Multi‐ATOM: Ultrahigh‐throughput single‐cell quantitative phase imaging with subcellular resolution

A growing body of evidence has substantiated the significance of quantitative phase imaging (QPI) in enabling cost‐effective and label‐free cellular assays, which provides useful insights into understanding the biophysical properties of cells and their roles in cellular functions. However, available QPI modalities are limited by the loss of imaging resolution at high throughput and thus run short of sufficient statistical power at the single‐cell precision to define cell identities in a large and heterogeneous population of cells—hindering their utility in mainstream biomedicine and biology. Here we present a new QPI modality, coined multiplexed asymmetric‐detection time‐stretch optical microscopy (multi‐ATOM) that captures and processes quantitative label‐free single‐cell images at ultrahigh throughput without compromising subcellular resolution. We show that multi‐ATOM, based upon ultrafast phase‐gradient encoding, outperforms state‐of‐the‐art QPI in permitting robust phase retrieval at a QPI throughput of >10 000 cell/sec, bypassing the need for interferometry which inevitably compromises QPI quality under ultrafast operation. We employ multi‐ATOM for large‐scale, label‐free, multivariate, cell‐type classification (e.g. breast cancer subtypes, and leukemic cells vs peripheral blood mononuclear cells) at high accuracy (>94%). Our results suggest that multi‐ATOM could empower new strategies in large‐scale biophysical single‐cell analysis with applications in biology and enriching disease diagnostics.      

Reflection‐mode switchable subwavelength Bessel‐beam and Gaussian‐beam photoacoustic microscopy in vivo

We have developed a reflection‐mode switchable subwavelength Bessel‐beam (BB) and Gaussian‐beam (GB) photoacoustic microscopy (PAM) system. To achieve both reflection‐mode and high resolution, we tightly attached a very small ultrasound transducer to an optical objective lens with numerical aperture of 1.0 and working distance of 2.5 mm. We used axicon and an achromatic doublet in our system to obtain the extended depth of field (DOF) of the BB. To compare the DOF performance achieved with our BB‐PAM system against GB‐PAM system, we designed our system so that the GB can be easily generated by simply removing the lenses. Using a 532 nm pulse laser, we achieved the lateral resolutions of 300 and 270 nm for BB‐PAM and GB‐PAM, respectively. The measured DOF of BB‐PAM was approximately 229 μm, which was about 7× better than that of GB‐PAM. We imaged the vasculature of a mouse ear using BB‐PAM and GB‐PAM and confirmed that the DOF of BB‐PAM is much better than the DOF of GB‐PAM. Thus, we believe that the high resolution achieved at the extended DOF by our system is very practical for wide range of biomedical research including red blood cell (RBC) migration in blood vessels at various depths and observation of cell migration or cell culture.      

Wide‐field color imaging of scatter‐based tissue contrast using both high spatial frequency illumination and cross‐polarization gating

This study characterizes the scatter‐specific tissue contrast that can be obtained by high spatial frequency (HSF) domain imaging and cross‐polarization (CP) imaging, using a standard color imaging system, and how combining them may be beneficial. Both HSF and CP approaches are known to modulate the sensitivity of epi‐illumination reflectance images between diffuse multiply scattered and superficially backscattered photons, providing enhanced contrast from microstructure and composition than what is achieved by standard wide‐field imaging. Measurements in tissue‐simulating optical phantoms show that CP imaging returns localized assessments of both scattering and absorption effects, while HSF has uniquely specific sensitivity to scatter‐only contrast, with a strong suppression of visible contrast from blood. The combination of CP and HSF imaging provided an expanded sensitivity to scatter compared with CP imaging, while rejecting specular reflections detected by HSF imaging. ex vivo imaging of an atlas of dissected rodent organs/tissues demonstrated the scatter‐based contrast achieved with HSF, CP and HSF‐CP imaging, with the white light spectral signal returned by each approach translated to a color image for intuitive encoding of scatter‐based contrast within images of tissue. The results suggest that visible CP‐HSF imaging could have the potential to aid diagnostic imaging of lesions in skin or mucosal tissues and organs, where just CP is currently the standard practice imaging modality.      

Two‐ and three‐photon absorption cross‐section characterization for high‐brightness,cell‐specific multiphoton fluorescence brain imaging

We demonstrate an accurate quantitative characterization of absolute two‐ and three‐photon absorption (2PA and 3PA) action cross sections of a genetically encodable fluorescent marker Sypher3s. Both 2PA and 3PA action cross sections of this marker are found to be remarkably high, enabling high‐brightness, cell‐specific two‐ and three‐photon fluorescence brain imaging. Brain imaging experiments on sliced samples of rat's cortical areas are presented to demonstrate these imaging modalities. The 2PA action cross section of Sypher3s is shown to be highly sensitive to the level of pH, enabling pH measurements via a ratiometric readout of the two‐photon fluorescence with two laser excitation wavelengths, thus paving the way toward fast optical pH sensing in deep‐tissue experiments.     

Dual‐color STED super‐resolution microscope using a single laser source

STED (stimulated emission depletion) microscopy is one of the most promising super‐resolution fluorescence microscopies，due to its fast imaging and ultra‐high resolution. In this paper, we present a dual‐color STED microscope with a single laser source. Polarization beam splitters are used to separate the output from a supercontinuum laser source into four laser beams, including two excitation beams (488, 635 nm) and two depletion beams (592, 775 nm). These four laser beams are then used to build a low cost dual‐color STED system to achieve a spatial resolution of 75 nm in cell samples.     

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.      

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.     

Two‐photon imaging of fiber‐coupled neurons

Optical coupling between a single, individually addressable neuron and a properly designed optical fiber is demonstrated. Two‐photon imaging is shown to enable a quantitative in situ analysis of such fiber–single‐neuron coupling in the live brain of transgenic mice. Fiber‐optic interrogation of single pyramidal neurons in mouse brain cortex is performed with the positioning of the fiber probe relative to the neuron accurately mapped by means of two‐photon imaging. These results pave the way for fiber‐optic interfaces to single neurons for a stimulation and interrogation of individually addressable brain cells in chronic in vivo studies on freely behaving transgenic animal models, as well as the integration of fiber‐optic single‐neuron stimulation into the optical imaging framework.

     

The whither of bacteriophytochrome‐based near‐infrared fluorescent proteins: Insights from two‐photon absorption spectroscopy

We present one‐ and two‐photon‐absorption fluorescence spectroscopic analysis of biliverdin (BV) chromophore–based single‐domain near‐infrared fluorescent proteins (iRFPs). The results of these studies are used to estimate the internal electric fields acting on BV inside iRFPs and quantify the electric dipole properties of this chromophore, defining the red shift of excitation and emission spectra of BV‐based iRFPs. The iRFP studied in this work is shown to fit well the global diagram of the red‐shift tunability of currently available BV‐based iRFPs as dictated by the quadratic Stark effect, suggesting the existence of the lower bound for the strongest red shifts attainable within this family of fluorescent proteins. The absolute value of the two‐photon absorption (TPA) cross section of a fluorescent calcium sensor based on the studied iRFP is found to be significantly larger than the TPA cross sections of other widely used genetically encodable fluorescent calcium sensors.      

Prostate‐specific membrane antigen‐targeted photoacoustic imaging of prostate cancer in vivo

A sensitive, noninvasive method to detect localized prostate cancer, particularly for early detection and repetitive study in patients undergoing active surveillance, remains an unmet need. Here, we propose a molecular photoacoustic (PA) imaging approach by targeting the prostate‐specific membrane antigen (PSMA), which is over‐expressed in the vast majority of prostate cancers. We performed spectroscopic PA imaging in an experimental model of prostate cancer, namely, in immunocompromised mice bearing PSMA+ (PC3 PIP) and PSMA? (PC3 flu) tumors through administration of the known PSMA‐targeted fluorescence agent, YC‐27. Differences in contrast between PSMA+ and isogenic control tumors were observed upon PA imaging, with PSMA+ tumors showing higher contrast in average of 66.07‐fold with 5 mice at the 24‐hour postinjection time points. These results were corroborated using standard near‐infrared fluorescence imaging with YC‐27, and the squared correlation between PA and fluorescence intensities was 0.89. Spectroscopic PA imaging is a new molecular imaging modality with sufficient sensitivity for targeting PSMA in vivo, demonstrating the potential applications for other saturable targets relevant to cancer and other disorders.      

Towards correlative super‐resolution fluorescence and electron cryo‐microscopy

Correlative light and electron microscopy (CLEM) has become a powerful tool in life sciences. Particularly cryo‐CLEM, the combination of fluorescence cryo‐microscopy (cryo‐FM) permitting for non‐invasive specific multi‐colour labelling, with electron cryo‐microscopy (cryo‐EM) providing the undisturbed structural context at a resolution down to the Ångstrom range, has enabled a broad range of new biological applications. Imaging rare structures or events in crowded environments, such as inside a cell, requires specific fluorescence‐based information for guiding cryo‐EM data acquisition and/or to verify the identity of the structure of interest. Furthermore, cryo‐CLEM can provide information about the arrangement of specific proteins in the wider structural context of their native nano‐environment. However, a major obstacle of cryo‐CLEM currently hindering many biological applications is the large resolution gap between cryo‐FM (typically in the range of ～400 nm) and cryo‐EM (single nanometre to the Ångstrom range). Very recently, first proof of concept experiments demonstrated the feasibility of super‐resolution cryo‐FM imaging and the correlation with cryo‐EM. This opened the door towards super‐resolution cryo‐CLEM, and thus towards direct correlation of structural details from both imaging modalities.     

Label‐free in vivo cellular‐level detection and imaging of apoptosis

Cell death plays a critical role in health and homeostasis as well as in the pathogenesis and treatment of a broad spectrum of diseases and can be broadly divided into two main categories: apoptosis, or programmed cell death, and necrosis, or acute cell death. While these processes have been characterized extensively in vitro, label‐free detection of apoptosis and necrosis at the cellular level in vivo has yet to be shown. In this study, for the first time, fluorescence lifetime imaging microscopy (FLIM) of intracellular reduced nicotinamide adenine dinucleotide (NADH) was utilized to assess the metabolic response of in vivo mouse epidermal keratinocytes following induction of apoptosis and necrosis. Results show significantly elevated levels of both the mean lifetime of NADH and the intracellular ratio of protein bound‐to‐free NADH in the apoptotic compared to the necrotic tissue. In addition, the longitudinal profiles of these two cell death processes show remarkable differences. By identifying and extracting these temporal metabolic signatures, apoptosis in single cells can be studied in native tissue environments within the living organism.

     

Multicontrast endomyocardial imaging by single‐channel high‐resolution cross‐polarization optical coherence tomography

A single‐channel high‐resolution cross‐polarization (CP) optical coherence tomography (OCT) system is presented for multicontrast imaging of human myocardium in one‐shot measurement. The intensity and functional contrasts, including the ratio between the cross‐ and co‐polarization channels as well as the cumulative retardation, are reconstructed from the CP‐OCT readout. By comparing the CP‐OCT results with histological analysis, it is shown that the system can successfully delineate microstructures in the myocardium and differentiate the fibrotic myocardium from normal or ablated myocardium based on the functional contrasts provided by the CP‐OCT system. The feasibility of using A‐line profiles from the 2 orthogonal polarization channels to identify fibrotic myocardium, normal myocardium and ablated lesion is also discussed.      

Luminescent properties of 4‐aminobenzo‐15‐crown‐5 after preferential binding of ferric ions in aqueous solutions

We report a combined approach that introduces the use of 4‐aminobenzo‐15‐crown‐5 (4AB15C5) for the detection of ferric(III) ions by colorimetric, ultraviolet (UV)–visible light absorption, fluorescence, and live‐cell imaging techniques along with density functional theory (DFT) calculations. We have found that 4AB15C5 is sensitive and selective for binding ferric(III) ions in aqueous solutions. DFT calculations using the polarizable continuum model have been used to explain the strong binding of the ferric ion by 4AB15C5 in aqueous solutions. The detection limit in the fluorescence quenching measurements was found to be as low as 50 μM for the ferric ion with a determined Stern–Volmer constant of 1.52 × 10⁴ M^?1. Fluorescence intensity did not change for other ions tested, Fe²⁺, Co²⁺, Mn²⁺, Mg²⁺, Zn²⁺, Ca²⁺, NH₄⁺, Na⁺, and K⁺ ions. Live‐cell fluorescence imaging was also used to check the intracellular variations in ferric ion levels. Our spectroscopic data indicated that 4AB15C5 can bind ferric ions selectively in aqueous solutions.     

Real‐time mid‐infrared imaging of living microorganisms

The speed and efficiency of quantum cascade laser‐based mid‐infrared microspectroscopy are demonstrated using two different model organisms as examples. For the slowly moving Amoeba proteus, a quantum cascade laser is tuned over the wavelength range of 7.6 µm to 8.6 µm (wavenumbers 1320 cm^–1 and 1160 cm^–1, respectively). The recording of a hyperspectral image takes 11.3 s whereby an average signal‐to‐noise ratio of 29 is achieved. The limits of time resolution are tested by imaging the fast moving Caenorhabditis elegans at a discrete wavenumber of 1265 cm^–1. Mid‐infrared imaging is performed with the 640 × 480 pixel video graphics array (VGA) standard and at a full‐frame time resolution of 0.02 s (i.e. well above the most common frame rate standards). An average signal‐to‐noise ratio of 16 is obtained. To the best of our knowledge, these findings constitute the first mid‐infrared imaging of living organisms at VGA standard and video frame rate.

     

Flexible wide‐field optical micro‐angiography based on Fourier‐domain multiplexed dual‐beam swept source optical coherence tomography

Wide‐field optical coherence tomography angiography (OCTA) is gaining interest in clinical imaging applications. In this pursuit, it is challenging to maintain the imaging resolution and sensitivity throughout the wide field of view (FoV). Here, we propose a novel method/system of dual‐beam arrangement and Fourier‐domain multiplexing to achieve wide‐field OCTA when imaging the uneven surface samples. The proposed system provides 2 separate FoVs, with flexibility that the imaging area, focus of the imaging beam and imaging depth range can be individually adjusted for each FoV, leading to either (1) increased system imaging FoV or (2) capability of targeting 2 regions of interests that locate at depths with large difference between each other. We demonstrate this novel method by employing 100 kHz laser source in a swept source OCTA to achieve an effective 200 kHz sweeping rate, covering a 22 × 22 mm FoV. The results are verified by a SS‐OCTA system employing a 200 kHz laser source, together with the experimental demonstrations when imaging whole brain vasculature in rodent models and skin blood perfusion in human fingers, show‐casing the capability of proposed system to image live large samples with complex surface topography.      

Simultaneous multi‐site two‐photon photostimulation in three dimensions

We demonstrate simultaneous multi‐site two‐photon photolysis of caged neurotransmitters with close to diffraction‐limited resolution in all three dimensions (3D). We use holographic projection of multiple focal spots, which allows full control over the 3D positions of uncaging sites with a high degree of localized excitation. Our system incorporates a two‐photon imaging setup to visualize the 3D morphology of the neurons in order to accurately determine the photostimulation sites. We show its application to studies of synaptic integration by performing simultaneous and controlled glutamate delivery at multiple locations on dendritic trees. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

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.      

Extended‐release PEG–luciferin allows for long‐term imaging of firefly luciferase activity in vivo

Bioluminescence has gained favour in the last decade as an approach for observing tumours in vivo in a non‐destructive manner. This very sensitive technique is based on light emission by the reaction of luciferin with the enzyme luciferase, as measured by a photodetector. Ever since the development of recombinant tumour cell lines that have been engineered to produce luciferase, a vast number of experiments have been carried out examining tumour growth, tumour metastasis and the effect of therapeutic regimens in such cases. A primary stumbling block, however, is the relatively short circulatory half‐life of luciferin. In this paper, we propose the PEGylation of 6‐amino‐d ‐luciferin to extend its in vivo circulatory half‐life, thus making the possibility of long‐term observations in animals possible. The covalent attachment was through a carbamate linker that is known to hydrolyse in vivo, releasing the parent compound. Based on our studies, longer emission of the PEGylated luciferin was observed, as compared to free luciferin in mice bearing PC3 prostate tumours expressing luciferase. This result suggests that this reagent can be used in applications requiring extended monitoring of luciferase activation in vivo. Copyright © 2008 John Wiley & Sons, Ltd.