The DIVER Microscope for Imaging in Scattering Media

Dvornikov, Alexander; Malacrida, Leonel; Gratton, Enrico

doi:10.3390/mps2020053

Cited by 27 publications

(20 citation statements)

References 41 publications

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“…One of the latest advancements includes implementation of frequency-domain FLIM in a multiphoton microscope capable of imaging deeper than conventional systems. 125 Finally, frequency-domain FLIM can be implemented without the use of costly pulsed lasers.…”

Section: Instrumentationmentioning

confidence: 99%

Fluorescence lifetime imaging microscopy: fundamentals and advances in instrumentation, analysis, and applications

et al. 2020

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Significance: Fluorescence lifetime imaging microscopy (FLIM) is a powerful technique to distinguish the unique molecular environment of fluorophores. FLIM measures the time a fluorophore remains in an excited state before emitting a photon, and detects molecular variations of fluorophores that are not apparent with spectral techniques alone. FLIM is sensitive to multiple biomedical processes including disease progression and drug efficacy. Aim: We provide an overview of FLIM principles, instrumentation, and analysis while highlighting the latest developments and biological applications. Approach: This review covers FLIM principles and theory, including advantages over intensitybased fluorescence measurements. Fundamentals of FLIM instrumentation in time-and frequencydomains are summarized, along with recent developments. Image segmentation and analysis strategies that quantify spatial and molecular features of cellular heterogeneity are reviewed. Finally, representative applications are provided including high-resolution FLIM of cell-and organelle-level molecular changes, use of exogenous and endogenous fluorophores, and imaging protein-protein interactions with Förster resonance energy transfer (FRET). Advantages and limitations of FLIM are also discussed. Conclusions: FLIM is advantageous for probing molecular environments of fluorophores to inform on fluorophore behavior that cannot be elucidated with intensity measurements alone. Development of FLIM technologies, analysis, and applications will further advance biological research and clinical assessments.

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Section: Instrumentationmentioning

confidence: 99%

Fluorescence lifetime imaging microscopy: fundamentals and advances in instrumentation, analysis, and applications

et al. 2020

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“…Phasor-mapped FLIM and SHG images were acquired with an Olympus FVMPE-RS (Olympus, Waltham, MA) upright microscope equipped with an Insight X3 laser (Spectra-Physics, Santa Clara, CA) and a DIVER (Deep Imaging Via Enhanced Recovery) detector [ 36 , 37 ]. Samples were excited with a 740 nm laser in a two-photon excitation scheme at a laser repetition rate of 80 MHz.…”

Section: Methodsmentioning

confidence: 99%

Reduction of fibrosis and immune suppressive cells in ErbB2-dependent tumorigenesis by an LXR agonist

et al. 2021

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One of the central challenges for cancer therapy is the identification of factors in the tumor microenvironment that increase tumor progression and prevent immune surveillance. One such element associated with breast cancer is stromal fibrosis, a histopathologic criterion for invasive cancer and poor survival. Fibrosis is caused by inflammatory factors and remodeling of the extracellular matrix that elicit an immune tolerant microenvironment. To address the role of fibrosis in tumorigenesis, we developed NeuT/ATTAC transgenic mice expressing a constitutively active NeuT/erbB2 transgene, and an inducible, fat-directed caspase-8 fusion protein, which upon activation results in selective and partial ablation of mammary fat and its replacement with fibrotic tissue. Induction of fibrosis in NeuT/ATTAC mice led to more rapid tumor development and an inflammatory and fibrotic stromal environment. In an effort to explore therapeutic options that could reduce fibrosis and immune tolerance, mice were treated with the oxysterol liver X receptor (LXR) pan agonist, N,N-dimethyl-3-β-hydroxy-cholenamide (DMHCA), an agent known to reduce fibrosis in non-malignant diseases. DMHCA reduced tumor progression, tumor multiplicity and fibrosis, and improved immune surveillance by reducing infiltrating myeloid-derived suppressor cells and increasing CD4 and CD8 effector T cells. These effects were associated with downregulation of an LXR-dependent gene network related to reduced breast cancer survival that included Spp1, S100a9, Anxa1, Mfge8 and Cd14. These findings suggest that the use of DMHCA may be a potentially effective approach to reduce desmoplasia and immune tolerance and increase the efficacy of cancer therapy.

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“…64,65 By expanding this setup to multiphoton excitation on a microscope, phototoxicity of the sample is decreased and deeper penetration is possible for large tissue samples of up to 3 mm. 66 This technique can also be coupled with confocal imaging methods to measure cell morphological parameters or fluorescent markers of cell death. Walsh and Skala et al have shown that by measuring the optical redox index (OMI), a parameter that depends on the lifetime of NADH and FAD, they were able to see heterogeneous drug response within their human patient-and mousederived tumor organoids.…”

Section: What Assays Are Necessary To Interrogate This Complexity?mentioning

confidence: 99%

Section: Main Text/sectionsmentioning

confidence: 99%

A Perspective on Expanding Our Understanding of Cancer Treatments by Integrating Approaches from the Biological and Physical Sciences

2020

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Multicellular systems such as cancer suffer from immense complexity. It is imperative to capture the heterogeneity of these systems across scales to achieve a deeper understanding of the underlying biology and develop effective treatment strategies. In this perspective article, we will discuss how recent technologies and approaches from the biological and physical sciences have transformed traditional ways of measuring, interpreting, and treating cancer. During the SLAS 2019 Annual Meeting, SBI2 hosted a Special Interest Group (SIG) on this topic. Academic and industry leaders engaged in discussions surrounding what biological model systems are appropriate to study cancer complexity, what assays are necessary to interrogate this complexity, and how physical sciences approaches may be useful to detangle this complexity. In particular, we examined the utility of mathematical models in predicting cancer progression and treatment response when tightly integrated with reproducible, quantitative, and dynamic biological measurements achieved using high-content imaging and analysis. The dialogue centered around the impetus for convergent biosciences, bringing new perspectives to cancer research to further understand this complex adaptive system and successfully intervene therapeutically.

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The DIVER Microscope for Imaging in Scattering Media

Cited by 27 publications

References 41 publications

Fluorescence lifetime imaging microscopy: fundamentals and advances in instrumentation, analysis, and applications

Fluorescence lifetime imaging microscopy: fundamentals and advances in instrumentation, analysis, and applications

Reduction of fibrosis and immune suppressive cells in ErbB2-dependent tumorigenesis by an LXR agonist

A Perspective on Expanding Our Understanding of Cancer Treatments by Integrating Approaches from the Biological and Physical Sciences

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