scholarly journals In Vivo Simultaneous Nonlinear Absorption Raman and Fluorescence (SNARF) Imaging of Mouse Brain Cortical Structures

2021 ◽  
Author(s):  
Andrew T Francis ◽  
Bryce Manifold ◽  
Elena C Thomas ◽  
Ruoqian Hu ◽  
Andrew H Hill ◽  
...  
Keyword(s):  
Nonlinear Absorption ◽  
Transient Absorption ◽  
Stimulated Raman Scattering ◽  
Multiphoton Microscopy ◽  
Label Free ◽  
Two Photon ◽  
Age Dependent ◽  
Vessel Structure ◽  
Microscopy Techniques

Two photon excited fluorescence (TPEF) microscopy is a widely used optical imaging technique that has revolutionized neurophotonics through a diverse palette of dyes, specialized transgenic models, easy implementation, and straightforward data analysis. However, in vivo TPEF imaging is often limited in the number of contrasts available to distinguish different cells, structures, or functions. We propose using two label free multiphoton microscopy techniques: stimulated Raman scattering (SRS) microscopy and transient absorption microscopy (TAM) as complementary and orthogonal imaging modalities to TPEF for in vivo brain imaging. In this study, we construct a simultaneous nonlinear absorption, Raman, and fluorescence (SNARF) microscope and image several cortical structures up to 250-300 μm below the pial surface, the highest reported in vivo imaging depth for SRS or TAM. We further demonstrate the capabilities of our SNARF microscope through the quantification of age-dependent myelination, hemodynamics, vessel structure, cell density, and cell identity in vivo. Using machine learning, we report the use of label free SRS and TAM features to predict capillary lining cell identities with 90% accuracy. The SNARF microscope and methodology outlined herein provide a powerful platform to study several research topics, including neurovascular coupling, blood brain barrier, neuronal and axonal degeneration in aging, and neurodegenerative diseases.

scholarly journals Label-free concurrent 5-modal microscopy (Co5M) resolves unknown spatio-temporal processes in wound healing

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Markus Seeger ◽  
Christoph Dehner ◽  
Dominik Jüstel ◽  
Vasilis Ntziachristos
Keyword(s):  
Wound Healing ◽  
Label Free ◽  
Third Harmonic ◽  
Two Photon ◽  
Non Invasive ◽  
Photon Excitation ◽  
Free Mode ◽  
Spatio Temporal ◽  
And Function

AbstractThe non-invasive investigation of multiple biological processes remains a methodological challenge as it requires capturing different contrast mechanisms, usually not available with any single modality. Intravital microscopy has played a key role in dynamically studying biological morphology and function, but it is generally limited to resolving a small number of contrasts, typically generated by the use of transgenic labels, disturbing the biological system. We introduce concurrent 5-modal microscopy (Co5M), illustrating a new concept for label-free in vivo observations by simultaneously capturing optoacoustic, two-photon excitation fluorescence, second and third harmonic generation, and brightfield contrast. We apply Co5M to non-invasively visualize multiple wound healing biomarkers and quantitatively monitor a number of processes and features, including longitudinal changes in wound shape, microvascular and collagen density, vessel size and fractality, and the plasticity of sebaceous glands. Analysis of these parameters offers unique insights into the interplay of wound closure, vasodilation, angiogenesis, skin contracture, and epithelial reformation in space and time, inaccessible by other methods. Co5M challenges the conventional concept of biological observation by yielding multiple simultaneous parameters of pathophysiological processes in a label-free mode.

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

2020 ◽  
Vol 45 (10) ◽  
pp. 2704
Author(s):  
Ting Wu ◽  
Jiuling Liao ◽  
Jia Yu ◽  
Yufeng Gao ◽  
Hui Li ◽  
...  
Keyword(s):  
Fiber Laser ◽  
Label Free ◽  
Two Photon ◽  
Photon Excitation ◽  
Femtosecond Fiber Laser ◽  
Two Photon Excitation

scholarly journals Application of multiphoton microscopy in dermatological studies: A mini-review

2014 ◽  
Vol 07 (05) ◽  
pp. 1330010 ◽  
Author(s):  
Elijah Yew ◽  
Christopher Rowlands ◽  
Peter T. C. So
Keyword(s):  
Near Infrared ◽  
Second Harmonic ◽  
Multiphoton Microscopy ◽  
Infrared Light ◽  
Future Research ◽  
Two Photon ◽  
Recent Developments ◽  
Future Research Directions ◽  
Microscopy Techniques ◽  
Penetration Depths

This review summarizes the historical and more recent developments of multiphoton microscopy, as applied to dermatology. Multiphoton microscopy offers several advantages over competing microscopy techniques: there is an inherent axial sectioning, penetration depths that compete well with confocal microscopy on account of the use of near-infrared light, and many two-photon contrast mechanisms, such as second-harmonic generation, have no analogue in one-photon microscopy. While the penetration depths of photons into tissue are typically limited on the order of hundreds of microns, this is of less concern in dermatology, as the skin is thin and readily accessible. As a result, multiphoton microscopy in dermatology has generated a great deal of interest, much of which is summarized here. The review covers the interaction of light and tissue, as well as the various considerations that must be made when designing an instrument. The state of multiphoton microscopy in imaging skin cancer and various other diseases is also discussed, along with the investigation of aging and regeneration phenomena, and finally, the use of multiphoton microscopy to analyze the transdermal transport of drugs, cosmetics and other agents is summarized. The review concludes with a look at potential future research directions, especially those that are necessary to push these techniques into widespread clinical acceptance.

scholarly journals In vivo multiphoton fluorescence imaging with polymer dots

2017 ◽  
Author(s):  
Ahmed M. Hassan ◽  
Xu Wu ◽  
Jeremy W. Jarrett ◽  
Shihan Xu ◽  
David R. Miller ◽  
...  
Keyword(s):  
Multiphoton Microscopy ◽  
Multiphoton Imaging ◽  
Two Photon ◽  
Photon Excitation ◽  
Deep Imaging ◽  
Polymer Dots ◽  
Versatile Tool ◽  
Cortical Imaging ◽  
Semiconducting Polymer Dots

AbstractDeep in vivo imaging of vasculature requires small, bright, and photostable fluorophores suitable for multiphoton microscopy (MPM). Although semiconducting polymer dots (pdots) are an emerging class of highly fluorescent contrast agents with favorable advantages for the next generation of in vivo imaging, their use for deep multiphoton imaging has never before been demonstrated. Here we characterize the multiphoton properties of three pdot variants (CNPPV, PFBT, and PFPV) and demonstrate deep imaging of cortical microvasculature in C57 mice. Specifically, we measure the two-versus three-photon power dependence of these pdots and observe a clear three-photon excitation signature at wavelengths longer than 1300 nm, and a transition from two-photon to three-photon excitation within a 1060 – 1300 nm excitation range. Furthermore, we show that pdots enable in vivo two-photon imaging of cerebrovascular architecture in mice up to 850 μm beneath the pial surface using 800 nm excitation. In contrast with traditional multiphoton probes, we also demonstrate that the broad multiphoton absorption spectrum of pdots permits imaging at longer wavelengths (λex = 1,060 and 1225 nm). These wavelengths approach an ideal biological imaging wavelength near 1,300 nm and confer compatibility with a high-power ytterbium-fiber laser and a high pulse energy optical parametric amplifier, resulting in substantial improvements in signal-to-background ratio (>3.5-fold) and greater cortical imaging depths of 900 μm and 1300 μm. Ultimately, pdots are a versatile tool for MPM due to their extraordinary brightness and broad absorption, which will undoubtedly unlock the ability to interrogate deep structures in vivo.

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