Deep tissue imaging with multiphoton fluorescence microscopy

Miller, David R.; Jarrett, Jeremy W.; Hassan, Ahmed M.; Dunn, Andrew K.

doi:10.1016/j.cobme.2017.09.004

Cited by 126 publications

(88 citation statements)

References 51 publications

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“…We predict that the development of GPCR sensors absorbing in this wavelength range, that could for instance be engineered based on bacterial phytochromes (e.g., BphP1; Yao et al, 2016), will enable successful application of the probes with optoacoustic imaging, a scalable imaging modality capable of resolving whole-brain activity three-dimensionally at millisecond time scale and 100 µm resolution non-invasively (i.e., without the need for optical fiber or gradient index lense implantation) (Gottschalk et al, 2019). Another advantage of red/NIR FPs is that their photophysical properties are optimal for threephoton absorption (3PA) in the 1700 nm optical window (Horton et al, 2013;Deng et al, 2019), which is recently emerging as a superior technique for deep in vivo imaging (Horton et al, 2013;Miller et al, 2017). Due to the much reduced scattering and absorption, 3PA at these wavelengths is limited only by signalto-noise ratio (which depends on protein brightness) up to more than 3 mm depth (Horton et al, 2013), and structural imaging of neurons has already been demonstrated at a depth of 1.4 mm (Horton et al, 2013), reaching the subcortical region of the mouse brain.…”

Section: The Push Toward Red-shifted Wavelengthsmentioning

confidence: 99%

A Bright and Colorful Future for G-Protein Coupled Receptor Sensors

Ravotto

Duffet

Zhou

et al. 2020

Front. Cell. Neurosci.

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Neurochemicals have a large impact on brain states and animal behavior but are notoriously hard to detect accurately in the living brain. Recently developed genetically encoded sensors obtained from engineering a circularly permuted green fluorescent protein into G-protein coupled receptors (GPCR) provided a vital boost to neuroscience, by innovating the way we monitor neural communication. These new probes are becoming widely successful due to their flexible combination with state of the art optogenetic tools and in vivo imaging techniques, mainly fiber photometry and 2-photon microscopy, to dissect dynamic changes in brain chemicals with unprecedented spatial and temporal resolution. Here, we highlight current approaches and challenges as well as novel insights in the process of GPCR sensor development, and discuss possible future directions of the field.

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Section: The Push Toward Red-shifted Wavelengthsmentioning

confidence: 99%

A Bright and Colorful Future for G-Protein Coupled Receptor Sensors

Ravotto

Duffet

Zhou

et al. 2020

Front. Cell. Neurosci.

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“…The two-photon fluorescence signal is very specific to the excitation and emission wavelengths of fluorophores. More information about the fluorophores in the local environment in tissue sections and biopsies can be obtained from the dynamics of the fluorescence signal [27][28][29][30].…”

Section: Two-photon Fluorescence Microscopymentioning

confidence: 99%

Label-Free Non-linear Multimodal Optical Microscopy—Basics, Development, and Applications

et al. 2019

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Non-linear optical (NLO) microscopy has proven to be a powerful tool especially for tissue imaging with sub-cellular resolution, high penetration depth, endogenous contrast specificity, pinhole-less optical sectioning capability. In this review, we discuss label-free non-linear optical microscopes including the two-photon fluorescence (TPF), fluorescence lifetime imaging microscopy (FLIM), polarization-resolved second harmonic generation (SHG) and coherent anti-Stokes Raman scattering (CARS) techniques with various samples. The non-linear signals are generated from collagen in tissue (SHG), amylopectin from starch granules (SHG), sarcomere structure of fresh muscle (SHG), elastin in skin (TPF), nicotinamide adenine dinucleotide (NADH) in cells (TPF), and lipid droplets in cells (CARS). Again, the non-linear signals are very specific to the molecular structure of the sample and its relative orientation to the polarization of the incident light. Thus, polarization-resolved non-linear optical microscopy provides high image contrast and quantitative estimate of sample orientation. An overview of the advancements on polarization-resolved SHG microscopy including Stokes vector based polarimetry, circular dichroism, and susceptibility are also presented in this review article. The working principles and corresponding implements of above-mentioned microscopy techniques are elucidated. The potential of time-resolved TPF lifetime imaging microscopy (TP-FLIM) is explored by imaging endogenous fluorescence of NAD(P)H, a key coenzyme in cellular metabolic processes. We also discuss single laser source time-resolved multimodal CARS-FLIM microscopy using time-correlated single-photon counting (TCSPC) in combination with continuum generation from photonic crystal fiber (PCF). Using examples, we demonstrate that the multimodal NLO microscopy is a powerful tool to assess the molecular specificity with high resolution.

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“…The fraction of excitation light reaching the focal volume at a depth, z, can be approximated as exp[-(µ a (λ)+µ s (λ))z], where µ a (λ) and µ s (λ) are wavelength-dependent absorption and scattering coefficients. 37 This function reveals that there is an ideal biological imaging wavelength situated at 1,300 nm 23 where photo-attenuation is minimized in brain tissue (Fig. S6).…”

Section: Polymer Dot Excitability At Longer Wavelengths Extends Vascumentioning

confidence: 99%

“…Moreover, the ability to excite polymer dots at longer wavelengths allows us to approach an ideal biological imaging wavelength situated at 1,300 nm where absorption and tissue scattering events are minimized. 8,23 For instance, the photophysical advantages of longer wavelength excitation of PFPV (λ ex = 1,060 nm) coupled with the ytterbium-fiber laser's intrinsic pulse characteristics results in a 3.5-fold improvement in SBR, and an overall 50 µm gain in penetration depth in vivo. Multiphoton imaging of CNPPV-labeled vasculature using an OPA (λ ex = 1225 nm) increases SBR by ~8.2 fold (z = 700 µm), and extends imaging depth 450 µm further into the brain.…”

Section: Introductionmentioning

confidence: 99%

In vivo multiphoton fluorescence imaging with polymer dots

Hassan

Jarrett

et al. 2017

Preprint

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Deep 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 (λ ex = 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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Deep tissue imaging with multiphoton fluorescence microscopy

Cited by 126 publications

References 51 publications

A Bright and Colorful Future for G-Protein Coupled Receptor Sensors

A Bright and Colorful Future for G-Protein Coupled Receptor Sensors

Label-Free Non-linear Multimodal Optical Microscopy—Basics, Development, and Applications

In vivo multiphoton fluorescence imaging with polymer dots

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