Deep learning-based autofocus method enhances image quality in light-sheet fluorescence microscopy

Light-sheet fluorescence microscopy (LSFM) is a minimally invasive and high throughput imaging technique ideal for capturing large volumes of tissue with sub-cellular resolution. A fundamental requirement for LSFM is a seamless overlap of the light-sheet that excites a selective plane in the specimen, with the focal plane of the objective lens. However, spatial heterogeneity in the refractive index of the specimen often results in violation of this requirement when imaging deep in the tissue. To address this issue, autofocus methods are commonly used to refocus the focal plane of the objective-lens on the light-sheet. Yet, autofocus techniques are slow since they require capturing a stack of images and tend to fail in the presence of spherical aberrations that dominate volume imaging. To address these issues, we present a deep learning-based autofocus framework that can estimate the position of the objective-lens focal plane relative to the light-sheet, based on two defocused images. This approach outperforms or provides comparable results with the best traditional autofocus method on small and large image patches respectively. When the trained network is integrated with a custom-built LSFM, a certainty measure is used to further refine the network’s prediction. The network performance is demonstrated in real-time on cleared genetically labeled mouse forebrain and pig cochleae samples. Our study provides a framework that could improve light-sheet microscopy and its application toward imaging large 3D specimens with high spatial resolution.     

A compact Airy beam light sheet microscope with a tilted cylindrical lens

Light-sheet imaging is rapidly gaining importance for imaging intact biological specimens. Many of the latest innovations rely on the propagation-invariant Bessel or Airy beams to form an extended light sheet to provide high resolution across a large field of view. Shaping light to realize propagation-invariant beams often relies on complex programming of spatial light modulators or specialized, custom made, optical elements. Here we present a straightforward and low-cost modification to the traditional light-sheet setup, based on the open-access light-sheet microscope OpenSPIM, to achieve Airy light-sheet illumination. This brings wide field single-photon light-sheet imaging to a broader range of endusers. Fluorescent microspheres embedded in agarose and a zebrafish larva were imaged to demonstrate how such a microscope can have a minimal footprint and cost without compromising on imaging quality.OCIS codes: (170.0170) Medical optics and biotechnology, (180.6900) Three-dimensional microscopy, (140.3300) Laser beam shaping, (220.1000) Aberration compensation, (070.0070) Fourier optics and signal processing, (110.0180) Microscopy, (110.1758) Computational imaging, (110.4850) Optical transfer functions, (110.7348) Wavefront encoding     

Scanning thin-sheet laser imaging microscopy (sTSLIM) with structured illumination and HiLo background rejection

We report replacement of one side of a static illumination, dual sided, thin-sheet laser imaging microscope (TSLIM) with an intensity modulated laser scanner in order to implement structured illumination (SI) and HiLo image demodulation techniques for background rejection. The new system is equipped with one static and one scanned light-sheet and is called a scanning thin-sheet laser imaging microscope (sTSLIM). It is an optimized version of a light-sheet fluorescent microscope that is designed to image large specimens (<15 mm in diameter). In this paper we describe the hardware and software modifications to TSLIM that allow for static and uniform light-sheet illumination with SI and HiLo image demodulation. The static light-sheet has a thickness of 3.2 μm; whereas, the scanned side has a light-sheet thickness of 4.2 μm. The scanned side images specimens with subcellular resolution (<1 μm lateral and <4 μm axial resolution) with a size up to 15 mm. SI and HiLo produce superior contrast compared to both the uniform static and scanned light-sheets. HiLo contrast was greater than SI and is faster and more robust than SI because as it produces images in two-thirds of the time and exhibits fewer intensity streaking artifacts.     

High-speed 3D imaging flow cytometry with optofluidic spatial transformation

Three-dimensional (3D) fluorescence imaging is important to accurately capture and understand biological structures and phenomena. However, because of its slow acquisition speed, it was difficult to implement 3D fluorescence imaging for imaging flow cytometry. Especially, modern flow cytometers operate at a flow velocity of 1–10 m/s, and no 3D fluorescence imaging technique was able to capture cells at such high velocity. Here, we present a high-speed 3D fluorescence imaging technique in which a set of optical cross sections of a cell is captured within a single frame of a camera by combining strobe light-sheet excitation and optofluidic spatial transformation. Using this technique, we demonstrated 3D fluorescence imaging of cells flowing at a velocity of over 10 m/s, which is the fastest to our knowledge. Such technology can allow integration of 3D imaging with flow systems of common flow cytometers and cell sorters.     

Quantitative analysis of illumination and detection corrections in adaptive light sheet fluorescence microscopy

Light-sheet fluorescence microscopy (LSFM) is a high-speed, high-resolution and minimally phototoxic technique for 3D imaging of in vivo and in vitro specimens. LSFM exhibits optical sectioning and when combined with tissue clearing techniques, it facilitates imaging of centimeter scale specimens with micrometer resolution. Although LSFM is ubiquitous, it still faces two main challenges that effect image quality especially when imaging large volumes with high-resolution. First, the light-sheet illumination plane and detection lens focal plane need to be coplanar, however sample-induced aberrations can violate this requirement and degrade image quality. Second, introduction of sample-induced optical aberrations in the detection path. These challenges intensify when imaging whole organisms or structurally complex specimens like cochleae and bones that exhibit many transitions from soft to hard tissue or when imaging deep (> 2 mm). To resolve these challenges, various illumination and aberration correction methods have been developed, yet no adaptive correction in both the illumination and the detection path have been applied to improve LSFM imaging. Here, we bridge this gap, by implementing the two correction techniques on a custom built adaptive LSFM. The illumination beam angular properties are controlled by two galvanometer scanners, while a deformable mirror is positioned in the detection path to correct for aberrations. By imaging whole porcine cochlea, we compare and contrast these correction methods and their influence on the image quality. This knowledge will greatly contribute to the field of adaptive LSFM, and imaging of large volumes of tissue cleared specimens.     

Versatile,do-it-yourself,low-cost spinning disk confocal microscope

Confocal microscopy is an invaluable tool for 3D imaging of biological specimens, however, accessibility is often limited to core facilities due to the high cost of the hardware. We describe an inexpensive do-it-yourself (DIY) spinning disk confocal microscope (SDCM) module based on a commercially fabricated chromium photomask that can be added on to a laser-illuminated epifluorescence microscope. The SDCM achieves strong performance across a wide wavelength range (∼400-800 nm) as demonstrated through a series of biological imaging applications that include conventional microscopy (immunofluorescence, small-molecule stains, and fluorescence in situ hybridization) and super-resolution microscopy (single-molecule localization microscopy and expansion microscopy). This low-cost and simple DIY SDCM is well-documented and should help increase accessibility to confocal microscopy for researchers.     

Cellular imaging of deep organ using two-photon Bessel light-sheet nonlinear structured illumination microscopy

In vivo fluorescent cellular imaging of deep internal organs is highly challenging, because the excitation needs to penetrate through strong scattering tissue and the emission signal is degraded significantly by photon diffusion induced by tissue-scattering. We report that by combining two-photon Bessel light-sheet microscopy with nonlinear structured illumination microscopy (SIM), live samples up to 600 microns wide can be imaged by light-sheet microscopy with 500 microns penetration depth, and diffused background in deep tissue light-sheet imaging can be reduced to obtain clear images at cellular resolution in depth beyond 200 microns. We demonstrate in vivo two-color imaging of pronephric glomeruli and vasculature of zebrafish kidney, whose cellular structures located at the center of the fish body are revealed in high clarity by two-color two-photon Bessel light-sheet SIM.OCIS codes: (170.2520) Fluorescence microscopy, (170.6900) Three-dimensional microscopy     

High resolution optical projection tomography platform for multispectral imaging of the mouse gut

Optical projection tomography (OPT) is a powerful tool for three-dimensional imaging of mesoscopic biological samples with great use for biomedical phenotyping studies. We present a fluorescent OPT platform that enables direct visualization of biological specimens and processes at a centimeter scale with high spatial resolution, as well as fast data throughput and reconstruction. We demonstrate nearly isotropic sub-28 µm resolution over more than 60 mm³ after reconstruction of a single acquisition. Our setup is optimized for imaging the mouse gut at multiple wavelengths. Thanks to a new sample preparation protocol specifically developed for gut specimens, we can observe the spatial arrangement of the intestinal villi and the vasculature network of a 3-cm long healthy mouse gut. Besides the blood vessel network surrounding the gastrointestinal tract, we observe traces of vasculature at the villi ends close to the lumen. The combination of rapid acquisition and a large field of view with high spatial resolution in 3D mesoscopic imaging holds an invaluable potential for gastrointestinal pathology research.     

Single-objective high-resolution confocal light sheet fluorescence microscopy for standard biological sample geometries

Three-dimensional fluorescence-based imaging of living cells and organisms requires the sample to be exposed to substantial excitation illumination energy, typically causing phototoxicity and photobleaching. Light sheet fluorescence microscopy dramatically reduces phototoxicity, yet most implementations are limited to objective lenses with low numerical aperture and particular sample geometries that are built for specific biological systems. To overcome these limitations, we developed a single-objective light sheet fluorescence system for biological imaging based on axial plane optical microscopy and digital confocal slit detection, using either Bessel or Gaussian beam shapes. Compared to spinning disk confocal microscopy, this system displays similar optical resolution, but a significantly reduced photobleaching at the same signal level. This single-objective light sheet technique is built as an add-on module for standard research microscopes and the technique is compatible with high-numerical aperture oil immersion objectives and standard samples mounted on coverslips. We demonstrate the performance of this technique by imaging three-dimensional dynamic processes, including bacterial biofilm dispersal, the response of biofilms to osmotic shocks, and macrophage phagocytosis of bacterial cells.     

Continuous imaging of large-volume tissues with a machinable optical clearing method at subcellular resolution

Optical clearing methods are widely used for three-dimensional biological information acquisition in the whole organ. However, the imaging quality of cleared tissues is often limited by ununiformed tissue clearing. By combining tissue clearing with mechanical sectioning based whole organ imaging system, we can reduce the influence of light scattering and absorption on the tissue to get isotropic and high resolution in both superficial and deep layers. However, it remains challenging for optical cleared biological tissue to maintain good sectioning property. Here, we developed a clearing method named M-CUBIC (machinable CUBIC), which combined a modified CUBIC method with PNAGA (poly-N-acryloyl glycinamide) hydrogel embedding to transparentize tissue while improving its sectioning property. With high-throughput light-sheet tomography platform (HLTP) and fluorescent micro-optical sectioning tomography (fMOST), we acquired continuous datasets with subcellular resolution from intact mouse brains for single neuron tracing, as well as the fine vascular structure of kidneys. This method can be used to acquire microstructures of multiple types of biological organs with subcellular resolutions, which can facilitate biological research.     

Enhancement of image quality and imaging depth with Airy light-sheet microscopy in cleared and non-cleared neural tissue

We have investigated the effect of Airy illumination on the image quality and depth penetration of digitally scanned light-sheet microscopy in turbid neural tissue. We used Fourier analysis of images acquired using Gaussian and Airy light-sheets to assess their respective image quality versus penetration into the tissue. We observed a three-fold average improvement in image quality at 50 μm depth with the Airy light-sheet. We also used optical clearing to tune the scattering properties of the tissue and found that the improvement when using an Airy light-sheet is greater in the presence of stronger sample-induced aberrations. Finally, we used homogeneous resolution probes in these tissues to quantify absolute depth penetration in cleared samples with each beam type. The Airy light-sheet method extended depth penetration by 30% compared to a Gaussian light-sheet.OCIS codes: (070.0070) Fourier optics and signal processing, (100.2960) Image analysis, (100.2980) Image enhancement, (170.3880) Medical and biological imaging, (180.0180) Microscopy, (180.2520) Fluorescence microscopy     

Multimodal microscopy for the simultaneous visualization of five different imaging modalities using a single light source

Optical microscopy has been widely used in biomedical research as it provides photophysical and photochemical information of the target in subcellular spatial resolution without requiring physical contact with the specimen. To obtain a deeper understanding of biological phenomena, several efforts have been expended to combine such optical imaging modalities into a single microscope system. However, the use of multiple light sources and detectors through separated beam paths renders previous systems extremely complicated or slow for in vivo imaging. Herein, we propose a novel high-speed multimodal optical microscope system that simultaneously visualizes five different microscopic contrasts, i.e., two-photon excitation, second-harmonic generation, backscattered light, near-infrared fluorescence, and fluorescence lifetime, using a single femtosecond pulsed laser. Our proposed system can visualize five modal images with a frame rate of 3.7 fps in real-time, thereby providing complementary optical information that enhances both structural and functional contrasts. This highly photon-efficient multimodal microscope system enables various properties of biological tissues to be assessed.     

Optimization of the excitation light sheet in selective plane illumination microscopy

Selective plane illumination microscopy (SPIM) allows rapid 3D live fluorescence imaging on biological specimens with high 3D spatial resolution, good optical sectioning capability and minimal photobleaching and phototoxic effect. SPIM gains its advantage by confining the excitation light near the detection focal plane, and its performance is determined by the ability to create a thin, large and uniform excitation light sheet. Several methods have been developed to create such an excitation light sheet for SPIM. However, each method has its own strengths and weaknesses, and tradeoffs must be made among different aspects in SPIM imaging. In this work, we present a strategy to select the excitation light sheet among the latest SPIM techniques, and to optimize its geometry based on spatial resolution, field of view, optical sectioning capability, and the sample to be imaged. Besides the light sheets discussed in this work, the proposed strategy is also applicable to estimate the SPIM performance using other excitation light sheets.OCIS codes: (180.2520) Fluorescence microscopy, (180.6900) Three-dimensional microscopy     

Dual-view light-sheet imaging through a tilted glass interface using a deformable mirror

Light-sheet microscopy has become indispensable for imaging developing organisms, and imaging from multiple directions (views) is essential to improve its spatial resolution. We combine multi-view light-sheet microscopy with microfluidics using adaptive optics (deformable mirror) which corrects aberrations introduced by the 45^o-tilted glass coverslip. The optimal shape of the deformable mirror is computed by an iterative algorithm that optimizes the point-spread function in two orthogonal views. Simultaneous correction in two optical arms is achieved via a knife-edge mirror that splits the excitation path and combines the detection paths. Our design allows multi-view light-sheet microscopy with microfluidic devices for precisely controlled experiments and high-content screening.     

Label-free imaging of zebrafish larvae in vivo by photoacoustic microscopy

Zebrafish play an important role in biological and biomedical research. Traditional in vivo imaging methods for studying zebrafish larvae primarily require fluorescence labeling. In this work, relying on tissue intrinsic optical absorption contrast, we acquired high resolution label-free 3D images of zebrafish larvae by using photoacoustic microscopy (PAM) in vivo. The spatial resolution reaches several microns, allowing the study of microstructures in various living organs. We demonstrated that our method has the potential to be a powerful non-invasive imaging method for studying various small animal models, including zebrafish larvae, Caenorhabditis elegans, frogs and drosophila larvae.     

Rapid spontaneous Raman light sheet microscopy using cw-lasers and tunable filters

We perform rapid spontaneous Raman 2D imaging in light-sheet microscopy using continuous wave lasers and interferometric tunable filters. By angularly tuning the filter, the cut-on/off edge transitions are scanned along the excited Stokes wavelengths. This allows obtaining cumulative intensity profiles of the scanned vibrational bands, which are recorded on image stacks; resembling a spectral version of the knife-edge technique to measure intensity profiles. A further differentiation of the stack retrieves the Raman spectra at each pixel of the image which inherits the 3D resolution of the host light sheet system. We demonstrate this technique using solvent solutions and composites of polystyrene beads and lipid droplets immersed in agar and by imaging the C–H (2800-3100cm⁻¹) region in a C. elegans worm. The image acquisition time results in 4 orders of magnitude faster than confocal point scanning Raman systems, allowing the possibility of performing fast spontaneous Raman·3D-imaging on biological samples.OCIS codes: (170.3880) Medical and biological imaging, (180.5655) Raman microscopy, (300.6450) Spectroscopy, Raman, (110.4234) Multispectral and hyperspectral imaging     

Wavefront engineered light needle microscopy for axially resolved rapid volumetric imaging

Increasing the acquisition speed of three-dimensional volumetric images is important—particularly in biological imaging—to unveil the structural dynamics and functionalities of specimens in detail. In conventional laser scanning fluorescence microscopy, volumetric images are constructed from optical sectioning images sequentially acquired by changing the observation plane, limiting the acquisition speed. Here, we present a novel method to realize volumetric imaging from two-dimensional raster scanning of a light needle spot without sectioning, even in the traditional framework of laser scanning microscopy. Information from multiple axial planes is simultaneously captured using wavefront engineering for fluorescence signals, allowing us to readily survey the entire depth range while maintaining spatial resolution. This technique is applied to real-time and video-rate three-dimensional tracking of micrometer-sized particles, as well as the prompt visualization of thick fixed biological specimens, offering substantially faster volumetric imaging.     

High-resolution 3D optical microscopy inside the beating zebrafish heart using prospective optical gating

3D fluorescence imaging is a fundamental tool in the study of functional and developmental biology, but effective imaging is particularly difficult in moving structures such as the beating heart. We have developed a non-invasive real-time optical gating system that is able to exploit the periodic nature of the motion to acquire high resolution 3D images of the normally-beating zebrafish heart without any unnecessary exposure of the sample to harmful excitation light. In order for the image stack to be artefact-free, it is essential to use a synchronization source that is invariant as the sample is scanned in 3D. We therefore describe a scheme whereby fluorescence image slices are scanned through the sample while a brightfield camera sharing the same objective lens is maintained at a fixed focus, with correction of sample drift also included. This enables us to maintain, throughout an extended 3D volume, the same standard of synchronization we have previously demonstrated in and near a single 2D plane. Thus we are able image the complete beating zebrafish heart exactly as if the heart had been artificially stopped, but sidestepping this undesirable interference with the heart and instead allowing the heart to beat as normal.OCIS codes: (170.2520) Fluorescence microscopy, (110.2960) Image analysis, (170.3880) Medical and biological imaging, (180.6900) Three-dimensional microscopy, (110.6880) Three-dimensional image acquisition     

Compact plane illumination plugin device to enable light sheet fluorescence imaging of multi-cellular organisms on an inverted wide-field microscope

We developed a compact plane illumination plugin (PIP) device which enabled plane illumination and light sheet fluorescence imaging on a conventional inverted microscope. The PIP device allowed the integration of microscope with tunable laser sheet profile, fast image acquisition, and 3-D scanning. The device is both compact, measuring approximately 15 by 5 by 5 cm, and cost-effective, since we employed consumer electronics and an inexpensive device molding method. We demonstrated that PIP provided significant contrast and resolution enhancement to conventional microscopy through imaging different multi-cellular fluorescent structures, including 3-D branched cells in vitro and live zebrafish embryos. Imaging with the integration of PIP greatly reduced out-of-focus contamination and generated sharper contrast in acquired 2-D plane images when compared with the stand-alone inverted microscope. As a result, the dynamic fluid domain of the beating zebrafish heart was clearly segmented and the functional monitoring of the heart was achieved. Furthermore, the enhanced axial resolution established by thin plane illumination of PIP enabled the 3-D reconstruction of the branched cellular structures, which leads to the improvement on the functionality of the wide field microscopy.OCIS codes: (170.3880) Medical and biological imaging, (170.2520) Fluorescence microscopy, (110.3010) Image reconstruction techniques, (110.2945) Illumination design, (170.6900) Three-dimensional microscopy