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Article ; Online: More than double the fun with two-photon excitation microscopy.

Luu, Peter / Fraser, Scott E / Schneider, Falk

Communications biology

2024 Volume 7, Issue 1, Page(s) 364

Abstract: For generations researchers have been observing the dynamic processes of life through the lens of a microscope. This has offered tremendous insights into biological phenomena that span multiple orders of time- and length-scales ranging from the pure ... ...

Abstract	For generations researchers have been observing the dynamic processes of life through the lens of a microscope. This has offered tremendous insights into biological phenomena that span multiple orders of time- and length-scales ranging from the pure magic of molecular reorganization at the membrane of immune cells, to cell migration and differentiation during development or wound healing. Standard fluorescence microscopy techniques offer glimpses at such processes in vitro, however, when applied in intact systems, they are challenged by reduced signal strengths and signal-to-noise ratios that result from deeper imaging. As a remedy, two-photon excitation (TPE) microscopy takes a special place, because it allows us to investigate processes in vivo, in their natural environment, even in a living animal. Here, we review the fundamental principles underlying TPE aimed at basic and advanced microscopy users interested in adopting TPE for intravital imaging. We focus on applications in neurobiology, present current trends towards faster, wider and deeper imaging, discuss the combination with photon counting technologies for metabolic imaging and spectroscopy, as well as highlight outstanding issues and drawbacks in development and application of these methodologies.
MeSH term(s)	Animals ; Microscopy, Fluorescence, Multiphoton/methods ; Microscopy, Fluorescence/methods ; Intravital Microscopy ; Spectrum Analysis ; Photons
Language	English
Publishing date	2024-03-26
Publishing country	England
Document type	Journal Article ; Review
ISSN	2399-3642
ISSN (online)	2399-3642
DOI	10.1038/s42003-024-06057-0
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article ; Online: VoDEx: a Python library for time annotation and management of volumetric functional imaging data.

Nadtochiy, Anna / Luu, Peter / Fraser, Scott E / Truong, Thai V

ArXiv

2023

Abstract: In functional imaging studies, accurately synchronizing the time course of experimental manipulations and stimulus presentations with resulting imaging data is crucial for analysis. Current software tools lack such functionality, requiring manual ... ...

Abstract	In functional imaging studies, accurately synchronizing the time course of experimental manipulations and stimulus presentations with resulting imaging data is crucial for analysis. Current software tools lack such functionality, requiring manual processing of the experimental and imaging data, which is error-prone and potentially non-reproducible. We present VoDEx, an open-source Python library that streamlines the data management and analysis of functional imaging data. VoDEx synchronizes the experimental timeline and events (eg. presented stimuli, recorded behavior) with imaging data. VoDEx provides tools for logging and storing the timeline annotation, and enables retrieval of imaging data based on specific time-based and manipulation-based experimental conditions. Availability and Implementation: VoDEx is an open-source Python library and can be installed via the "pip install" command. It is released under a BSD license, and its source code is publicly accessible on GitHub https://github.com/LemonJust/vodex. A graphical interface is available as a napari-vodex plugin, which can be installed through the napari plugins menu or using "pip install." The source code for the napari plugin is available on GitHub https://github.com/LemonJust/napari-vodex.
Language	English
Publishing date	2023-05-11
Publishing country	United States
Document type	Preprint
ISSN	2331-8422
ISSN (online)	2331-8422
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Article ; Online: VoDEx: a Python library for time annotation and management of volumetric functional imaging data.

Nadtochiy, Anna / Luu, Peter / Fraser, Scott E / Truong, Thai V

Bioinformatics (Oxford, England)

2023 Volume 39, Issue 9

Abstract: Summary: In functional imaging studies, accurately synchronizing the time course of experimental manipulations and stimulus presentations with resulting imaging data is crucial for analysis. Current software tools lack such functionality, requiring ... ...

Abstract	Summary: In functional imaging studies, accurately synchronizing the time course of experimental manipulations and stimulus presentations with resulting imaging data is crucial for analysis. Current software tools lack such functionality, requiring manual processing of the experimental and imaging data, which is error-prone and potentially non-reproducible. We present VoDEx, an open-source Python library that streamlines the data management and analysis of functional imaging data. VoDEx synchronizes the experimental timeline and events (e.g. presented stimuli, recorded behavior) with imaging data. VoDEx provides tools for logging and storing the timeline annotation, and enables retrieval of imaging data based on specific time-based and manipulation-based experimental conditions. Availability and implementation: VoDEx is an open-source Python library and can be installed via the "pip install" command. It is released under a BSD license, and its source code is publicly accessible on GitHub (https://github.com/LemonJust/vodex). A graphical interface is available as a napari-vodex plugin, which can be installed through the napari plugins menu or using "pip install." The source code for the napari plugin is available on GitHub (https://github.com/LemonJust/napari-vodex). The software version at the time of submission is archived at Zenodo (version v1.0.18, https://zenodo.org/record/8061531).
MeSH term(s)	Software ; Image Processing, Computer-Assisted/methods ; Animals ; Programming Languages
Language	English
Publishing date	2023-09-12
Publishing country	England
Document type	Journal Article
ZDB-ID	1422668-6
ISSN	1367-4811 ; 1367-4803
ISSN (online)	1367-4811
ISSN	1367-4803
DOI	10.1093/bioinformatics/btad568
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Article ; Online: A single-shot hyperspectral phasor camera for fast, multi-color fluorescence microscopy.

Wang, Pu / Kitano, Masahiro / Keomanee-Dizon, Kevin / Truong, Thai V / Fraser, Scott E / Cutrale, Francesco

Cell reports methods

2023 Volume 3, Issue 4, Page(s) 100441

Abstract: Hyperspectral fluorescence imaging improves multiplexed observations of biological samples by utilizing multiple color channels across the spectral range to compensate for spectral overlap between labels. Typically, spectral resolution comes at a cost of ...

Abstract	Hyperspectral fluorescence imaging improves multiplexed observations of biological samples by utilizing multiple color channels across the spectral range to compensate for spectral overlap between labels. Typically, spectral resolution comes at a cost of decreased detection efficiency, which both hampers imaging speed and increases photo-toxicity to the samples. Here, we present a high-speed, high-efficiency snapshot spectral acquisition method, based on optical compression of the fluorescence spectra via Fourier transform, that overcomes the challenges of discrete spectral sampling: single-shot hyperspectral phasor camera (SHy-Cam). SHy-Cam captures fluorescence spatial and spectral information in a single exposure with a standard scientific CMOS camera, with photon efficiency of over 80%, easily and with acquisition rates exceeding 30 datasets per second, making it a powerful tool for multi-color
MeSH term(s)	Data Compression ; Hyperspectral Imaging ; Microscopy, Fluorescence ; Optical Devices
Language	English
Publishing date	2023-03-31
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't ; Research Support, U.S. Gov't, Non-P.H.S.
ISSN	2667-2375
ISSN (online)	2667-2375
DOI	10.1016/j.crmeth.2023.100441
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Article: Application of fluorescence lifetime imaging microscopy to monitor glucose metabolism in pancreatic islets

Wang, Zhongying / Archang, Maani / Gurlo, Tatyana / Wong, Elaine / Fraser, Scott E / Butler, Peter C

Biomedical optics express

2023 Volume 14, Issue 8, Page(s) 4170–4178

Abstract: Glucose stimulated insulin secretion is mediated by glucose metabolism via oxidative phosphorylation generating ATP that triggers membrane depolarization and exocytosis of insulin. In stressed beta cells, glucose metabolism is remodeled, with enhanced ... ...

Abstract	Glucose stimulated insulin secretion is mediated by glucose metabolism via oxidative phosphorylation generating ATP that triggers membrane depolarization and exocytosis of insulin. In stressed beta cells, glucose metabolism is remodeled, with enhanced glycolysis uncoupled from oxidative phosphorylation, resulting in the impaired glucose-mediated insulin secretion characteristic of diabetes. Relative changes in glycolysis and oxidative phosphorylation can be monitored in living cells using the 3-component fitting approach of fluorescence lifetime imaging microscopy (FLIM). We engrafted pancreatic islets onto the iris to permit in vivo FLIM monitoring of the trajectory of glucose metabolism. The results show increased oxidative phosphorylation of islet cells (∼90% beta cells) in response to hyperglycemia; in contrast red blood cells traversing the islets maintained exclusive glycolysis as expected in the absence of mitochondria.
Language	English
Publishing date	2023-07-19
Publishing country	United States
Document type	Journal Article
ZDB-ID	2572216-5
ISSN	2156-7085
ISSN	2156-7085
DOI	10.1364/BOE.493722
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Article ; Online: Insights into Metabolic Activity and Structure of the Retina through Multiphoton Fluorescence Lifetime Imaging Microscopy in Mice.

Kesavamoorthy, Niranjana / Junge, Jason A / Fraser, Scott E / Ameri, Hossein

Cells

2022 Volume 11, Issue 15

Abstract: Fluorescence lifetime imaging microscopy (FLIM) evaluates the metabolic state of tissue based on reduced nicotinamide adenine dinucleotide (NAD(P)H) and flavin adenine dinucleotide (FAD). Fluorescence lifetime imaging ophthalmoscopy (FLIO) can image the ... ...

Abstract	Fluorescence lifetime imaging microscopy (FLIM) evaluates the metabolic state of tissue based on reduced nicotinamide adenine dinucleotide (NAD(P)H) and flavin adenine dinucleotide (FAD). Fluorescence lifetime imaging ophthalmoscopy (FLIO) can image the fundus of the eyes, but cannot detect NAD(P)H. We used multiphoton FLIM to study the metabolic state of the retina in fixed eyes of wild-type mice C57BL6/J. We sectioned the eye using a polyacrylamide gel-embedding technique and estimated the percentage of bound NAD(P)H. We found that oxidative phosphorylation was the predominant metabolic state, particularly in the inner retina, when a fixed retina was used. We also demonstrated the feasibility of FAD imaging of the retina. In addition, we demonstrated that autofluorescence and various FLIM channels, such as hemoglobin, melanin and collagen, can be used to evaluate the structure of the retina and other parts of the eye without any special staining.
MeSH term(s)	Animals ; Flavin-Adenine Dinucleotide/metabolism ; Mice ; Microscopy, Fluorescence ; NAD/metabolism ; Oxidative Phosphorylation ; Retina/diagnostic imaging ; Retina/metabolism
Chemical Substances	NAD (0U46U6E8UK) ; Flavin-Adenine Dinucleotide (146-14-5)
Language	English
Publishing date	2022-07-22
Publishing country	Switzerland
Document type	Journal Article ; Research Support, N.I.H., Extramural
ZDB-ID	2661518-6
ISSN	2073-4409 ; 2073-4409
ISSN (online)	2073-4409
ISSN	2073-4409
DOI	10.3390/cells11152265
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Article ; Online: SHR and SCR coordinate root patterning and growth early in the cell cycle.

Winter, Cara M / Szekely, Pablo / Popov, Vladimir / Belcher, Heather / Carter, Raina / Jones, Matthew / Fraser, Scott E / Truong, Thai V / Benfey, Philip N

Nature

2024 Volume 626, Issue 7999, Page(s) 611–616

Abstract: Precise control of cell division is essential for proper patterning and growth during the development of multicellular organisms. Coordination of formative divisions that generate new tissue patterns with proliferative divisions that promote growth is ... ...

Abstract	Precise control of cell division is essential for proper patterning and growth during the development of multicellular organisms. Coordination of formative divisions that generate new tissue patterns with proliferative divisions that promote growth is poorly understood. SHORTROOT (SHR) and SCARECROW (SCR) are transcription factors that are required for formative divisions in the stem cell niche of Arabidopsis roots
MeSH term(s)	Arabidopsis/cytology ; Arabidopsis/genetics ; Arabidopsis/growth & development ; Arabidopsis/metabolism ; Arabidopsis Proteins/genetics ; Arabidopsis Proteins/metabolism ; Cell Cycle/genetics ; Cell Division/genetics ; Gene Expression Regulation, Plant ; Plant Roots/cytology ; Plant Roots/growth & development ; Plant Roots/metabolism ; Microscopy, Confocal ; Mutation
Chemical Substances	Arabidopsis Proteins ; SCR protein, Arabidopsis ; SHORT ROOT protein, Arabidopsis
Language	English
Publishing date	2024-01-31
Publishing country	England
Document type	Journal Article
ZDB-ID	120714-3
ISSN	1476-4687 ; 0028-0836
ISSN (online)	1476-4687
ISSN	0028-0836
DOI	10.1038/s41586-023-06971-z
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Article ; Online: Development of Highly Fluorogenic Styrene Probes for Visualizing RNA in Live Cells.

Kim, Moon Jung / Li, Yida / Junge, Jason A / Kim, Nathan K / Fraser, Scott E / Zhang, Chao

ACS chemical biology

2023 Volume 18, Issue 7, Page(s) 1523–1533

Abstract: Styrene dyes are useful imaging probes and fluorescent sensors due to their strong fluorogenic responses to environmental changes or binding macromolecules. Previously, indole-containing styrene dyes have been reported to selectively bind RNA in the ... ...

Abstract	Styrene dyes are useful imaging probes and fluorescent sensors due to their strong fluorogenic responses to environmental changes or binding macromolecules. Previously, indole-containing styrene dyes have been reported to selectively bind RNA in the nucleolus and cytoplasm. However, the application of these indole-based dyes in cell imaging is limited by their moderate fluorescence enhancement and quantum yields, as well as relatively high background associated with these green-emitting dyes. In this work, we have investigated the positional and electronic effects of the electron donor by generating regioisomeric and isosteric analogues of the indole ring. Select probes exhibited large Stokes shifts, enhanced molar extinction coefficients, and bathochromic shifts in their absorption and fluorescence wavelengths. In particular, the indolizine analogues displayed high membrane permeability, strong fluorogenic responses upon binding RNA, compatibility with fluorescence lifetime imaging microscopy (FLIM), low cytotoxicity, and excellent photostability. These indolizine dyes not only give rise to rapid, sensitive, and intense staining of nucleoli in live cells but can also resolve subnucleolar structures enabling highly detailed studies of nucleolar morphology. Furthermore, our dyes can partition into RNA coacervates and resolve the formation of multiphase complex coacervate droplets. These indolizine-containing styrene probes offer the highest fluorescence enhancement among the RNA-selective dyes reported in the literature; thus, these new dyes are excellent alternatives to the commercially available RNA dye, SYTO RNASelect, for visualizing RNA in live cells and
MeSH term(s)	Humans ; Fluorescent Dyes/chemistry ; HeLa Cells ; Microscopy, Fluorescence ; RNA/chemistry ; Styrenes
Chemical Substances	Fluorescent Dyes ; RNA (63231-63-0) ; Styrenes ; SYTO RNASelect
Language	English
Publishing date	2023-05-18
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ISSN	1554-8937
ISSN (online)	1554-8937
DOI	10.1021/acschembio.3c00141
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Book ; Online: VoDEx

Nadtochiy, Anna / Luu, Peter / Fraser, Scott E. / Truong, Thai V.

a Python library for time annotation and management of volumetric functional imaging data

2023

Abstract	Summary: In functional imaging studies, accurately synchronizing the time course of experimental manipulations and stimulus presentations with resulting imaging data is crucial for analysis. Current software tools lack such functionality, requiring manual processing of the experimental and imaging data, which is error-prone and potentially non-reproducible. We present VoDEx, an open-source Python library that streamlines the data management and analysis of functional imaging data. VoDEx synchronizes the experimental timeline and events (eg. presented stimuli, recorded behavior) with imaging data. VoDEx provides tools for logging and storing the timeline annotation, and enables retrieval of imaging data based on specific time-based and manipulation-based experimental conditions. Availability and Implementation: VoDEx is an open-source Python library and can be installed via the "pip install" command. It is released under a BSD license, and its source code is publicly accessible on GitHub https://github.com/LemonJust/vodex. A graphical interface is available as a napari-vodex plugin, which can be installed through the napari plugins menu or using "pip install." The source code for the napari plugin is available on GitHub https://github.com/LemonJust/napari-vodex.
Keywords	Quantitative Biology - Quantitative Methods
Subject code	005
Publishing date	2023-05-11
Publishing country	us
Document type	Book ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: A versatile, multi-laser twin-microscope system for light-sheet imaging.

Keomanee-Dizon, Kevin / Fraser, Scott E / Truong, Thai V

The Review of scientific instruments

2020 Volume 91, Issue 5, Page(s) 53703

Abstract: Light-sheet microscopy offers faster imaging and reduced phototoxicity in comparison to conventional point-scanning microscopy, making it a preferred technique for imaging biological dynamics for durations of hours or days. Such extended imaging sessions ...

Abstract	Light-sheet microscopy offers faster imaging and reduced phototoxicity in comparison to conventional point-scanning microscopy, making it a preferred technique for imaging biological dynamics for durations of hours or days. Such extended imaging sessions pose a challenge, as it reduces the number of specimens that can be imaged in a given day. Here, we present a versatile light-sheet imaging instrument that combines two independently controlled microscope-twins, built so that they can share an ultrafast near-infrared laser and a bank of continuous-wave visible lasers, increasing the throughput and decreasing the cost. To permit a wide variety of specimens to be imaged, each microscope-twin provides flexible imaging parameters, including (i) operation in one-photon and/or two-photon excitation modes, (ii) delivery of one to three light-sheets via a trio of orthogonal excitation arms, (iii) sub-micron to micron imaging resolution, (iv) multicolor compatibility, and (v) upright (with provision for inverted) detection geometry. We offer a detailed description of the twin-microscope design to aid instrument builders who wish to construct and use similar systems. We demonstrate the instrument's versatility for biological investigation by performing fast imaging of the beating heart in an intact zebrafish embryo, deep imaging of thick patient-derived tumor organoids, and gentle whole-brain imaging of neural activity in behaving larval zebrafish.
MeSH term(s)	Equipment Design ; Lasers ; Light ; Microscopy/instrumentation
Language	English
Publishing date	2020-05-29
Publishing country	United States
Document type	Journal Article
ZDB-ID	209865-9
ISSN	1089-7623 ; 0034-6748
ISSN (online)	1089-7623
ISSN	0034-6748
DOI	10.1063/1.5144487
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