Front Cover: Monitoring implantable immunoisolation devices with intrinsic fluorescence of genipin (J. Biophotonics 4/2019)

Incorporation of genipin into design of immunoisolation devices is harnessed for in vivo imaging. Using cell‐compatible in situ crosslinking reactions, a fast, efficient and noncytotoxic procedure is shown to maximize fluorescence of microcapsules. By injecting increasing doses of microcapsules in mice, good linearity of signal to microcapsule dose is obtained over several weeks. This allows quantitative assessment of the actual injected dose and monitoring of its position over time, thereby significantly enhancing the efficacy and biosafety of the therapy. Further details can be found in the article by Edorta Santos‐Vizcaino, Henry Haley, Ainhoa Gonzalez‐Pujana, et al. ( e201800170 ).

     

Widefield fluorescence lifetime imaging of protoporphyrin IX for fluorescence‐guided neurosurgery: An ex vivo feasibility study

Achieving a maximal safe extent of resection during brain tumor surgery is the goal for improved patient prognosis. Fluorescence‐guided neurosurgery using 5‐aminolevulinic acid (5‐ALA) induced protoporphyrin IX has thereby become a valuable tool enabling a high frequency of complete resections and a prolonged progression‐free survival in glioblastoma patients. We present a widefield fluorescence lifetime imaging device with 250 mm working distance, working under similar conditions such as surgical microscopes based on a time‐of‐flight dual tap CMOS camera. In contrast to intensity‐based fluorescence imaging, our method is invariant to light scattering and absorption while being sensitive to the molecular composition of the tissue. We evaluate the feasibility of lifetime imaging of protoporphyrin IX using our system to analyze brain tumor phantoms and fresh 5‐ALA‐labeled human tissue samples. The results demonstrate the potential of our lifetime sensing device to go beyond the limitation of current intensity‐based fluorescence‐guided neurosurgery.      

Fiber‐based fluorescence lifetime imaging of recellularization processes on vascular tissue constructs

New techniques able to monitor the maturation of tissue engineered constructs over time are needed for a more efficient control of developmental parameters. Here, a label‐free fluorescence lifetime imaging (FLIm) approach implemented through a single fiber‐optic interface is reported for nondestructive in situ assessment of vascular biomaterials. Recellularization processes of antigen removed bovine pericardium scaffolds with endothelial cells and mesenchymal stem cells were evaluated on the serous and the fibrous sides of the scaffolds, 2 distinct extracellular matrix niches, over the course of a 7 day culture period. Results indicated that fluorescence lifetime successfully report cell presence resolved from extracellular matrix fluorescence. The recellularization process was more rapid on the serous side than on the fibrous side for both cell types, and endothelial cells expanded faster than mesenchymal stem cells on antigen‐removed bovine pericardium. Fiber‐based FLIm has the potential to become a nondestructive tool for the assessment of tissue maturation by allowing in situ imaging of intraluminal vascular biomaterials.      

Rapid one‐step purification of single‐cells encapsulated in alginate microcapsules from oil to aqueous phase using a hydrophobic filter paper: Implications for single‐cell experiments

By virtue of the biocompatibility and physical properties of hydrogel, picoliter‐sized hydrogel microcapsules have been considered to be a biometric signature containing several features similar to that of encapsulated single cells, including phenotype, viability, and intracellular content. To maximize the experimental potential of encapsulating cells in hydrogel microcapsules, a method that enables efficient hydrogel microcapsule purification from oil is necessary. Current methods based on centrifugation for the conventional stepwise rinsing of oil, are slow and laborious and decrease the monodispersity and yield of the recovered hydrogel microcapsules. To remedy these shortcomings we have developed a simple one‐step method to purify alginate microcapsules, containing a single live cell, from oil to aqueous phase. This method employs oil impregnation using a commercially available hydrophobic filter paper without multistep centrifugal purification and complicated microchannel networks. The oil‐suspended alginate microcapsules encapsulating single cells from mammalian cancer cell lines (MCF–7, HepG2, and U937) and microorganisms (Chlorella vulgaris) were successfully exchanged to cell culture media by quick (～10 min) depletion of the surrounding oil phase without coalescence of neighboring microcapsules. Cell proliferation and high integrity of the microcapsules were also demonstrated by long‐term incubation of microcapsules containing a single live cell. We expect that this method for the simple and rapid purification of encapsulated single‐cell microcapsules will attain widespread adoption, assisting cell biologists and clinicians in the development of single‐cell experiments.     

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.      

Multiparametric evaluation of hindlimb ischemia using time‐series indocyanine green fluorescence imaging

Peripheral arterial disease (PAD) can further cause lower limb ischemia. Quantitative evaluation of the vascular perfusion in the ischemic limb contributes to diagnosis of PAD and preclinical development of new drug. In vivo time‐series indocyanine green (ICG) fluorescence imaging can noninvasively monitor blood flow and has a deep tissue penetration. The perfusion rate estimated from the time‐series ICG images is not enough for the evaluation of hindlimb ischemia. The information relevant to the vascular density is also important, because angiogenesis is an essential mechanism for post‐ischemic recovery. In this paper, a multiparametric evaluation method is proposed for simultaneous estimation of multiple vascular perfusion parameters, including not only the perfusion rate but also the vascular perfusion density and the time‐varying ICG concentration in veins. The target method is based on a mathematical model of ICG pharmacokinetics in the mouse hindlimb. The regression analysis performed on the time‐series ICG images obtained from a dynamic reflectance fluorescence imaging system. The results demonstrate that the estimated multiple parameters are effective to quantitatively evaluate the vascular perfusion and distinguish hypo‐perfused tissues from well‐perfused tissues in the mouse hindlimb. The proposed multiparametric evaluation method could be useful for PAD diagnosis.

The estimated perfusion rate and vascular perfusion density maps (left) and the time‐varying ICG concentration in veins of the ankle region (right) of the normal and ischemic hindlimbs.     

Comparison of optically‐derived biomarkers in healthy and brain tumor tissue under one‐ and two‐photon excitation

The surgical outcome of brain tumor resection and needle biopsy is significantly correlated to the patient's prognosis. Brain tumor surgery is limited to resecting the solid portion of the tumor as current intraoperative imaging modalities are incapable of delineating infiltrative regions. For accurate delineation, in situ tissue interrogation at the submicron scale is warranted. Additionally, multimodal detection is required to remediate the genetically and molecularly heterogeneous nature of brain tumors, notably, that of gliomas, meningioma and brain metastasis. Multimodal detection, such as spectrally‐ and temporally‐resolved fluorescence under one‐ and two‐photon excitation, enables characterizing tissue based on several endogenous optical contrasts. In order to assign the optically‐derived parameters to different tissue types, construction of an optical database obtained from biopsied tissue is warranted. This report showcases the different quantitative and semi‐quantitative optical markers that may comprise the tissue discrimination database. These include: the optical index ratio, the optical redox ratio, the relative collagen density, spectrally‐resolved fluorescence lifetime parameters, two‐photon fluorescence imaging and second harmonic generation imaging.     

Inside Front Cover: Increasing fluorescence lifetime for resolution improvement in stimulated emission depletion nanoscopy (J. Biophotonics 5/2019)

A STED‐FLIM system is developed to observe the changes of fluorescence lifetime. The pictures show increased lifetime of fluorescent microspheres samples with laser illumination time in both confocal and STED imaging modes. Due to the saturation power of fluorophores is correlated with fluorescence lifetime, the lifetime increase is beneficial for the reduction of the saturation power, indicating the same imaging resolution can be achieved in a lower depletion power. Further details can be found in the article by Lu‐Wei Wang, Yue Chen, Wei Yan, et al. ( e201800315 ).

     

Front Cover: Dynamic observation and quantification of type I/II collagen in chondrogenesis of mesenchymal stem cells by second‐order susceptibility microscopy (J. Biophotonics 2/2019)

Top row is time‐lapse, multiphoton imaging of induced chondrogenesis from stem cells. The second row shows the time‐lapse second harmonic generation imaging of generated collagen. However, only the third row of second order susceptibility imaging allows the differentiation of the content of the two types of collagen (I and II) that were produced over time. The method allows the noninvasive and label‐free discrimination of different collagen species in real time and can be used for quality control in tissue engineering. Further details can be found in the article by Chiu‐Mei Hsueh, Hung‐Ming Lin, Te‐Yu Tseng, et al. ( e201800097 ).

     

Front Cover: Widefield fluorescence lifetime imaging of protoporphyrin IX for fluorescence‐guided neurosurgery: An ex vivo feasibility study (J. Biophotonics 6/2019)

To optimize the resection of gliomas during neurosurgery we present an imaging system capable of wide field fluorescence lifetime mapping with 11 mm field of view and 250 mm working distance. Based on a time of flight dual‐tap CMOS camera and a modulated laser at 405 nm we show unobserved quenching effects in tissue phantoms and demonstrate life‐time imaging on 5‐ALA labeled human ex vivo brain tumor samples. Further details can be found in the article by Mikael T. Erkkilä, Bianca Bauer, Nancy Hecker‐Denschlag, et al. ( e201800378 ).

     

Saturated two‐photon excitation fluorescence microscopy for the visualization of cerebral neural networks at millimeters deep depth

Optical imaging is a key modality for observing biological specimen with higher spatial resolution. However, scattering and absorption of light in tissues are inherent barriers in maximizing imaging depth in biological tissues. To achieve this goal, use of light at near‐infrared spectrum can improve the present situation. Here, the capability of saturated two‐photon saturated excitation (TP‐SAX) fluorescence microscopy to image at depths of >2.0 mm, with submicron resolution in transparent mouse brain imaging, is demonstrated. At such depths with scattering‐enlarged point spread function (PSF), we find that TP‐SAX is capable to provide spatial resolution improvement compared to its corresponding TPFM, which is on the other hand already providing a much improved resolution compared with single‐photon confocal fluorescence microscopy. With the capability to further improve spatial resolution at such deep depth with scattering‐enlarged PSF, TP‐SAX can be used for exquisite visualization of delicate cerebral neural structure in the scattering regime with a submicron spatial resolution inside intact mouse brain.      

Real‐time imaging of photodynamic action in bacteria

Fluorescence imaging studies of the processes leading to photodynamic inactivation of bacteria have been limited due to the small size of microorganisms as well as by the faint fluorescence of most photosensitizers. A versatile method based on highly‐sensitive fluorescence microscopy is presented which allows to study, in real time, the incorporation of photosensitizers inside S. aureus upon photodynamic action. The method takes advantage of the fluorescence enhancement of phenothiazine and porphyrin photosensitizers upon entering the bacterial cytosol after the cell wall has been compromised. In combination with typical assays, such as the addition of specific enhancers of reactive oxygen species, it is possible to extract mechanistic information about the pathway of photodynamic damage at the single‐cell level. Imaging experiments in deuterated buffer strongly support a Type‐I mechanism for methylene blue and a very minor role of singlet oxygen.

     

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.      

Visualizing astrocytes in the deep mouse brain in vivo

Astrocytes play a key role in the central nervous system. However, methods of visualizing astrocytes in the deep brain in vivo have been lacking. 3‐photon fluorescence imaging of astrocytes labeled by sulforhodamine 101 (SR101) is demonstrated in deep mouse brain in vivo. Excitation wavelength selection was guided by wavelength‐dependent 3‐photon action cross section (ησ ₃) measurement of SR101. 3‐photon fluorescence imaging of the SR101‐labeled vasculature enabled an imaging depth of 1340‐μm into the mouse brain. This justifies the deep imaging capability of the technique and indicates that the imaging depth is not determined by the signal‐to‐background ratio limit encountered in 2‐photon fluorescence imaging. Visualization of astrocytes 910 μm below the surface of the mouse brain in vivo is demonstrated, 30% deeper than that using 2‐photon fluorescence microscopy. Through quantitative comparison of the signal difference between the SR101‐labeled blood vessels and astrocytes, the challenges of visualizing astrocytes below the white matter is further elucidated.      

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.      

Increasing fluorescence lifetime for resolution improvement in stimulated emission depletion nanoscopy

Super‐resolution microscopy (SRM) has had a substantial impact on the biological sciences due to its ability to observe tiny objects less than 200 nm in size. Stimulated emission depletion (STED) microscopy represents a major category of these SRM techniques that can achieve diffraction‐unlimited resolution based on a purely optical modulation of fluorescence behaviors. Here, we investigated how the laser beams affect fluorescence lifetime in both confocal and STED imaging modes. The results showed that with increasing illumination time, the fluorescence lifetime in two kinds of fluorescent microspheres had an obvious change in STED imaging mode, compared with that in confocal imaging mode. As a result, the reduction of saturation intensity induced by the increase of fluorescence lifetime can improve the STED imaging resolution at the same depletion power. The phenomenon was also observed in Star635P‐labeled human Nup153 in fixed HeLa cells, which can be treated as a reference for the synthesis of fluorescent labels with the sensitivity to the surrounding environment for resolution improvement in STED nanoscopy.      

In vivo autofluorescence in the biological windows: the role of pigmentation

Small animal deep‐tissue fluorescence imaging in the second Biological Window (II‐BW, 1000–1350 nm) is limited by the presence of undesirable infrared‐excited, infrared‐emitted (900–1700 nm) autofluorescence whose origin, spectral properties and dependence on strains is still unknown. In this work, the infrared autofluorescence and laser‐induced whole body heating of five different mouse strains with distinct coat colors (black, grey, agouti, white and nude) has been systematically investigated. While neither the spectral properties nor the magnitude of organ autofluorescence vary significantly between mouse strains, the coat color has been found to strongly determine both the autofluorescence intensity as well as the laser‐induced whole body heating. Results included in this work reveal mouse strain as a critical parameter that has to be seriously considered in the design and performance of small animal imaging experiments based on infrared‐emitting fluorescent markers.

     

easySTORM: a robust,lower‐cost approach to localisation and TIRF microscopy

TIRF and STORM microscopy are super‐resolving fluorescence imaging modalities for which current implementations on standard microscopes can present significant complexity and cost. We present a straightforward and low‐cost approach to implement STORM and TIRF taking advantage of multimode optical fibres and multimode diode lasers to provide the required excitation light. Combined with open source software and relatively simple protocols to prepare samples for STORM, including the use of Vectashield for non‐TIRF imaging, this approach enables TIRF and STORM imaging of cells labelled with appropriate dyes or expressing suitable fluorescent proteins to become widely accessible at low cost.