Volumetric bioimaging based on light field microscopy with temporal focusing illumination

Diffraction-Assisted Light Field Microscopy for Microtomography and Digital Volume Correlation with Improved Spatial Resolution

Fourier light field microscopy (FLFM) shows great potential in high-speed volumetric imaging of biodynamics. However, due to the inherent disadvantage of wide-field illumination, it suffers from intense background, arising from out of the depth-of-field signal and tissue scattered noise. The background will not only deteriorate the image contrast, making quantitative measurement difficult, but also introduce artifacts, especially in functional imaging of the neuronal network activity in vivo. Here, we propose the robust Fourier light field microscopy (RFLFM), which suppresses the background in FLFM by introducing structured illumination and computational reconstruction based on HiLo. The superior performance of RFLFM is verified by volumetric imaging of biological dynamics in larval zebrafish and mouse in vivo, at a volumetric imaging rate up to 33.3 Hz. The statistical results show that the fluorescence background can be significantly depressed, with the signal-to-background ratio improved by orders of magnitude and the whole image contrast improved by as much as ∼ 10.4 times. Moreover, we stress that, in functional imaging of neuronal network activity in turbid brain tissues, our system can avoid artifacts resulting from background fluctuations, while conventional light field microscopy fails. As a simple but powerful tool, we anticipate our technique to be widely adopted in robust, high-contrast, high-speed volumetric imaging.

In the past few years the integral-imaging, or lightfield, concept has been applied successfully to microscopy. More specifically, in the case of fluorescent samples integral, or lightfield, microscopy offers the advantage of capturing the 3D information in a single shot. Due to its potential utility integral microscopy is now facing many challenges, like improving the resolution and depth of field, the development and optimization of specially-adapted reconstruction algorithms, or the search of applications in which lightfield microscopy is superior to existing techniques. This contribution is devoted to review the recent advances of integral microscopy and enunciate the right questions about the progress of the technique.

Precision measurement of three-dimensional (3D) microstructures has drawn great interests from researchers and industries. Currently there are a bunch of high precision measurement methods such as contour-graphs, interferometers and optical computed tomography used in industrial applications. Nonetheless, the loss of information, low efficiency and possible surface damaging of traditional ways still exist. This paper presents a new way to measure micro/nano structures based on light field microscope. The light field information of the microstructures is acquired by using a microlens array which is inserted between the camera sensor and the objective lens. Then a series of regular sub-images are recorded by the photosensor, which is used to reconstruct a 3D image by the developed algorithms. The non-contact shooting process is realized through only one exposure which is much more efficient than the traditional ways. Microlens array with aspheric surface is also designed and used in the developed system, to eliminate aberrations and compensate the loss of spatial resolution. A series of simulation and experimental studies have been undertaken to measure microstructures, and the feasibility of the developed system is validated.

In this paper, a novel microscopic imaging technology, termed light field Fourier ptychographic microscopy (LFFPM), is presented which enables the reconstruction of wide-field, high-resolution images with the depth information by utilizing a light-emitting-diode (LED) illumination matrix.Conventional light field microscope lacks the ability of high lateral resolution because it sacrifices its lateral resolution to its angular resolution. Compared to conventional light field microscope, LFFPM enhances the lateral resolution with no need for the object to be in focus. Using LFFPM, the result shows that the lateral resolution is more than 2 times better than light field microscopy in simulation and 1.5 times in the experiment. Furthermore, the simulation result also shows that the ability of LFFPM to recovery the information of the phase and amplitude.Thus, this technology has a wide prospect of various biomedical applications such as neuroanatomy and haematology.

Fourier integral microscopy (FiMic), also referred to as Fourier light field microscopy (FLFM) in the literature, was recently proposed as an alternative to conventional light field microscopy (LFM). FiMic is designed to overcome the non-uniform lateral resolution limitation specific to LFM. By inserting a micro-lens array at the aperture stop of the microscope objective, the Fourier integral microscope directly captures in a single-shot a series of orthographic views of the scene from different viewpoints. We propose an algorithm for the deconvolution of FiMic data by combining the well known Maximum Likelihood Expectation (MLEM) method with total variation (TV) regularization to cope with noise amplification in conventional Richardson-Lucy deconvolution.

In this paper, a laser speckle contrast imaging (LSCI) system using light field (LF) microscope approach is proposed. As far as we known, it is first time to combine LSCI with LF. To verify this idea, a prototype consists of a modified LF microscope imaging system and an experimental device was built. A commercially used Lytro camera was modified for microscope imaging. Hollow glass tubes with different depth fixed in glass dish were used to simulate the vessels in brain and test the performance of the system. Compared with conventional LSCI, three new functions can be realized by using our system, which include refocusing, extending the depth of field (DOF) and gathering 3D information. Experiments show that the principle is feasible and the proposed system works well.

We present a novel approach to three-dimensional optical microscopy, named correlation light-field microscopy (CLM). This approach is based on correlation plenoptic imaging and exploits correlations between intensity fluctuations, intrinsic in chaotic light, to retrieve both spatial information about the intensity distribution of light on the sample and angular information about the directions of propagation of the light rays. Such a plenoptic (or light-field) information about the sample enables an extension of the natural depth of field, while avoiding the intrinsic loss of spatial resolution occurring in conventional light-field microscopy. We discuss the capability of CLM of refocusing out-of-focus planes of the sample, paving the way to scanning-free three-dimensional reconstruction while keeping the at-focus resolution at the diffraction limit showing a brief comparison with light-field microscopy. Finally we discuss the perspective of improvements in CLM acquisition speed by the integration of SPAD array sensors in the setup.

We applied an unmatched back projector based deconvolution algorithm to reduce image reconstruction time in light field microscopy. 3D laser written microstructures were fabricated and used to characterize the light field imaging.

In 1985, Omura, Y. discovered that, when specific molecules were placed anywhere in the close vicinity of the path of a light beam (laser), their molecular information, as well as information on electrical & magnetic fields, is transmitted bi-directionally along the path of this light beam. Namely, this information is transmitted in the direction the light beam is projected and towards the direction from which the light beam is coming. This finding was applied to the following clinical and basic research: 1) In the past, using indirect Bi-Digital O-Ring Test, human or animal patients were diagnosed through an intermediate third person holding a good electrical conducting probe, the tip of which was touching the part of the patient to be examined. However, in order to diagnose the patient in isolation from a distance, or a dangerous or unmanagable unanesthesized animal, such as a lion or tiger, the author succeeded in making a diagnosis by replacing the metal conducting probe with a soft laser beam which is held by the one hand of the third person whose index finger is placed in close vicinity of the laser beam generated by a battery-powered penlight-type solid state laser generator. Thus, diagnosis within visible distance, without direct patient contact, became a reality. 2) Using a projection light microscope, by giving indirect Bi-Digital O-Ring Test while contacting with a fine electro-conductive probe on the magnified fine structure of normal and abnormal cells, various normal and abnormal intracellular substances were localized through a third person holding a pure reference control substance with the same hand that is holding the probe as an intermediary for the indirect Bi-Digital O-Ring Test. Instead of the photon beam in a light microscope, the author found that, using an electron beam passing through the close vicinity of specific molecules of specimens in an electron microscope, the molecular information is transmitted to the magnified fluorescent screen, and an indirect Bi-Digital O-Ring Test could be performed through a projected penlight-type solid state soft laser beam on the magnified intracellular structure through an observation glass window. Using the magnified fine structure of the cells, by either a light projection microscopic field or electron microscope, in various cancer cells of both humans and animals, Oncogen C-fos (AB2) and mercury were found inside of the nucleus. Integrin alpha 5 beta 1 was found on cell membranes and nuclear cell membranes of cancer cells. Acetylcholine was not found anywhere within cancer cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Light Field Microscopy (LFM) is an imaging technique that captures 3D spatial information in a single 2D image. LFM is attractive because of its relatively simple implementation and fast acquisition rate. However, classic 3D reconstruction typically suffers from high computational cost, low lateral resolution, and reconstruction artifacts. In this work, we propose a new physics-based learning approach to improve the performance of the reconstruction under realistic conditions, these being lack of training data, background noise, and high data dimensionality. First, we propose a novel description of the system using a linear convolutional neural network. This description is complemented by a method that compacts the number of views of the acquired light field. Then, this model is used to solve the inverse problem under two scenarios. If labelled data is available, we train an end-to-end network that uses the Learned Iterative Shrinkage and Thresholding Algorithm (LISTA). If no labelled data is available, we propose an unsupervised technique that uses only unlabelled data to train LISTA by making use of Wasserstein Generative Adversarial Networks (WGANs). We experimentally show that our approach performs better than classic strategies in terms of artifact reduction and image quality.

In this paper, we proposed a hexagonal liquid crystal micro‐lens array (HLC‐MLA) to instead of fixed micro‐lens array for 3D light field microscope. The focal length of liquid crystal lens can be adjusted electrically. With this property, the depth of the field (DoF) of light field microscopy was well extended. Besides, the dense packing design of hexagonal liquid crystal micro‐lens array can reduce pixels consuming.

Optical microscopy has witnessed notable advancements but has also become more costly and complex. Conventional wide field microscopy (WFM) has low resolution and shallow depth-of-field (DOF), which limits its applications in practical biological experiments. Recently, confocal and light sheet microscopy become major workhorses for biology that incorporate high-precision scanning to perform imaging within an extended DOF but at the sacrifice of expense, complexity, and imaging speed. Here, we propose deep focus microscopy, an efficient framework optimized both in hardware and algorithm to address the tradeoff between resolution and DOF. Our deep focus microscopy achieves large-DOF and high-resolution projection imaging by integrating a deep focus network (DFnet) into light field microscopy (LFM) setups. Based on our constructed dataset, deep focus microscopy features a significantly enhanced spatial resolution of ∼260 nm, an extended DOF of over 30 µm, and broad generalization across diverse sample structures. It also reduces the computational costs by four orders of magnitude compared to conventional LFM technologies. We demonstrate the excellent performance of deep focus microscopy in vivo, including long-term observations of cell division and migrasome formation in zebrafish embryos and mouse livers at high resolution without background contamination.

3D microscopy is a useful tool to visualize the detailed structures and mechanisms of biomedical specimens. In particular, biophysical phenomena such as neural activity require fast 3D volumetric imaging because fluorescence signals degrade quickly. A light-field microscope (LFM) has recently attracted attention as a high-speed volumetric imaging technique by recording 3D information in a single-snapshot. This review highlighted recent progress in LFM techniques for 3D biomedical applications. In detail, various image reconstruction algorithms according to LFM configurations are explained, and several biomedical applications such as neuron activity localization, live-cell imaging, locomotion analysis, and single-molecule visualization are introduced. We also discuss deep learning-based LFMs to enhance image resolution and reduce reconstruction artifacts.

Three-dimensional (3D) observation of moving cells offers critical insights into fundamental biological processes. However, the limited temporal resolution of traditional scanning-based 3D imaging techniques constrains their use in live-cell imaging. Light-field microscopy (LFM) has emerged as a scanning-free alternative, enabling rapid 3D volumetric data acquisition. Despite this advantage, LFM's spatial resolution and reconstruction artifacts remain challenges that hinder its broader application in biological imaging. This study proposes a custom-designed staggered bifocal microlens array (MLA), fabricated through a cost-effective, high-throughput method, to overcome these limitations. This approach enhances spatial resolution while minimizing reconstruction artifacts compared to conventional uniform MLAs. We demonstrate the technique through 3D imaging of microbeads at varying depths and live cells suspended in droplets. This bifocal MLA presents a promising advancement for extending LFM's applications in biological research.