Seeing the Wood through the Trees: A Review of Techniques for Distinguishing Green Fluorescent Protein from Endogenous Autofluorescence

Billinton, Nicholas; Knight, Andrew W.

doi:10.1006/abio.2000.5006

Cited by 459 publications

(399 citation statements)

References 108 publications

(106 reference statements)

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“…The green fluorescent protein that has made its way into common usage was originally derived from the jellyfish Aequora aequorea and was subsequently modified for more rapid folding, improved stability, and greater brightness [36]. However, the initial green fluorescent protein variants suffered from limited expression in some organisms.…”

Section: Gfpmentioning

confidence: 99%

“…Its primary limitation is its rapid diffusion rate out of unfixed cells, a feature requiring special consideration and methodology. In addition, even though GFP has no endogenous counterpart, concerns have been expressed that the excitation and emission wavelengths of GFP are similar to the autofluorescent emissions of a few endogenous molecules such as the flavins and NAD(P)H [36]. Because the levels of these molecules can vary substantially among cell types, this could result in the false identification of GFP within certain cell types.…”

Section: Evaluation Of Gfp In Tissue Requires Fixation Before Sectioningmentioning

confidence: 99%

“…Billinton and Knight [36] have written an excellent review on this topic. The distinctive hue and saturation of these autofluorescent molecules can resemble those of specific fluorophores, but the intensity of emissions from these molecules is considerably lower than that of typical fluorophores.…”

Section: Primer On Imaging and Autofluorescencementioning

confidence: 99%

“…However, in some cases, such wide BP filters may be necessary to capture enough light to detect a signal. In this case, special techniques [36] must be used to reduce background signal.…”

Section: (C-f)mentioning

confidence: 99%

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Optimizing Techniques for Tracking Transplanted Stem Cells In Vivo

2005

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The potential for bone marrow-derived cells (BMDCs) to contribute to nonhematopoietic tissues has generated considerable debate in recent years. Causes for the controversies include disparities in the techniques used to track engraftment of BMDCs, inappropriate tissue preparation, a lack of appropriate positive and negative controls, and basic misunderstandings about how to properly collect and interpret images from epifluorescent and confocal microscopes. Our laboratory was among the first to use bone marrow transplants from transgenic mice constitutively expressing enhanced green fluorescent protein (GFP) to study the ability of BMDCs to give rise to nonhematopoietic tissue types, a system that is now in widespread use. During our 6 years of experience using GFP, as well as beta-galactosidase and the Y chromosome, to track BMDCs in vivo, we have identified many difficulties and have developed techniques to resolve them. We discuss several of these methods, and, in particular, we describe ratiometric analysis techniques for improving detection of transplanted cells derived from genetically modified bone marrow. Finally, to help resolve reported discrepancies regarding the frequency with which BMDCs contribute to skeletal myofibers, we demonstrate that the pattern of highly autofluorescent myofibers in skeletal muscle is clearly distinct from that of GFP-expressing myofibers and describe how unambiguous conclusions can be drawn from such data. Stem Cells

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Section: Gfpmentioning

confidence: 99%

Section: Evaluation Of Gfp In Tissue Requires Fixation Before Sectioningmentioning

confidence: 99%

Section: Primer On Imaging and Autofluorescencementioning

confidence: 99%

“…However, in some cases, such wide BP filters may be necessary to capture enough light to detect a signal. In this case, special techniques [36] must be used to reduce background signal.…”

Section: (C-f)mentioning

confidence: 99%

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Optimizing Techniques for Tracking Transplanted Stem Cells In Vivo

2005

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“…In comparison, nanodiamonds with NV centres are photostable, have a low toxicity to cells and also display a quantum yield close to unity. The emission wavelength of NV-containing nanodiamonds (625-800 nm) can also penetrate into tissues and is well beyond the range of cellular autofluorescence (300-500 nm) (Billinton and Knight 2001). Although a single quantum dot is threefold brighter than a nanodiamond containing a single NV centre, it is likely that with optimised fabrication, nanodiamonds will have fluorescence intensities comparable to or brighter than quantum dots (Faklaris et al 2009a).…”

Section: Nitrogen-vacancy Centres In Nanodiamondmentioning

confidence: 99%

Luminescent nanodiamonds for biomedical applications

et al. 2011

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In recent years, nanodiamonds have emerged from primarily an industrial and mechanical applications base, to potentially underpinning sophisticated new technologies in biomedical and quantum science. Nanodiamonds are relatively inexpensive, biocompatible, easy to surface functionalise and optically stable. This combination of physical properties are ideally suited to biological applications, including intracellular labelling and tracking, extracellular drug delivery and adsorptive detection of bioactive molecules. Here we describe some of the methods and challenges for processing nanodiamond materials, detection schemes and some of the leading applications currently under investigation.

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Single‐Molecule Spectroscopy of Motor Proteins

Prevo¹,

Açar²,

Kruijssen³

et al. 2000

Encyclopedia of Analytical Chemistry

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Single‐molecule fluorescence spectroscopy has become a crucial tool to study the behavior of biomolecules and their roles in complex biological processes. Single‐molecule methods have in particular contributed to our understanding of the mechanism of force generation and motility of motor proteins. Motor proteins are enzymes that convert the chemical free energy obtained from the hydrolysis of adenosine triphosphate (ATP) into mechanical work in order to drive processes such as muscle contraction and intracellular transport. Here, we review how single‐molecule fluorescence spectroscopy can be applied to motor proteins of the kinesin, myosin, and dynein superfamilies and how such studies have advanced our knowledge of the molecular basis of motor protein action.

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Seeing the Wood through the Trees: A Review of Techniques for Distinguishing Green Fluorescent Protein from Endogenous Autofluorescence

Cited by 459 publications

References 108 publications

Optimizing Techniques for Tracking Transplanted Stem Cells In Vivo

Optimizing Techniques for Tracking Transplanted Stem Cells In Vivo

Luminescent nanodiamonds for biomedical applications

Single‐Molecule Spectroscopy of Motor Proteins

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