Efficient and cost‐effective 3D cellular imaging by sub‐voxel‐resolving light‐sheet add‐on microscopy

Light‐sheet fluorescence microscopy (LSFM) allows volumetric live imaging at high‐speed and with low photo‐toxicity. Various LSFM modalities are commercially available, but their size and cost limit their access by the research community. A new method, termed sub‐voxel‐resolving (SVR) light‐sheet add‐on microscopy (SLAM), is presented to enable fast, resolution‐enhanced light‐sheet fluorescence imaging from a conventional wide‐field microscope. This method contains two components: a miniature add‐on device to regular wide‐field microscopes, which contains a horizontal laser light‐sheet illumination path to confine fluorophore excitation at the vicinity of the focal plane for optical sectioning; an off‐axis scanning strategy and a SVR algorithm that utilizes sub‐voxel spatial shifts to reconstruct the image volume that results in a twofold increase in resolution. SLAM method has been applied to observe the muscle activity change of crawling C. elegans, the heartbeat of developing zebrafish embryo, and the neural anatomy of cleared mouse brains, at high spatiotemporal resolution. It provides an efficient and cost‐effective solution to convert the vast number of in‐service microscopes for fast 3D live imaging with voxel‐super‐resolved capability.     

Multiparameter wide‐field integrated optical imaging system‐based spatially modulated illumination and laser speckles in model of tissue injuries

In this report, an integrated optical platform based on spatial illumination together with laser speckle contrast technique was utilized to measure multiple parameters in live tissue including absorption, scattering, saturation, composition, metabolism, and blood flow. Measurements in three models of tissue injury including drug toxicity, artery occlusion, and acute hyperglycemia were used to test the efficacy of this system. With this hybrid apparatus, a series of structured light patterns at low and high spatial frequencies are projected onto the tissue surface and diffuse reflected light is captured by a CCD camera. A six position filter wheel, equipped with four bandpass filters centered at wavelengths of 650, 690, 800 and 880 nm is placed in front of the camera. Then, light patterns are blocked and a laser source at 650 nm illuminates the tissue while the diffusely reflected light is captured by the camera through the two remaining open holes in the wheel. In this manner, near‐infrared (NIR) and laser speckle images are captured and stored together in the computer for off‐line processing to reconstruct the tissue's properties. Spatial patterns are used to differentiate the effects of tissue scattering from those of absorption, allowing accurate quantification of tissue hemodynamics and morphology, while a coherent light source is used to study blood flow changes, a feature which cannot be measured with the NIR structured light. This combined configuration utilizes the strengths of each system in a complementary way, thus collecting a larger range of sample properties. In addition, once the flow and hemodynamics are measured, tissue oxygen metabolism can be calculated, a property which cannot be measured independently. Therefore, this merged platform can be considered a multiparameter wide‐field imaging and spectroscopy modality. Overall, experiments demonstrate the capability of this spatially coregistered imaging setup to provide complementary, useful information of various tissue metrics in a simple and noncontact manner, making it attractive for use in a variety of biomedical applications.     

Artifact‐free objective‐type multicolor total internal reflection fluorescence microscopy with light‐emitting diode light sources—Part I

Total internal reflection fluorescence excitation (TIRF) microscopy allows the selective observation of fluorescent molecules in immediate proximity to an interface between different refractive indices. Objective‐type or prism‐less TIRF excitation is typically achieved with laser light sources. We here propose a simple, yet optically advantageous light‐emitting diode (LED)‐based implementation of objective‐type TIRF (LED‐TIRF). The proposed LED‐TIRF condenser is affordable and easy to set up at any epifluorescence microscope to perform multicolor TIRF and/or combined TIRF‐epifluorescence imaging with even illumination of the entire field of view. Electrical control of LED light sources replaces mechanical shutters or optical modulators. LED‐TIRF microscopy eliminates safety burdens that are associated with laser sources, offers favorable instrument lifetime and stability without active cooling. The non‐coherent light source and the type of projection eliminate interference fringing and local scattering artifacts that are associated with conventional laser‐TIRF. Unlike azimuthal spinning laser‐TIRF, LED‐TIRF does not require synchronization between beam rotation and the camera and can be monitored with either global or rolling shutter cameras. Typical implementations, such as live cell multicolor imaging in TIRF and epifluorescence of imaging of short‐lived, localized translocation events of a Ca²⁺‐sensitive protein kinase C α fusion protein are demonstrated.     

Lightsheet fluorescence lifetime imaging microscopy with wide‐field time‐correlated single photon counting

We report on wide‐field time‐correlated single photon counting (TCSPC)‐based fluorescence lifetime imaging microscopy (FLIM) with lightsheet illumination. A pulsed diode laser is used for excitation, and a crossed delay line anode image intensifier, effectively a single‐photon sensitive camera, is used to record the position and arrival time of the photons with picosecond time resolution, combining low illumination intensity of microwatts with wide‐field data collection. We pair this detector with the lightsheet illumination technique, and apply it to 3D FLIM imaging of dye gradients in human cancer cell spheroids, and C. elegans.     

Quantitative phase microscopy with enhanced contrast and improved resolution through ultra‐oblique illumination (UO‐QPM)

Recent developments in phase contrast microscopy have enabled the label‐free visualization of certain organelles due to their distinct morphological features, making this method an attractive alternative in the study of cellular dynamics. However tubular structures such as endoplasmic reticulum (ER) networks and complex dynamics such as the fusion and fission of mitochondria, due to their low phase contrast, still need fluorescent labeling to be adequately imaged. In this article, we report a quantitative phase microscope with ultra‐oblique illumination that enables us to see those structures and their dynamics with high contrast for the first time without labeling. The imaging capability was validated through comparison to the fluorescence images with the same field‐of‐view. The high image resolution (~270 nm) was validated using both beads and cellular structures. Furthermore, we were able to record the vibration of ER networks at a frame rate of 250 Hz. We additionally show complex cellular processes such as remodeling of the mitochondria networks through fusion and fission and vesicle transportation along the ER without labels. Our high spatial and temporal resolution allowed us to observe mitochondria “spinning”, which has not been reported before, further demonstrating the advantages of the proposed method.      

Development a flexible light‐sheet fluorescence microscope for high‐speed 3D imaging of calcium dynamics and 3D imaging of cellular microstructure

We report a flexible light‐sheet fluorescence microscope (LSFM) designed for studying dynamic events in cardiac tissue at high speed in 3D and the correlation of these events to cell microstructure. The system employs two illumination‐detection modes: the first uses angle‐dithering of a Gaussian light sheet combined with remote refocusing of the detection plane for video‐rate volumetric imaging; the second combines digitally‐scanned light‐sheet illumination with an axially‐swept light‐sheet waist and stage‐scanned acquisition for improved axial resolution compared to the first mode. We present a characterisation of the spatial resolution of the system in both modes. The first illumination‐detection mode achieves dual spectral‐channel imaging at 25 volumes per second with 1024 × 200 × 50 voxel volumes and is demonstrated by time‐lapse imaging of calcium dynamics in a live cardiomyocyte. The second illumination‐detection mode is demonstrated through the acquisition of a higher spatial resolution structural map of the t‐tubule network in a fixed cardiomyocyte cell.     

Bessel‐beam illumination in dual‐axis confocal microscopy mitigates resolution degradation caused by refractive heterogeneities

One of the main challenges for laser‐scanning microscopy of biological tissues with refractive heterogeneities is the degradation in spatial resolution that occurs as a result of beam steering and distortion. This challenge is particularly significant for dual‐axis confocal (DAC) microscopy, which achieves improved spatial‐filtering and optical‐sectioning performance over traditional confocal microscopy through off‐axis illumination and collection of light with low‐numerical aperture (NA) beams that must intersect precisely at their foci within tissues. DAC microscope image quality is sensitive to positional changes and distortions of these illumination‐ and collection‐beam foci. Previous studies have shown that Bessel beams display improved positional stability and beam quality than Gaussian beams when propagating through tissues with refractive heterogeneities, which suggests that Bessel‐beam illumination may enhance DAC microscopy of such tissues. Here, we utilize both Gaussian and Bessel illumination in a point‐scanned DAC microscope and quantify the resultant degradation in resolution when imaging within heterogeneous optical phantoms and fresh tissues. Results indicate that DAC microscopy with Bessel illumination exhibits reduced resolution degradation from microscopic tissue heterogeneities compared to DAC microscopy with conventional Gaussian illumination.

     

A compact high‐speed full‐field optical coherence microscope for high‐resolution in vivo skin imaging

A compact high‐speed full‐field optical coherence microscope has been developed for high‐resolution in vivo imaging of biological tissues. The interferometer, in the Linnik configuration, has a size of 11 × 11 × 5 cm³ and a weight of 210 g. Full‐field illumination with low‐coherence light is achieved with a high‐brightness broadband light‐emitting diode. High‐speed full‐field detection is achieved by using part of the image sensor of a high‐dynamic range CMOS camera. En face tomographic images are acquired at a rate of 50 Hz, with an integration time of 0.9 ms. The image spatial resolution is 0.9 μm × 1.2 μm (axial × transverse), over a field of view of 245 × 245 μm². Images of human skin, revealing in‐depth cellular‐level structures, were obtained in vivo and in real‐time without the need for stabilization of the subject. The system can image larger fields, up to 1 × 1 mm², but at a reduced depth.      

Structured illumination imaging with quasi‐periodic patterns

Structured illumination microscopy (SIM) is a well‐established method for optical sectioning and super‐resolution. The core of structured illumination is using a periodic pattern to excite image signals. This work reports a method for estimating minor pattern distortions from the raw image data and correcting these distortions during SIM image processing. The method was tested with both simulated and experimental image data from two‐photon Bessel light‐sheet SIM. The results proves the method is effective in challenging situations, where strong scattering background exists, signal‐to‐noise ratio (SNR) is low and the sample structure is sparse. Experimental results demonstrate restoring synaptic structures in deep brain tissue, despite the presence of strong light scattering and tissue‐induced SIM pattern distortion.     

Real‐time functional optical‐resolution photoacoustic microscopy using high‐speed alternating illumination at 532 and 1064 nm

Optical‐resolution photoacoustic microscopy (OR‐PAM), which has been widely used and studied as a noninvasive and in vivo imaging technique, can yield high‐resolution and absorption contrast images. Recently, metallic nanoparticles and dyes, such as gold nanoparticles, methylene blue, and indocyanine green, have been used as contrast agents of OR‐PAM. This study demonstrates real‐time functional OR‐PAM images with high‐speed alternating illumination at 2 wavelengths. To generate 2 wavelengths, second harmonic generation at 532 nm with an LBO crystal and a pump wavelength of 1064 nm is applied at a pulse repetition rate of 300 kHz. For alternating illumination, an electro‐optical modulator is used as an optical switch. Therefore, the A‐line rate for the functional image is 150 kHz, which is half of the laser repetition rate. To enable fast signal processing and real‐time displays, parallel signal processing using a graphics processing unit (GPU) is performed. OR‐PAM images of the distribution of blood vessels and gold nanorods in a BALB/c‐nude mouse's ear can be simultaneously obtained with 500 × 500 pixels and real‐time display at 0.49 fps.      

Dual light‐emitting diode‐based multichannel microscopy for whole‐slide multiplane,multispectral and phase imaging

We report the development of a multichannel microscopy for whole‐slide multiplane, multispectral and phase imaging. We use trinocular heads to split the beam path into 6 independent channels and employ a camera array for parallel data acquisition, achieving a maximum data throughput of approximately 1 gigapixel per second. To perform single‐frame rapid autofocusing, we place 2 near‐infrared light‐emitting diodes (LEDs) at the back focal plane of the condenser lens to illuminate the sample from 2 different incident angles. A hot mirror is used to direct the near‐infrared light to an autofocusing camera. For multiplane whole‐slide imaging (WSI), we acquire 6 different focal planes of a thick specimen simultaneously. For multispectral WSI, we relay the 6 independent image planes to the same focal position and simultaneously acquire information at 6 spectral bands. For whole‐slide phase imaging, we acquire images at 3 focal positions simultaneously and use the transport‐of‐intensity equation to recover the phase information. We also provide an open‐source design to further increase the number of channels from 6 to 15. The reported platform provides a simple solution for multiplexed fluorescence imaging and multimodal WSI. Acquiring an instant focal stack without z‐scanning may also enable fast 3‐dimensional dynamic tracking of various biological samples.      

Label‐free optical projection tomography for quantitative three‐dimensional anatomy of mouse embryo

Recent progress in three‐dimensional optical imaging techniques allows visualization of many comprehensive biological specimens. Optical clearing methods provide volumetric and quantitative information by overcoming the limited depth of light due to scattering. However, current imaging technologies mostly rely on the synthetic or genetic fluorescent labels, thus limits its application to whole‐body visualization of generic mouse models. Here, we report a label‐free optical projection tomography (LF‐OPT) technique for quantitative whole mouse embryo imaging. LF‐OPT is based on the attenuation contrast of light rather than fluorescence, and it utilizes projection imaging technique similar to computed tomography for visualizing the volumetric structure. We demonstrate this with a collection of mouse embryo morphologies in different stages using LF‐OPT. Additionally, we extract quantitative organ information applicable toward high‐throughput phenotype screening. Our results indicate that LF‐OPT can provide multi‐scale morphological information in various tissues including bone, which can be difficult in conventional optical imaging technique.     

Self‐referenced axial chromatic dispersion measurement in multiphoton microscopy through 2‐color third‐harmonic generation imaging

With tunable excitation light, multiphoton microscopy is widely used for imaging biological structures at subcellular resolution. Axial chromatic dispersion, present in virtually every transmissive optical system including the multiphoton microscope, leads to focal (and the resultant image) plane separation. Here, we experimentally demonstrate a technique to measure the axial chromatic dispersion in a multiphoton microscope, using simultaneous 2‐color third‐harmonic generation imaging excited by a 2‐color soliton source with tunable wavelength separation. Our technique is self‐referenced, eliminating potential measurement error when 1‐color tunable excitation light is used which necessitates reciprocating motion of the mechanical translation stage. Using this technique, we demonstrate measured axial chromatic dispersion with 2 different objective lenses in a multiphoton microscope. Further measurement in a biological sample also indicates that this axial chromatic dispersion, in combination with 2‐color imaging, may open up opportunity for simultaneous imaging of 2 different axial planes.      

Advanced photobioreactor LED illumination system: Scale‐down approach to study microalgal growth kinetics

A newly designed and constructed LED illumination device for commercial cylindrical bioreactors is presented for application in microalgal cultivations and investigation of growth kinetics. An ideally illuminated volume is achieved by focusing the light toward the center of the reactor and thereby compensating the mutual shading of the cells. The relevant biomass concentration for homogeneous illumination depending on reactor radius was determined by light distribution measurements for Chlamydomonas to 0.2 g/L (equal 0.435 optical density at 750 nm). It is shown that cultivation experiments with the newly designed illumination device operated in batch mode can be successfully applied for determination of growth rates and photo conversion efficiencies. The exact knowledge of physiological reactions of specific strain(s) and the estimation of relevant parameters for scale‐up can be used for construction of economic pilot plant photobioreactors. The determination of light‐dependent kinetics of growth and product formation is the first necessary step to achieve this. A wide variety of different parameters can be examined like the effect of different illumination conditions (light intensity, frequency of day/night cycles, flashing light, light color…) and thereby for each single application specific, relevant, and interesting parameters will be examined.     

Low‐photobleaching line‐scanning confocal microscopy using dual inclined beams

Confocal microscopy is an indispensable tool for biological imaging due to its high resolution and optical sectioning capability. However, its slow imaging speed and severe photobleaching have largely prevented further applications. Here, we present dual inclined beam line‐scanning (LS) confocal microscopy. The reduced excitation intensity of our imaging method enabled a 2‐fold longer observation time of fluorescence compared to traditional LS microscopy while maintaining a good sectioning capability and single‐molecule sensitivity. We characterized the performance of our method and applied it to subcellular imaging and three‐dimensional single‐molecule RNA imaging in mammalian cells.      

Uniform light delivery in volumetric optoacoustic tomography

Accurate image reconstruction in volumetric optoacoustic tomography implies the efficient generation and collection of ultrasound signals around the imaged object. Non‐uniform delivery of the excitation light is a common problem in optoacoustic imaging often leading to a diminished field of view, limited dynamic range and penetration, as well as impaired quantification abilities. Presented here is an optimized illumination concept for volumetric tomography that utilizes additive manufacturing via 3D printing in combination with custom‐made optical fiber illumination. The custom‐designed sample chamber ensures convenient access to the imaged object along with accurate positioning of the sample and a matrix array ultrasound transducer used for collection of the volumetric image data. Ray tracing is employed to optimize the positioning of the individual fibers in the chamber. Homogeneity of the generated light excitation field was confirmed in tissue‐mimicking agar spheres. Applicability of the system to image entire mouse organs ex vivo has been showcased. The new approach showed a clear advantage over conventional, single‐sided illumination strategies by eliminating the need to correct for illumination variances and resulting in enhancement of the effective field of view, greater penetration depth and significant improvements in the overall image quality.      

Low‐coherent optical diffraction tomography by angle‐scanning illumination

Temporally low‐coherent optical diffraction tomography (ODT) is proposed and demonstrated based on angle‐scanning Mach‐Zehnder interferometry. Using a digital micromirror device based on diffractive tilting, the full‐field interference of incoherent light is successfully maintained during every angle‐scanning sequences. Further, current ODT reconstruction principles for temporally incoherent illuminations are thoroughly reviewed and developed. Several limitations of incoherent illumination are also discussed, such as the nondispersive assumption, optical sectioning capacity and illumination angle limitation. Using the proposed setup and reconstruction algorithms, low‐coherent ODT imaging of plastic microspheres, human red blood cells and rat pheochromocytoma cells is experimentally demonstrated.      

Video microsurgery: evaluation of standard laparoscopic equipment for the practice of microsurgery

Traditional microsurgery involves the use of bulky and expensive stereo microscopes that have limited portability. Recent advances in video technology have enabled the exploration of alternative visualization methods. The purpose of this study was to evaluate standard laparoscopic equipment for microvascular anastomoses. Eight surgeons completed anastomoses on rat femoral and synthetic vessels using stereo microsurgery and video microsurgery visualization systems. All surgeons had previous experience with stereo microsurgery and none had ever used video microsurgery. Data were collected on overall anastomosis and individual suture times. A sample of completed anastomoses was placed in a video database and evaluated by use of a quality rating scale (8 to 10, excellent; 6 to 7, adequate; less than 6, poor). All surgeons subjectively evaluated the video microsurgery system. A total of 48 anastomoses were completed. The average total anastomosis time for the stereo microsurgery was 1018.9 +/- 463.2 seconds versus 1738.9 +/- 460.1 seconds for the video microsurgery. The average individual suture placement time was 114.6 +/- 60.6 seconds for the stereo microsurgery versus 211.7 +/- 128.4 seconds for the video microsurgery (p < 0.05). Twenty-five of the anastomoses underwent quality review. The overall score of the stereo microsurgery group was 8.1 +/- 1.7, and the video microsurgery group had an overall score of 7.3 +/- 1.6. Survey results revealed that 75 percent of the participants thought that the video microsurgery would be useful for human operations and would improve surgeon comfort, but 87.5 percent would not use the present video microsurgery system over stereo microsurgery in their practice. Although significant differences exist in overall anastomosis and individual suture completion times, no difference was found in the overall quality. Video microsurgery could become a useful tool on the basis of surgeon ergonomics; however, optical parameters require further refinement.     

A high‐throughput all‐optical laser‐scanning imaging flow cytometer with biomolecular specificity and subcellular resolution

Image‐based cellular assay advances approaches to dissect complex cellular characteristics through direct visualization of cellular functional structures. However, available technologies face a common challenge, especially when it comes to the unmet need for unraveling population heterogeneity at single‐cell precision: higher imaging resolution (and thus content) comes at the expense of lower throughput, or vice versa. To overcome this challenge, a new type of imaging flow cytometer based upon an all‐optical ultrafast laser‐scanning imaging technique, called free‐space angular‐chirp‐enhanced delay (FACED) is reported. It enables an imaging throughput (>20 000 cells s^?1) 1 to 2 orders of magnitude higher than the camera‐based imaging flow cytometers. It also has 2 critical advantages over optical time‐stretch imaging flow cytometry, which achieves a similar throughput: (1) it is widely compatible to the repertoire of biochemical contrast agents, favoring biomolecular‐specific cellular assay and (2) it enables high‐throughput visualization of functional morphology of individual cells with subcellular resolution. These capabilities enable multiparametric single‐cell image analysis which reveals cellular heterogeneity, for example, in the cell‐death processes demonstrated in this work—the information generally masked in non‐imaging flow cytometry. Therefore, this platform empowers not only efficient large‐scale single‐cell measurements, but also detailed mechanistic analysis of complex cellular processes.      

The utility of far‐infrared illumination in oxygenation dynamics as measured with near‐infrared spectroscopy

Near‐infrared spectroscopy (NIRS) is a noninvasive method for measuring the oxygenation in muscle and other tissues in vivo. For quantitative NIRS measurement of oxygenation dynamics, the vessel‐occlusion test was usually applied as physiological intervention. There are several drawbacks of the vessel‐occlusion method that include skin contact, uncomfortable and microcirculation block of patients. Thus, we propose the far‐infrared (FIR) illumination as a new physiological intervention method in this paper. Our preliminary result shows a linear correlation of oxygenation dynamic signals between FIR illumination and arterial‐occlusion test (AOT) that implies the FIR illumination could be applied for hemodynamic response measurement in clinical diagnosis. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)