Working with GFP in the Brain

Doyle, Kristian P.; Simon, Roger P.; Snyder, Aurelie; Stenzel-Poore, Mary P.

doi:10.2144/03343bm08

Cited by 9 publications

(5 citation statements)

References 8 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The most common endogenous fluorophores, flavins and flavoproteins [1], strongly absorb in the 450–500 nm range, and their emission overlaps that of fluorescein and rhodamine dyes, the dyes most commonly used in cell and tissue imaging. The heterogeneous complexes of lipids and proteins found in brain tissue are also excitable in the 400–550 nm range, and their emission spectra (550–750 nm) are broad and overlap those of many commonly used fluorescence probes used for imaging, including green fluorescence protein (GFP) [2–4]. The relative composition of the various endogenous fluorophores depends on the cell and/or tissue type and the autofluorescence level is contingent on the cell or tissue type and their physiological status [5–7].…”

Section: Introductionmentioning

confidence: 99%

Elimination of autofluorescence background from fluorescence tissue images by use of time-gated detection and the AzaDiOxaTriAngulenium (ADOTA) fluorophore

et al. 2012

View full text Add to dashboard Cite

Sample autofluorescence (fluorescence of inherent components of tissue and fixative-induced fluorescence) is a significant problem in direct imaging of molecular processes in biological samples. A large variety of naturally occurring fluorescent components in tissue results in broad emission that overlaps the emission of typical fluorescent dyes used for tissue labeling. In addition, autofluorescence is characterized by complex fluorescence intensity decay composed of multiple components whose lifetimes range from sub-nanoseconds to a few nanoseconds. For these reasons, the real fluorescence signal of the probe is difficult to separate from the unwanted autofluorescence. Here we present a method for reducing the autofluorescence problem by utilizing an azadioxatriangulenium (ADOTA) dye with a fluorescence lifetime of approximately 15 ns, much longer than those of most of the components of autofluorescence. A probe with such a long lifetime enables us to use time-gated intensity imaging to separate the signal of the targeting dye from the autofluorescence. We have shown experimentally that by discarding photons detected within the first 20 ns of the excitation pulse, the signal-to-background ratio is improved fivefold. This time-gating eliminates over 96 % of autofluorescence. Analysis using a variable time-gate may enable quantitative determination of the bound probe without the contributions from the background.

show abstract

Section: Introductionmentioning

confidence: 99%

Elimination of autofluorescence background from fluorescence tissue images by use of time-gated detection and the AzaDiOxaTriAngulenium (ADOTA) fluorophore

et al. 2012

View full text Add to dashboard Cite

show abstract

“…Observation of GFP emission in the presence of autofluorescence or other impurity emissions is an outstanding problem in bioimaging. [25][26][27] This example clearly highlights the advantage of combining hyperspectral imaging with multivariate data analysis tools for bioimaging applications.…”

Section: Selected Resultsmentioning

confidence: 92%

Design, construction, characterization, and application of a hyperspectral microarray scanner

Sinclair¹,

Timlin²,

Haaland³

et al. 2004

Appl. Opt.

View full text Add to dashboard Cite

We describe the design, construction, and operation of a hyperspectral microarray scanner for functional genomic research. The hyperspectral instrument operates with spatial resolutions ranging from 3 to 30 microm and records the emission spectrum between 490 and 900 nm with a spectral resolution of 3 nm for each pixel of the microarray. This spectral information, when coupled with multivariate data analysis techniques, allows for identification and elimination of unwanted artifacts and greatly improves the accuracy of microarray experiments. Microarray results presented in this study clearly demonstrate the separation of fluorescent label emission from the spectrally overlapping emission due to the underlying glass substrate. We also demonstrate separation of the emission due to green fluorescent protein expressed by yeast cells from the spectrally overlapping autofluorescence of the yeast cells and the growth media.

show abstract

“…Herein, we observed an average uorescence lifetime of 1.4 ns for FG, comprised of a high-intensity uorescent decay component with a lifetime of less than 100 ps, and a low-intensity component having a lifetime of 2.3 ns. The uorescence lifetime of lipofuscin has been measured between 500-650 ps in tissue [49,[51][52][53]. The large difference between uorescence lifetimes of FG and lipofuscin indicates they are readily separable using FLIM, demonstrated herein by the improvement in signal-to-background ratio with FLIM of FG-labeled murine brainstem (Fig.…”

Section: Discussionmentioning

confidence: 99%

Two-photon Excitation Fluorescent Spectral and Decay Properties of Retrograde Neuronal Tracer Fluoro-Gold

Miller

Hernández

Chacko

et al. 2021

Preprint

View full text Add to dashboard Cite

Fluoro-Gold is a fluorescent neuronal tracer suitable for targeted deep imaging of the nervous system. Widefield fluorescence microscopy enables visualization of fluoro-gold, but lacks depth discrimination. Though scanning laser confocal microscopy yields volumetric data, imaging depth is limited, and optimal single-photon excitation of fluoro-gold requires an unconventional ultraviolet excitation line. Two-photon excitation microscopy employs ultrafast pulsed infrared lasers to image fluorophores at high-resolution at unparalleled depths in opaque tissue. Deep imaging of fluoro-gold-labeled neurons carries potential to advance understanding of the central and peripheral nervous systems, yet its two-photon spectral and temporal properties remain uncharacterized. Herein, we report the relative two-photon excitation spectrum of fluoro-gold between 720 nm and 1100 nm, and its fluorescence decay rate in aqueous solution and murine brainstem tissue. We demonstrate unprecedented imaging depth of whole-mounted murine brainstem via two-photon excitation microscopy of fluoro-gold-labeled facial motor nuclei. Optimal two-photon excitation of fluoro-gold occurred at 730 nm, while maximum lifetime contrast was observed at 760 nm with mean fluorescence lifetime of 1.4 ns. Whole-mount brainstem explants were readily imaged to depths in excess of 450 µm via immersion in refractive-index matching solution.

show abstract

Working with GFP in the Brain

Cited by 9 publications

References 8 publications

Elimination of autofluorescence background from fluorescence tissue images by use of time-gated detection and the AzaDiOxaTriAngulenium (ADOTA) fluorophore

Elimination of autofluorescence background from fluorescence tissue images by use of time-gated detection and the AzaDiOxaTriAngulenium (ADOTA) fluorophore

Design, construction, characterization, and application of a hyperspectral microarray scanner

Two-photon Excitation Fluorescent Spectral and Decay Properties of Retrograde Neuronal Tracer Fluoro-Gold

Contact Info

Product

Resources

About