Accurate piecewise centroid calculation algorithm for wavefront measurement in adaptive optics.

Adaptive optics using direct wavefront sensing (direct AO) is widely used in two-photon microscopy to correct sample-induced aberrations and restore diffraction-limited performance at high speeds. In general, the direct AO method employs a Sharked-Hartman wavefront sensor (SHWS) to directly measure the aberrations through a spot array. However, the signal-to-noise ratio (SNR) of spots in SHWS varies significantly within deep tissues, presenting challenges for accurately locating spot centroids over a large SNR range, particularly under extremely low SNR conditions. To address this issue, we propose a piecewise centroid calculation algorithm called GCP, which integrates three optimal algorithms for accurate spot centroid calculations under high-, medium-, and low-SNR conditions. Simulations and experiments demonstrate that the GCP can accurately measure aberrations over a large SNR range and exhibits robustness under extremely low-SNR conditions. Importantly, GCP improves the AO working depth by 150 µm compared to the conventional algorithm.

Probing cell metabolism using the two-photon excitation autofluorescence lifetime of tryptophan.

Compared to intensity detection, fluorescence lifetime has the advantage of being unaffected by variations in excitation intensity, fluorophore concentration, or attenuation due to biological absorption and scattering. In this Letter, to the best of our knowledge, we present the use of the two-photon excitation autofluorescence lifetime imaging of tryptophan (TRP) to probe cell metabolism for the first time. Tests of pure chemical samples showed that the fluorescence lifetime of TRP was highly sensitive to changes in molecular conformation and the environment. In in vitro cell experiments, we successfully utilized the fluorescence lifetime of TRP to distinguish tumor cells from healthy cells, track the therapeutic effect of the tumor immunotherapy drug 1-MT for HeLa cells, and monitor cells in response to carbonyl cyanide 3-chlorophenylhydrazone (CCCP)-induced cell apoptosis. These results reveal that the two-photon excitation autofluorescence lifetime of TRP could be a sensitive natural probe of cell metabolism in living cells.

In vivo identification of arteries and veins using two-photon excitation elastin autofluorescence.

Distinguishing arteries from veins in vivo has a great significance in clinical practices and preclinical studies. Optical imaging methods such as two-photon microscopy can provide high-resolution morphological information of tissue and are therefore extremely suitable for imaging small blood vessels. However, few optical imaging methods allow in vivo identification of arteries and veins merely utilizing the autofluorescence signal of blood vessels. In this report, we found the arterial wall generates a remarkably stronger two-photon excitation autofluorescence (TPEA) signal compared with the venous wall based on BALB/c mice. According to histological analysis and fluorescence characteristic measurement, the contrast signal is confirmed to be from elastin fibers. Employing this unique feature, we propose an objective and effective artery-vein separation strategy that considers the presence of the elastin-TPEA border as the indicator of arteries. Using this strategy, the arterial and venous networks of the dorsal skin and cerebral cortex of BALB/c mice are demonstrated to be excellently mapped and accurately separated in vivo without depending on any exogenous contrast agent, empirical knowledge, and algorithm. This study may provide a novel technique for mapping arterial and venous networks for anatomic research as well as an extra aid to basic researches on the mechanism, diagnosis, and treatment of blood vessel-related diseases.

Adaptive optics via pupil ring segmentation improves spherical aberration correction for two-photon imaging of optically cleared tissues.

Optical clearing methods reduce the optical scattering of biological samples and thereby extend optical imaging penetration depth. However, refractive index mismatch between the immersion media of objectives and clearing reagents induces spherical aberration (SA), causing significant degradation of fluorescence intensity and spatial resolution. We present an adaptive optics method based on pupil ring segmentation to correct SA in optically cleared samples. Our method demonstrates superior SA correction over a modal-based adaptive optics method and restores the fluorescence intensity and resolution at high imaging depth. Moreover, the method can derive an SA correction map for the whole imaging volume based on three representative measurements. It facilitates SA correction during image acquisition without intermittent SA measurements. We applied this method in mouse brain tissues treated with different optical clearing methods. The results illustrate that the synaptic structures of neurons within 900 µm depth can be clearly resolved after SA correction.

In vivo label-free two-photon excitation autofluorescence microscopy of microvasculature using a 520 nm femtosecond fiber laser.

Observing microvasculature in its native environment provides invaluable information to understand the initiation and development of microcirculatory related diseases. However, the lack of a high-resolution three-dimensional (3D) imaging technique hinders in vivo investigation of the microvasculature. Recently, we found that the red blood cells can emit autofluorescence signals with short-wavelength two-photon excitation. In this study, we exploited this property and developed a time-resolved two-photon excitation microscopy system using a homemade 520 nm femtosecond fiber laser as the excitation source. Using this system, we could achieve intravital high-resolution 3D imaging of a microvascular network noninvasively. In a mouse tumor model, tumorous blood vessels could be observed and distinguished clearly from the normal vessels.

Intravital confocal fluorescence lifetime imaging microscopy in the second near-infrared window.

We present confocal fluorescence lifetime imaging microscopy in the second near-infrared (NIR-II) window to assess the morphological and biochemical information of live samples. A home-built superconducting single-photon detector (SSPD) was used to facilitate the NIR-II fluorescence lifetime measurement. The SSPD has many advantages, including high sensitivity to NIR-II signals (detection efficiency >50%), fast temporal response (∼109ps), low timing jitter (∼50ps), and low dark count rate (<100cps). We demonstrate the feasibility of the developed microscopy system by comparing fluorescence lifetimes of a range of fluorophores with emission in the NIR-II window and by performing multicolor three-dimensional fluorescence lifetime imaging of a mouse ear in vivo. The biochemical properties of the cells and tissues probed by the fluorescence lifetimes of the fluorophores provide complementary information for biomedical studies, significantly benefiting diverse applications in life science.

Short-wavelength excitation two-photon intravital microscopy of endogenous fluorophores.

The noninvasive two-photon excitation autofluorescence imaging of cellular and subcellular structure and dynamics in live tissue could provide critical in vivo information for biomedical studies. However, the two-photon microscopy of short-wavelength endogenous fluorophores, such as tryptophan and hemoglobin, is extremely limited due to the lack of suitable imaging techniques. In this study, we developed a short-wavelength excitation time- and spectrum-resolved two-photon microscopy system. A 520-nm femtosecond fiber laser was used as the excitation source, and a time-correlated single-photon counting module connected with a spectrograph was used to provide time- and spectrum-resolved detection capability. The system was specially designed for measuring ultraviolet and violet-blue fluorescence signals and thus was very suitable for imaging short-wavelength endogenous fluorophores. Using the system, we systematically compared the fluorescence spectra and fluorescence lifetimes of short-wavelength endogenous fluorophores, including the fluorescent molecules tyrosine, tryptophan, serotonin (5-HT), niacin (vitamin B3), pyridoxine (vitamin B6), and NADH and the protein group (keratin, elastin, and hemoglobin). Then, high-resolution three-dimensional (3D) label-free imaging of different biological tissues, including rat esophageal tissue, rat oral cheek tissue, and mouse ear skin, was performed in vivo or ex vivo. Finally, we conducted time-lapse imaging of leukocyte migration in the lipopolysaccharide injection immunization model and a mechanical trauma immunization model. The results indicate that the system can specifically characterize short-wavelength endogenous fluorophores and provide noninvasive label-free 3D visualization of fine structures and dynamics in biological systems. The microscopy system developed here can empower more flexible imaging of endogenous fluorophores and provide a novel method for the 3D monitoring of biological events in their native environment.

Albumin-Consolidated AIEgens for Boosting Glioma and Cerebrovascular NIR-II Fluorescence Imaging.

The application of an exogenous polymer matrix to construct aggregation-induced emission (AIE) nanoprobes promotes the utility of AIE luminogens (AIEgens) in diagnosing brain diseases. However, the limited fluorescence (FL) and low active-targeting abilities of AIE-based nanoprobes impede their imaging application. Here, we employed endogenous albumin as an effective matrix to encapsulate AIEgens to enhance FL quantum yield (QY) and active-targeting ability. The albumin-consolidated strategy effectively inhibited the intramolecular vibration of AIEgens and enhanced endocytosis mediated by the gp60 receptor. The QYs of three kinds of albumin-based AIE nanoprobes with FL emissions ranging from the visible (400-650 nm) to the second near-infrared (NIR-II, 1000-1700 nm) region was at least 10% higher, and the tumor-targeting efficiency was ∼25% higher, compared with those of nanoprobes constructed by the exogenous polymer. Albumin-based AIE nanoprobes have achieved active-targeting NIR-II imaging of brain tumors and cerebrovascular imaging with a high signal-to-background ratio (SBR, ∼90) and high resolution (∼70 µm) in mouse models. Therefore, the albumin-based AIE nanoprobes will enable FL imaging-guided surgery of brain tumors and cerebral ischemia, which will improve surgical efficacy to prevent recurrence and side effects.

Depth-resolved NIR-II fluorescence mesoscope.

NIR-II fluorescence imaging is a promising method for visualizing biological structures in deep tissue, owing to the advantages of significantly suppressed optical scattering and diminished autofluorescence in biological tissues. However, few NIR-II fluorescence imaging approaches can simultaneously achieve a large field of view, high resolution and superior penetration depth, while exhibiting optical sectioning capability. In this paper, we present a novel NIR-II fluorescence mesoscopy system based on the f-θ scanning scheme and confocal detection to overcome these limitations. When used with NIR-II fluorescent dyes, our setup performs NIR-II fluorescence imaging on samples as large as 7.5×7.5 mm2 with a lateral resolution of 6.3 µm. In addition, our system provides a depth-resolved imaging ability and zooming function. We successfully demonstrate in vivo cerebrovascular imaging of a mouse with local ischemia. Thus, our system provides new opportunities to explore the mechanism of cerebrovascular disease.

Multicolor re-scan super-resolution imaging of live cells.

BACKGROUND: Multicolor fluorescence microscopy has proved essential in biological studies. However, the application of conventional multicolor microscopy to imaging subcellular organelles is restricted by its diffraction-limited spatial resolution. Re-scan confocal microscopy (RCM), a novel super-resolution imaging technique, can effectively address this problem. However, previous multicolor RCM imaging methods usually led to spatial mismatch in images due to the sequential scanning of the sample with multiple excitation lasers. METHODS: We present a new RCM system to achieve multicolor super-resolution imaging. A spectrograph was used as the multicolor detection system, and a linear spectral unmixing algorithm was applied to separate different fluorophores in the spectral image. Moreover, since the image reconstruction process induced an artificial resolution improvement, a gamma correction was introduced to restore the multicolor super-resolution image. RESULTS: By imaging phalloidin-labeled F-actin in breast cancer cells, we found that the lateral resolution of our system is approximately 171 nm, which is a 1.8-fold improvement over that of wide-field imaging. The successful identification of three types of fluorescent beads indicated that our multicolor RCM can resolve different fluorophores whose spectra largely overlap with each other. Finally, we demonstrated that our method is suitable for imaging multicolor-labeled organelles of live cells. CONCLUSIONS: Our novel RCM system can acquire multicolor super-resolution images of live cells without spatial mismatch, obvious photobleaching or photodamage. This system may provide a new imaging tool for monitoring dynamic events involving interactions between multiple molecules and organelles in cells.

The integrated high-resolution reflection-mode photoacoustic and fluorescence confocal microscopy.

A dual modality microscopy with the highest imaging resolution reported so far based on reflection-mode photoacoustic and confocal fluorescence is presented in this study. The unique design of the imaging head of the microscope makes it highly convenient for scalable high-resolution imaging by simply switching the optical objectives. The submicron resolution performance of the system is demonstrated via in vivo imaging of zebrafish, normal mouse ear, and a xenograft tumor model inoculated in the mouse ear. The imaging results confirm that the presented dual-modality microscopy imaging system could play a vital role in observing model organism, studying tumor angiogenesis and assessment of antineoplastic drugs.