High Spatiotemporal Near-Infrared II Fluorescence Lifetime Imaging for Quantitative Detection of Clinical Tumor Margins.

Accurate detection of tumor margins is essential for successful cancer surgery. While indocyanine green (ICG)-based near-infrared (NIR) fluorescence (FL) surgical navigation enhances the visual identification of tumor margins, its accuracy remains subjective, underscoring the need for quantitative approaches. In this study, a high spatiotemporal fluorescence lifetime (FLT) imaging technology is developed in the second near-infrared window (NIR-II, 1000-1700 nm) for quantitative tumor margin detection, utilizing folate receptor-targeted ICG nanoprobes (FPH-ICG).

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.

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.

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 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.

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.

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.

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 (∼109), low timing jitter (∼50), and low dark count rate (<100).

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- scanning scheme and confocal detection to overcome these limitations.

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 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.

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.

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 imaging of zebrafish, normal mouse ear, and a xenograft tumor model inoculated in the mouse ear.