Single-shot generalized Hanbury Brown-Twiss experiments using a polarization camera for target intensity reconstruction in scattering media.

To see through a random light field in real-time, single-shot generalized Hanbury Brown-Twiss experiments using a polarization camera are proposed. The target intensity distribution is obtained from a complex coherence function which is calculated from auto-correlation and cross correlation functions of phase-shifted speckle intensity distributions. The phase-shifted speckle intensity distributions are simultaneously obtained through a strategy of parallel phase-shifting digital holography. Experimental results show that the proposed method can image a moving object in a random light field using a measured complex coherence function through the van Cittert-Zernike theorem.

Single-shot common-path off-axis digital holography: applications in bioimaging and optical metrology [Invited].

The demand for single-shot and common-path holographic systems has become increasingly important in recent years, as such systems offer various advantages compared to their counterparts. Single-shot holographic systems, for example, reduce computational complexity as only a single hologram with the object information required to process, making them more suitable for the investigation of dynamic events; and common-path holographic systems are less vibration-sensitive, compact, inexpensive, and high in temporal phase stability. We have developed a single-shot common-path off-axis digital holographic setup based on a beam splitter and pinhole. In this paper, we present a concise review of the proposed digital holographic system for several applications, including the quantitative phase imaging to investigate the morphological and quantitative parameters, as a metrological tool for testing of micro-optics, industrial inspection and measurement, and sound field imaging and visualization.

Multimodal sound field imaging using digital holography [Invited].

Sound is an important invisible physical phenomenon that needs to be explained in several physical and biological processes, along with visual phenomena. For this purpose, multiparameter digital holography (DH) has been proposed to visualize both features simultaneously due to the phase and amplitude reconstruction properties of DH. In this paper, we present a brief review on sound field imaging techniques with special focus on the multiparameter imaging capability of DH for visualizing sound and visual features. The basic theory and several experimental results with very high-speed recordings are also presented to demonstrate sound field imaging for the audible range as well as in the ultrasound range.

Single-shot common-path off-axis dual-wavelength digital holographic microscopy.

A single-shot common-path off-axis self-interference dual-wavelength digital holographic microscopic (DHM) system based on a cube beam splitter is demonstrated to expand the phase range in a stepped microstructure and for simultaneous measurement of the refractive index and physical thickness of a specimen. In the system, two laser beams with wavelengths of 532 nm and 632.8 nm are used. These laser beams are combined to transilluminate the object under study, then the object beam is divided into two beams by using a beam splitter oriented in such a way that both the beams propagate in almost the same direction, with an appropriate lateral separation between them. One of the object beams is spatially filtered at its Fourier plane, using a pinhole to generate a reference spherical beam free from the object information. The reference beam interferes with the object beam to form a digital hologram at the faceplate of the image sensor. The phase information is extracted from a single recorded digital hologram using the phase aberration compensation method that is based on principal component analysis (PCA). Owing to the common-path configuration, the system shows high temporal phase stability and it is less vibration-sensitive compared to counterparts such as a Mach-Zehnder type DHM. The performance of the dual-wavelength DHM system is verified in two different application fields by conducting the experiments using microsphere beads and living plant cells.

Three-dimensional stimulation and imaging-based functional optical microscopy of biological cells.

A new type of functional optical microscope system called three-dimensional (3D) stimulation and imaging-based functional optical microscopy (SIFOM) is proposed, to the best of our knowledge. SIFOM can precisely stimulate user-defined targeted biological cells and can simultaneously record the volumetric fluorescence distribution in a single acquisition. Precise and simultaneous stimulation of fluorescent-labeled biological cells is achieved by multiple 3D spots generated by digital holograms displayed on a phase-mode spatial light modulator. Single-shot 3D acquisition of the fluorescence distribution is accomplished by common-path off-axis incoherent digital holographic microscopy in which a diffraction grating with a focusing lens is displayed on another phase-mode spatial light modulator. The effectiveness of the proposed functional microscope system was verified in experiments using fluorescent microbeads and human lung cancer cells located at various defocused positions. The system can be used for manipulating the states of cells in optogenetics.

Single-shot incoherent digital holography using a dual-focusing lens with diffraction gratings.

A new optical configuration of incoherent digital holography is presented to improve the quality of reconstructed images when the random polarization state of incoherent light is used. The proposed system improves the signal-to-noise ratio of the holograms by suppressing the unmodulated terms of a spatial light modulator. To generate the self-interference of a quasi-incoherent point-like source, we use a dual-focusing lens with diffraction gratings. The preliminary experimental results confirm the validity of the proposed method by reconstructing two point-like sources generated by a LED light source. When the pixel pitch of the phase-mode SLM is small enough, the off-axis hologram can be generated. The single-shot recording of the incoherent digital holography is expected.

Image recovery from defocused 2D fluorescent images in multimodal digital holographic microscopy.

A technique of three-dimensional (3D) intensity retrieval from defocused, two-dimensional (2D) fluorescent images in the multimodal digital holographic microscopy (DHM) is proposed. In the multimodal DHM, 3D phase and 2D fluorescence distributions are obtained simultaneously by an integrated system of an off-axis DHM and a conventional epifluorescence microscopy, respectively. This gives us more information of the target; however, defocused fluorescent images are observed due to the short depth of field. In this Letter, we propose a method to recover the defocused images based on the phase compensation and backpropagation from the defocused plane to the focused plane using the distance information that is obtained from a 3D phase distribution. By applying Zernike polynomial phase correction, we brought back the fluorescence intensity to the focused imaging planes. The experimental demonstration using fluorescent beads is presented, and the expected applications are suggested.

Holographic microscope and its biological application.

Holographic structured illumination combined with optogenetics enables patterned stimulation of neurons and glial cells in an intact living brain. Moreover, in vivo functional imaging of cellular activity with recent advanced microscope technologies allows for visualization of the cellular responses during learning, emotion and cognition. Integrating these techniques can be used to verify the link between cell function and behavior output. However, there are technical limitations to stimulate multiple cells with high spatial and temporal resolution with available techniques of optogenetic stimulation. Here, we summarized a two-photon microscope combined with holographic system to stimulate multiple cells with high spatial and temporal resolution for living mice and their biological application.

Evaluation and Manipulation of Neural Activity using Two-Photon Holographic Microscopy.

Recent advances in optical bioimaging and optogenetics have enabled the visualization and manipulation of biological phenomena, including cellular activities, in living animals. In the field of neuroscience, detailed neural activity related to brain functions, such as learning and memory, has now been revealed, and it has become feasible to artificially manipulate this activity to express brain functions. However, the conventional evaluation of neural activity by two-photon Ca2+ imaging has the problem of low temporal resolution. In addition, manipulation of neural activity by conventional optogenetics through the optic fiber can only simultaneously regulate the activity of neurons with the same genetic background, making it difficult to control the activity of individual neurons. To solve this issue, we recently developed a microscope with a high spatiotemporal resolution for biological applications by combining optogenetics with digital holographic technology that can modify femtosecond infrared laser beams. Here, we describe protocols for the visualization, evaluation, and manipulation of neural activity, including the preparation of samples and operation of a two-photon holographic microscope (Figure 1). These protocols provide accurate spatiotemporal information on neural activity, which may be useful for elucidating the pathogenesis of neuropsychiatric disorders that lead to abnormalities in neural activity.

Multi-omics analysis revealed the role of CCT2 in the induction of autophagy in Alzheimer's disease.

Chaperonin containing TCP1 subunit 2 (CCT2) is essential in various neurodegenerative diseases, albeit its role in the pathogenesis of Alzheimer's disease (AD) remains elusive. This study aimed to evaluate the role of CCT2 in Alzheimer's disease. First, bioinformatics database analysis revealed that CCT2 was significantly downregulated in patients with Alzheimer's disease and associated with autophagic clearance of ß-amyloid. The 789 differentially expressed genes overlapped in AD-group and CCT2-low/high group, and the CCT2-high-associated genes screened by Pearson coefficients were enriched in protein folding, autophagy, and messenger RNA stability regulation pathways. These results suggest that CCT2 is significantly and positively associated with multiple pathways linked to autophagy and negatively associated with neuronal death. The logistic prediction model with 13 key genes, such as CCT2, screened in this study better predicts Alzheimer's disease occurrence (AUC = 0.9671) and is a favorable candidate for predicting potential biological targets of Alzheimer's disease. Additionally, this study predicts reciprocal micro RNAs and small molecule drugs for hub genes. Our findings suggest that low CCT2 expression may be responsible for the autophagy suppression in Alzheimer's disease, providing an accurate explanation for its pathogenesis and new targets and small molecule inhibitors for its treatment.

Pain induces stable, active microcircuits in the somatosensory cortex that provide a therapeutic target.

Sustained neuropathic pain from injury or inflammation remains a major burden for society. Rodent pain models have informed some cellular mechanisms increasing neuronal excitability within the spinal cord and primary somatosensory cortex (S1), but how activity patterns within these circuits change during pain remains unclear. We have applied multiphoton in vivo imaging and holographic stimulation to examine single S1 neuron activity patterns and connectivity during sustained pain. Following pain induction, there is an increase in synchronized neuronal activity and connectivity within S1, indicating the formation of pain circuits. Artificially increasing neuronal activity and synchrony using DREADDs reduced pain thresholds. The expression of N-type voltage-dependent Ca2+ channel subunits in S1 was increased after pain induction, and locally blocking these channels reduced both the synchrony and allodynia associated with inflammatory pain. Targeting these S1 pain circuits, via inhibiting N-type Ca2+ channels or other approaches, may provide ways to reduce inflammatory pain.

Digital Holographic Multimodal Cross-Sectional Fluorescence and Quantitative Phase Imaging System.

We present a multimodal imaging system based on simple off-axis digital holography, for simultaneous recording and retrieval of cross-sectional fluorescence and quantitative phase imaging of the biological specimen. Synergism in the imaging capabilities can be achieved by incorporating two off-axis digital holographic microscopes integrated to record different information at the same time. The cross-sectional fluorescence imaging is realized by a common-path configuration of the single-shot off-axis incoherent digital holographic system. The quantitative phase imaging, on the other hand, is achieved by another off-axis coherent digital holographic microscopy operating in transmission mode. The fundamental characteristics of the proposed multimodal system are confirmed by performing various experiments on fluorescent beads and fluorescent protein-labeled living cells of the moss Physcomitrella patens lying at different axial depth positions. Furthermore, the cross-sectional live fluorescence and phase imaging of the fluorescent beads are demonstrated by the proposed multimodal system. The experimental results presented here corroborate the feasibility of the proposed system and indicate its potential in the applications to analyze the functional and structural behavior of biological cells and tissues.

Common-path multimodal three-dimensional fluorescence and phase imaging system.

A stable multimodal system is developed by combining two common-path digital holographic microscopes (DHMs): coherent and incoherent, for simultaneous recording and retrieval of three-dimensional (3-D) phase and 3-D fluorescence imaging (FI), respectively, of a biological specimen. The 3-D FI is realized by a single-shot common-path off-axis fluorescent DHM developed recently by our group. In addition, we accomplish, the phase imaging by another single-shot, highly stable common-path off-axis DHM based on a beam splitter. In this DHM configuration, a beam splitter is used to divide the incoming object beam into two beams. One beam serves as the object beam carrying the useful information of the object under study, whereas another beam is spatially filtered at its Fourier plane by using a pinhole and it serves as a reference beam. This DHM setup, owing to a common-path geometry, is less vibration-sensitive and compact, having a similar field of view but with high temporal phase stability in comparison to a two-beam Mach-Zehnder-type DHM. The performance of the proposed common-path DHM and the multimodal system is verified by conducting various experiments on fluorescent microspheres and fluorescent protein-labeled living cells of the moss Physcomitrella patens. Moreover, the potential capability of the proposed multimodal system for 3-D live fluorescence and phase imaging of the fluorescent beads is also demonstrated. The obtained experimental results corroborate the feasibility of the proposed multimodal system and indicate its potential applications for the analysis of functional and structural behaviors of a biological specimen and enhancement of the understanding of physiological mechanisms and various biological diseases.

Three-dimensional fluorescence imaging using the transport of intensity equation.

We propose a nonscanning three-dimensional (3-D) fluorescence imaging technique using the transport of intensity equation (TIE) and free-space Fresnel propagation. In this imaging technique, a phase distribution corresponding to defocused fluorescence images with a point-light-source-like shape is retrieved by a TIE-based phase retrieval algorithm. From the obtained phase distribution, and its corresponding amplitude distribution, of the defocused fluorescence image, various images at different distances can be reconstructed at the desired plane after Fresnel propagation of the complex wave function. Through the proposed imaging approach, the 3-D fluorescence imaging can be performed in multiple planes. The fluorescence intensity images are captured with the help of an electrically tunable lens; hence, the imaging technique is free from motion artifacts. We present experimental results corresponding to microbeads and a biological sample to demonstrate the proposed 3-D fluorescence imaging technique.

Digital holography and its multidimensional imaging applications: a review.

In this review, we introduce digital holographic techniques and recent progress in multidimensional sensing by using digital holography. Digital holography is an interferometric imaging technique that does not require an imaging lens and can be used to perform simultaneous imaging of multidimensional information, such as three-dimensional structure, dynamics, quantitative phase, multiple wavelengths and polarization state of light. The technique can also obtain a holographic image of nonlinear light and a three-dimensional image of incoherent light with a single-shot exposure. The holographic recording ability of this technique has enabled a variety of applications.