deep-tissue-three-photon-fluorescence-microscopy-intact-mouse

Deep-Tissue Three-Photon Fluorescence Microscopy in Intact Mouse and Zebrafish Brain

Multiphoton microscopy techniques, such as two-photon microscopy (2PM) and three-photon microscopy (3PM), are powerful tools for deep-tissue in vivo ...

murine-model-thoracic-aortic-dissection-induced-oral

Murine Model of Thoracic Aortic Dissection Induced by Oral &#946;-Aminopropionitrile and Subcutaneous Angiotensin II Infusion

Thoracic aortic dissection (TAD) is a highly fatal cardiovascular disease that lacks efficient medical treatment. Replication of animal models of TAD ...

Cornell University

Tianjin Medical University

Optical imaging through the intact mouse skull is challenging because of skull-induced aberrations and scattering. We found that three-photon excitation provided improved optical sectioning compared with that obtained with two-photon excitation, even when we used the same excitation wavelength and imaging system. Here we demonstrate three-photon imaging of vasculature through the adult mouse skull at >500-μm depth, as well as GCaMP6s calcium imaging over weeks in cortical layers 2/3 and 4 in awake mice, with 8.5 frames per second and a field of view spanning hundreds of micrometers.

Three-photon imaging of mouse brain structure and function through the intact skull.

Light attenuation in thick biological tissues, caused by a combination of absorption and scattering, limits the penetration depth in multiphoton microscopy (MPM). Both tissue scattering and absorption are dependent on wavelengths, which makes it essential to choose the excitation wavelength with minimum attenuation for deep imaging. Although theoretical models have been established to predict the wavelength dependence of light attenuation in brain tissues, the accuracy of these models in experimental settings needs to be verified. Furthermore, the water absorption contribution to the tissue attenuation, especially at 1450 nm where strong water absorption is predicted to be the dominant contributor in light attenuation, has not been confirmed. Here we performed a systematic study of  three-photon imaging at different excitation wavelengths, 1300 nm, 1450 nm, 1500 nm, 1550 nm, and 1700 nm, and quantified the tissue attenuation by calculating the effective attenuation length at each wavelength. The experimental data show that the effective attenuation length at 1450 nm is significantly shorter than that at 1300 nm or 1700 nm. Our results provide unequivocal validation of the theoretical estimations based on water absorption and tissue scattering in predicting the effective attenuation lengths for long wavelength  imaging.

Comparing the effective attenuation lengths for long wavelength  imaging of the mouse brain.

Optical microscopy is a valuable tool for  monitoring of biological structures and functions because of its non-invasiveness. However, imaging deep into biological tissues is challenging due to the scattering and absorption of light. Previous research has shown that 1300 nm and 1700 nm are the two best wavelength windows for deep brain imaging. Here, we combined long-wavelength illumination of ~1700 nm with reflectance confocal microscopy and achieved an imaging depth of ~1.3 mm with ~1-micrometer spatial resolution in adult mouse brains, which is 3-4 times deeper than that of conventional confocal microscopy using visible wavelength. We showed that the method can be added to any laser-scanning microscopy with simple and low-cost sources and detectors, such as continuous-wave diode lasers and InGaAs photodiodes. The long-wavelength, reflectance confocal imaging we demonstrated is label-free, and requires low illumination power. Furthermore, the imaging system is simple and low-cost, potentially creating new opportunities for biomedical research and clinical applications.

 label-free confocal imaging of the deep mouse brain with long-wavelength illumination.

Optical imaging is important for understanding brain function. However, established methods with high spatiotemporal resolution are limited by the potential for laser damage to living tissues. We describe an adaptive femtosecond excitation source that only illuminates the region of interest, which leads to a 30-fold reduction in the power requirement for two- or three-photon imaging of brain activity in awake mice for improved high-speed longitudinal neuroimaging.

An adaptive excitation source for high-speed multiphoton microscopy.

1300 nm three-photon calcium imaging has emerged as a useful technique to allow calcium imaging in deep brain regions. Application to large-scale neural activity imaging entails a careful balance between recording fidelity and perturbation to the sample. We calculated and experimentally verified the excitation pulse energy to achieve  required for the detection of calcium transients in GCaMP6s-expressing neurons for 920 nm two-photon and 1320 nm three-photon excitation. By considering the combined effects of in-focus signal attenuation and out-of-focus background generation, we quantified the cross-over depth beyond which three-photon microscopy outpeforms two-photon microscopy in recording fidelity. Brain tissue heating by continuous three-photon imaging was simulated with Monte Carlo method and experimentally validated with immunohistochemistry. Increased immunoreactivity was observed with 150 mW excitation power at 1 and 1.2 mm imaging depths. Our analysis presents a translatable model for the optimization of three-photon calcium imaging based on experimentally tractable parameters.

Quantitative analysis of 1300-nm three-photon calcium imaging in the mouse brain.

We present a platform for parallel production of standalone, untethered electronic sensors that are truly microscopic, i.e., smaller than the resolution of the naked eye. This platform heterogeneously integrates silicon electronics and inorganic microlight emitting diodes (LEDs) into a 100-μm-scale package that is powered by and communicates with light. The devices are fabricated, packaged, and released in parallel using photolithographic techniques, resulting in ∼10,000 individual sensors per square inch. To illustrate their use, we show proof-of-concept measurements recording voltage, temperature, pressure, and conductivity in a variety of environments.

Microscopic sensors using optical wireless integrated circuits.

, chronic neural recording is critical to understand the nervous system, while a tetherless, miniaturized recording unit can render such recording minimally invasive. We present a tetherless, injectable micro-scale opto-electronically transduced electrode (MOTE) that is ~60m × 30m × 330m, the smallest neural recording unit to date. The MOTE consists of an AlGaAs micro-scale light emitting diode (LED) heterogeneously integrated on top of conventional 180nm complementary metal-oxide-semiconductor (CMOS) circuit. The MOTE combines the merits of optics (AlGaAs LED for power and data uplink), and of electronics (CMOS for signal amplification and encoding). The optical powering and communication enable the extreme scaling while the electrical circuits provide a high temporal resolution (<100s). This paper elaborates on the heterogeneous integration in MOTEs, a topic that has been touted without much demonstration on feasibility or scalability. Based on photolithography, we demonstrate how to build heterogenous systems that are scalable as well as biologically stable - the MOTEs can function in saline water for more than six months, and in a mouse brain for two months (and counting). We also present handling/insertion techniques for users ( biologists) to deploy MOTEs with little or no extra training.

Fabrication of Injectable Micro-Scale Opto-Electronically Transduced Electrodes (MOTEs) for Physiological Monitoring.

Next-generation sequencing (NGS) technology is capable of sequencing millions or billions of DNA molecules simultaneously. Therefore, it represents a promising tool for the analysis of molecular targets for the initial diagnosis of disease, monitoring of disease progression, and identifying the mechanism of drug resistance. On behalf of the Tumor Biomarker Committee of the Chinese Society of Clinical Oncology (CSCO) and the China Actionable Genome Consortium (CAGC), the present expert group hereby proposes advisory guidelines on clinical applications of NGS technology for the analysis of cancer driver genes for precision cancer therapy. This group comprises an assembly of laboratory cancer geneticists, clinical oncologists, bioinformaticians, pathologists, and other professionals. After multiple rounds of discussions and revisions, the expert group has reached a preliminary consensus on the need of NGS in clinical diagnosis, its regulation, and compliance standards in clinical sample collection. Moreover, it has prepared NGS criteria, the sequencing standard operation procedure (SOP), data analysis, report, and NGS platform certification and validation.

Application of next-generation sequencing technology to precision medicine in cancer: joint consensus of the Tumor Biomarker Committee of the Chinese Society of Clinical Oncology.

Developing liquid biopsy technology with higher sensitivity and specificity especially for low-frequency mutations remains crucial. This study demonstrated superior performance of the newly developed digital PCR (dPCR) kit for ctDNA-based  p.T790M detection in metastatic non-small-cell lung cancer (NSCLC) against ARMS-PCR.

A large-scale, multicentered trial evaluating the sensitivity and specificity of digital PCR versus ARMS-PCR for detecting ctDNA-based  p.T790M in non-small-cell lung cancer patients.

Anaplastic lymphoma kinase (ALK) test in advanced non-small cell lung cancer (NSCLC) can help physicians provide target therapies for patients harboring ALK gene rearrangement. This study aimed to investigate the real-world test patterns and positive rates of ALK gene rearrangements in advanced NSCLC.

ArticleTotal : 2	Year
Publication title Cited by 7	2022
Publication title Cited by 1	2025

Article	Year
Three-photon imaging of mouse brain structure and function through the intact skull. Nature methods\| PubMed ID: 30202059	2018
Comparing the effective attenuation lengths for long wavelength imaging of the mouse brain. Biomedical optics express\| PubMed ID: 30338138	2018
label-free confocal imaging of the deep mouse brain with long-wavelength illumination. Biomedical optics express\| PubMed ID: 31065448	2018
An adaptive excitation source for high-speed multiphoton microscopy. Nature methods\| PubMed ID: 31792434	2020
Quantitative analysis of 1300-nm three-photon calcium imaging in the mouse brain. eLife\| PubMed ID: 31999253	2020
Microscopic sensors using optical wireless integrated circuits. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 32303653	2020
Fabrication of Injectable Micro-Scale Opto-Electronically Transduced Electrodes (MOTEs) for Physiological Monitoring. Journal of microelectromechanical systems : a joint IEEE and ASME publication on microstructures, microactuators, microsensors, and microsystems\| PubMed ID: 33071528	2020
Application of next-generation sequencing technology to precision medicine in cancer: joint consensus of the Tumor Biomarker Committee of the Chinese Society of Clinical Oncology. Cancer biology & medicine\| PubMed ID: 31119060	2019
A large-scale, multicentered trial evaluating the sensitivity and specificity of digital PCR versus ARMS-PCR for detecting ctDNA-based p.T790M in non-small-cell lung cancer patients. Translational lung cancer research\| PubMed ID: 34858779	2021
Real-world data on ALK rearrangement test in Chinese advanced non-small cell lung cancer (RATICAL): a nationwide multicenter retrospective study. Cancer communications (London, England)\| PubMed ID: 39016057	2024

Chunyan Wu

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