant-time deterministic algorithm which characterizes the symmetricity of the FT magnitude image in terms of a single parameter, named the fiber alignment anisotropy R ranging from 0 (randomized fibers) to 1 (perfect alignment). This represents an important improvement of the technology and may bring us one step closer to utilizing the technology for various applications in real time. In addition, we present a digital image phantom-based framework for characterizing and validating the algorithm, as well as assessing the robustness of the algorithm against different perturbations.

Structured illumination multimodal 3D-resolved quantitative phase and fluorescence sub-diffraction microscopy.

Sub-diffraction resolution imaging has played a pivotal role in biological research by visualizing key, but previously unresolvable, sub-cellular structures. Unfortunately, applications of far-field sub-diffraction resolution are currently divided between fluorescent and coherent-diffraction regimes, and a multimodal sub-diffraction technique that bridges this gap has not yet been demonstrated. Here we report that structured illumination (SI) allows multimodal sub-diffraction imaging of both coherent quantitative-phase (QP) and fluorescence. Due to SI's conventionally fluorescent applications, we first demonstrate the principle of SI-enabled three-dimensional (3D) QP sub-diffraction imaging with calibration microspheres. Image analysis confirmed enhanced lateral and axial resolutions over diffraction-limited QP imaging, and established striking parallels between coherent SI and conventional optical diffraction tomography. We next introduce an optical system utilizing SI to achieve 3D sub-diffraction, multimodal QP/fluorescent visualization of A549 biological cells fluorescently tagged for F-actin. Our results suggest that SI has a unique utility in studying biological phenomena with significant molecular, biophysical, and biochemical components.

Quantifying melanin concentration in retinal pigment epithelium using broadband photoacoustic microscopy.

Melanin is the dominant light absorber in retinal pigment epithelium (RPE). The loss of RPE melanin is a sign of ocular senescence and is both a risk factor and a symptom of age-related macular degeneration (AMD). Quantifying the RPE melanin concentration provides insight into the pathological role of RPE in ocular aging and the onset and progression of AMD. The main challenge in accurate quantification of RPE melanin concentration is to distinguish this ten-micrometer-thick cell monolayer from the underlying choroid, which also contains melanin but carries different pathognomonic information. In this work, we investigated a three-dimensional photoacoustic microscopic (PAM) method with high axial resolution, empowered by broad acoustic detection bandwidth, to distinguish RPE from choroid and quantify melanin concentrations in the RPE ex vivo. We first conducted numerical simulation on photoacoustic generation in the RPE, which suggested that a PAM system with at least 100-MHz detection bandwidth provided sufficient axial resolution to distinguish the melanin in RPE from that in choroid. Based on simulation results, we integrated a transparent broadband micro-ring resonator (MRR) based detector in a homebuilt PAM system. We imaged ex vivo RPE-choroid complexes (RCCs) from both porcine and human eyes and quantified the absolute melanin concentrations in the RPE and choroid, respectively. In our study, the measured melanin concentrations were 14.7 mg/mL and 17.0 mg/mL in human and porcine RPE, and 12 mg/mL and 61 mg/mL in human and porcine choroid, respectively. This study suggests that broadband PAM is capable of quantifying the RPE melanin concentration from RCCs ex vivo.

Cellular resolution multiplexed FLIM tomography with dual-color Bessel beam.

Fourier multiplexed FLIM (FmFLIM) tomography enables multiplexed 3D lifetime imaging of whole embryos. In our previous FmFLIM system, the spatial resolution was limited to 25 µm because of the trade-off between the spatial resolution and the imaging depth. In order to achieve cellular resolution imaging of thick specimens, we built a tomography system with dual-color Bessel beam. In combination with FmFLIM, the Bessel FmFLIM tomography system can perform parallel 3D lifetime imaging on multiple excitation-emission channels at a cellular resolution of 2.8 µm. The image capability of the Bessel FmFLIM tomography system was demonstrated by 3D lifetime imaging of dual-labeled transgenic zebrafish embryos.

Gabor fusion master slave optical coherence tomography.

This paper describes the application of the Gabor filtering protocol to a Master/Slave (MS) swept source optical coherence tomography (SS)-OCT system at 1300 nm. The MS-OCT system delivers information from selected depths, a property that allows operation similar to that of a time domain OCT system, where dynamic focusing is possible. The Gabor filtering processing following collection of multiple data from different focus positions is different from that utilized by a conventional swept source OCT system using a Fast Fourier transform (FFT) to produce an A-scan. Instead of selecting the bright parts of A-scans for each focus position, to be placed in a final B-scan image (or in a final volume), and discarding the rest, the MS principle can be employed to advantageously deliver signal from the depths within each focus range only. The MS procedure is illustrated on creating volumes of data of constant transversal resolution from a cucumber and from an insect by repeating data acquisition for 4 different focus positions. In addition, advantage is taken from the tolerance to dispersion of the MS principle that allows automatic compensation for dispersion created by layers above the object of interest. By combining the two techniques, Gabor filtering and Master/Slave, a powerful imaging instrument is demonstrated. The master/slave technique allows simultaneous display of three categories of images in one frame: multiple depth en-face OCT images, two cross-sectional OCT images and a confocal like image obtained by averaging the en-face ones. We also demonstrate the superiority of MS-OCT over its FFT based counterpart when used with a Gabor filtering OCT instrument in terms of the speed of assembling the fused volume. For our case, we show that when more than 4 focus positions are required to produce the final volume, MS is faster than the conventional FFT based procedure.

Selectable light-sheet uniformity using tuned axial scanning.

Light-sheet fluorescence microscopy (LSFM) is an optical sectioning technique capable of rapid three-dimensional (3D) imaging of a wide range of specimens with reduced phototoxicity and superior background rejection. However, traditional light-sheet generation approaches based on elliptical or circular Gaussian beams suffer an inherent trade-off between light-sheet thickness and area over which this thickness is preserved. Recently, an increase in light-sheet uniformity was demonstrated using rapid biaxial Gaussian beam scanning along the lateral and beam propagation directions. Here we apply a similar scanning concept to an elliptical beam generated by a cylindrical lens. In this case, only z-scanning of the elliptical beam is required and hence experimental implementation of the setup can be simplified. We introduce a simple dimensionless uniformity statistic to better characterize scanned light-sheets and experimentally demonstrate custom tailored uniformities up to a factor of 5 higher than those of unscanned elliptical beams. This technique offers a straightforward way to generate and characterize a custom illumination profile that provides enhanced utilization of the detector dynamic range and field of view, opening the door to faster and more efficient 2D and 3D imaging.

Single shot, three-dimensional fluorescence microscopy with a spatially rotating point spread function.

A wide-field fluorescence microscope with a double-helix point spread function (PSF) is constructed to obtain the specimen's three-dimensional distribution with a single snapshot. Spiral-phase-based computer-generated holograms (CGHs) are adopted to make the depth-of-field of the microscope adjustable. The impact of system aberrations on the double-helix PSF at high numerical aperture is analyzed to reveal the necessity of the aberration correction. A modified cepstrum-based reconstruction scheme is promoted in accordance with properties of the new double-helix PSF. The extended depth-of-field images and the corresponding depth maps for both a simulated sample and a tilted section slice of bovine pulmonary artery endothelial (BPAE) cells are recovered, respectively, verifying that the depth-of-field is properly extended and the depth of the specimen can be estimated at a precision of 23.4nm. This three-dimensional fluorescence microscope with a framerate-rank time resolution is suitable for studying the fast developing process of thin and sparsely distributed micron-scale cells in extended depth-of-field.

Optical projection tomography for rapid whole mouse brain imaging.

In recent years, three-dimensional mesoscopic imaging has gained significant importance in life sciences for fundamental studies at the whole-organ level. In this manuscript, we present an optical projection tomography (OPT) method designed for imaging of the intact mouse brain. The system features an isotropic resolution of ~50 µm and an acquisition time of four to eight minutes, using a 3-day optimized clearing protocol. Imaging of the brain autofluorescence in 3D reveals details of the neuroanatomy, while the use of fluorescent labels displays the vascular network and amyloid deposition in 5xFAD mice, an important model of Alzheimer's disease (AD). Finally, the OPT images are compared with histological slices.

Correlative three-dimensional fluorescence and refractive index tomography: bridging the gap between molecular specificity and quantitative bioimaging.

Optical diffraction tomography (ODT) provides label-free three-dimensional (3D) refractive index (RI) measurement of biological samples. However, due to the nature of the RI values of biological specimens, ODT has limited access to molecular specific information. Here, we present an optical setup combining ODT with three-channel 3D fluorescence microscopy, to enhance the molecular specificity of the 3D RI measurement. The 3D RI distribution and 3D deconvoluted fluorescence images of HeLa cells and NIH-3T3 cells are measured, and the cross-correlative analysis between RI and fluorescence of live cells are presented.

High axial resolution imaging system for large volume tissues using combination of inclined selective plane illumination and mechanical sectioning.

To resolve fine structures of biological systems like neurons, it is required to realize microscopic imaging with sufficient spatial resolution in three dimensional systems. With regular optical imaging systems, high lateral resolution is accessible while high axial resolution is hard to achieve in a large volume. We introduce an imaging system for high 3D resolution fluorescence imaging of large volume tissues. Selective plane illumination was adopted to provide high axial resolution. A scientific CMOS working in sub-array mode kept the imaging area in the sample surface, which restrained the adverse effect of aberrations caused by inclined illumination. Plastic embedding and precise mechanical sectioning extended the axial range and eliminated distortion during the whole imaging process. The combination of these techniques enabled 3D high resolution imaging of large tissues. Fluorescent bead imaging showed resolutions of 0.59 µm, 0.47µm, and 0.59 µm in the x, y, and z directions, respectively. Data acquired from the volume sample of brain tissue demonstrated the applicability of this imaging system. Imaging of different depths showed uniform performance where details could be recognized in either the near-soma area or terminal area, and fine structures of neurons could be seen in both the xy and xz sections.