Spectral Imaging for Separation of Fluorescent Signals from Autofluorescence

2000 ◽  
Vol 6 (S2) ◽  
pp. 838-839
Author(s):  
Richard M. Levenson
Keyword(s):  
Green Fluorescent Protein ◽  
Connective Tissue ◽  
Fluorescence Microscopy ◽  
Broad Spectrum ◽  
Fluorescent Protein ◽  
Fluorescence Signal ◽  
Post Processing ◽  
Neuronal Tissue ◽  
Green Fluorescent ◽  
Pathology Specimen

Autofluorescence , also known as adventitious fluorescence or background fluorescence, ofter poses a significant problem in many applications of fluorescence microscopy. It contributes to unwanted noise and can swamp the desired signal. Particularly difficult samples to image include many pathology specimen that have been processed using crosslinking fixatives (typically formaldehyde). This procedure dramatically increase the autofluorescence level, leading to bright, broad spectrum emissions, particularly from connective tissue components. Unprocessed plant tissue and neuronal tissue also have extremely high levels of endogenous autofluorescence that can make many convenient labeling strategies, including most (green fluorescent protein (GFP) labels, extremely problematic. Various solutions have been proposed for the reduction or elimination of autofluorescence. These include using narrow bandpass emission filters to try to isolate the desired fluorescence signal, the use of labels which can be excited at wavelengths that are much less likely to induce autofluorescence (moving the excitation towards the NIR is effective), and post-processing aldehyde-fixed samples with such reagents as sodium borohydride or toluidine blue to chemically suppress the autofluorescence signal.However, in many cases, these approaches are either infeasible or ineffective.

Spectral Imaging for Separation of Fluorescent Signals from Autofluorescence

2001 ◽  
Vol 7 (S2) ◽  
pp. 12-13
Author(s):  
Richard M. Levenson
Keyword(s):  
Fourier Transform ◽  
Liquid Crystal ◽  
Fluorescence Microscopy ◽  
Optical Spectrum ◽  
Fluorescent Protein ◽  
Spectral Imaging ◽  
Fluorescence Signal ◽  
Post Processing ◽  
Neuronal Tissue ◽  
Green Fluorescent

Autofluorescence, also known as adventitious fluorescence or background fluorescence, often poses a significant problem in many applications of fluorescence microscopy. It contributes to unwanted noise and can overwhelm the desired signal. Particularly difficult samples to image include many pathology specimens that have been processed using crosslinking fixatives (typically formaldehyde). This procedure dramatically increase the autofluorescence level, leading to bright, broad spectrum emissions, particularly from connective tissue components. Unprocessed plant tissue and neuronal tissue also have extremely high levels of endogenous autofluorescence that can make many convenient labeling strategies, including most green fluorescent protein (GFP) labels, extremely problematic. Various solutions have been proposed for the reduction or elimination of autofluorescence. These include using narrow bandpass emission filters to try to isolate the desired fluorescence signal, the use of labels that can be excited at wavelengths that are much less likely to induce autofluorescence (moving the excitation towards the NIR is effective), and post-processing aldehyde-fixed samples with such reagents as sodium borohydride or toluidine blue to chemically suppress the autofluorescence signal. However, in many cases, these approaches are either infeasible or ineffective. Spectral imaging, that is, the acquisition of a high-resolution optical spectrum at every pixel of an image, offers another approach to elimination of the contribution of autofluorescence. This has recently become possible due to the development of a number of technologies that allow the collection of spectral datasets from fluorescently labeled samples using fluorescence microscopy. Such technologies include liquid crystal tunable filters (LCTFs) and Fourier transform imaging spectrometers, both of which are now commercially available.

scholarly journals Phagocytosed Bordetella pertussis Fails To Survive in Human Neutrophils

2000 ◽  
Vol 68 (2) ◽  
pp. 956-959 ◽  
Author(s):  
Derrick H. Lenz ◽  
Christine L. Weingart ◽  
Alison A. Weiss
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescence Microscopy ◽  
Bordetella Pertussis ◽  
Fluorescent Protein ◽  
Survival Rates ◽  
Polymyxin B ◽  
Immune Serum ◽  
Human Neutrophils ◽  
Green Fluorescent

ABSTRACT Previous studies have reported that phagocytosed Bordetella pertussis survives in human neutrophils. This issue has been reexamined. Opsonized or unopsonized bacteria expressing green fluorescent protein (GFP) were incubated with adherent human neutrophils. Phagocytosis was quantified by fluorescence microscopy, and the viability of phagocytosed bacteria was determined by colony counts following treatment with polymyxin B to kill extracellular bacteria. Only 1 to 2% of the phagocytosed bacteria remained viable. Opsonization with heat-inactivated immune serum reduced the amount of attachment and phagocytosis of the bacteria but did not alter survival rates. In contrast to previous reports, these data suggest that phagocytosed B. pertussis bacteria are killed by human neutrophils.

scholarly journals Improved in Vivo Whole-Animal Detection Limits of Green Fluorescent Protein–Expressing Tumor Lines by Spectral Fluorescence Imaging

2007 ◽  
Vol 6 (4) ◽  
pp. 7290.2007.00023 ◽  
Author(s):  
Jenny M. Tam ◽  
Rabi Upadhyay ◽  
Mikael J. Pittet ◽  
Ralph Weissleder ◽  
Umar Mahmood
Keyword(s):  
Green Fluorescent Protein ◽  
Detection Limit ◽  
Cell Tracking ◽  
Fluorescent Protein ◽  
Spectral Imaging ◽  
Signal Interference ◽  
Fluorescence Signal ◽  
Emission Signal ◽  
Green Fluorescent

Green fluorescent protein (GFP) has been used for cell tracking and imaging gene expression in superficial or surgically exposed structures. However, in vivo murine imaging is often limited by several factors, including scatter and attenuation with depth and overlapping autofluorescence. The autofluorescence signals have spectral profiles that are markedly different from the GFP emission spectral profile. The use of spectral imaging allows separation and quantitation of these contributions to the total fluorescence signal seen in vivo by weighting known pure component profiles. Separation of relative GFP and autofluorescence signals is not readily possible using epifluorescent continuous-wave single excitation and emission bandpass imaging (EFI). To evaluate detection thresholds using these two methods, nude mice were subcutaneously injected with a series of GFP-expressing cells. For EFI, optimized excitation and emission bandpass filters were used. Owing to the ability to separate autofluorescence contributions from the emission signal using spectral imaging compared with the mixed contributions of GFP and autofluorescence in the emission signal recorded by the EFI system, we achieved a 300-fold improvement in the cellular detection limit. The detection limit was 3 × 103 cells for spectral imaging versus 1 × 106 cells for EFI. Despite contributions to image stacks from autofluorescence, a 100-fold dynamic range of cell number in the same image was readily visualized. Finally, spectral imaging was able to separate signal interference of red fluorescent protein from GFP images and vice versa. These findings demonstrate the utility of the approach in detecting low levels of multiple fluorescent markers for whole-animal in vivo applications.

scholarly journals pH of the Cytoplasm and Periplasm of Escherichia coli: Rapid Measurement by Green Fluorescent Protein Fluorimetry

2007 ◽  
Vol 189 (15) ◽  
pp. 5601-5607 ◽  
Author(s):  
Jessica C. Wilks ◽  
Joan L. Slonczewski
Keyword(s):  
Escherichia Coli ◽  
Green Fluorescent Protein ◽  
Time Resolution ◽  
Fluorescent Protein ◽  
Osmotic Shock ◽  
Yellow Fluorescent Protein ◽  
Fluorescence Signal ◽  
Cytoplasmic Ph ◽  
Green Fluorescent ◽  
External Ph

ABSTRACT Cytoplasmic pH and periplasmic pH of Escherichia coli cells in suspension were observed with 4-s time resolution using fluorimetry of TorA-green fluorescent protein mutant 3* (TorA-GFPmut3*) and TetR-yellow fluorescent protein. Fluorescence intensity was correlated with pH using cell suspensions containing 20 mM benzoate, which equalizes the cytoplasmic pH with the external pH. When the external pH was lowered from pH 7.5 to 5.5, the cytoplasmic pH fell within 10 to 20 s to pH 5.6 to 6.5. Rapid recovery occurred until about 30 s after HCl addition and was followed by slower recovery over the next 5 min. As a control, KCl addition had no effect on fluorescence. In the presence of 5 to 10 mM acetate or benzoate, recovery from external acidification was diminished. Addition of benzoate at pH 7.0 resulted in cytoplasmic acidification with only slow recovery. Periplasmic pH was observed using TorA-GFPmut3* exported to the periplasm through the Tat system. The periplasmic location of the fusion protein was confirmed by the observation that osmotic shock greatly decreased the periplasmic fluorescence signal by loss of the protein but had no effect on the fluorescence of the cytoplasmic protein. Based on GFPmut3* fluorescence, the pH of the periplasm equaled the external pH under all conditions tested, including rapid acid shift. Benzoate addition had no effect on periplasmic pH. The cytoplasmic pH of E. coli was measured with 4-s time resolution using a method that can be applied to any strain construct, and the periplasmic pH was measured directly for the first time.

scholarly journals Constitutive and Inducible Expression of Green Fluorescent Protein in Brucella suis

1999 ◽  
Vol 67 (12) ◽  
pp. 6695-6697 ◽  
Author(s):  
Stephan Köhler ◽  
Safia Ouahrani-Bettache ◽  
Marion Layssac ◽  
Jacques Teyssier ◽  
Jean-Pierre Liautard
Keyword(s):  
Flow Cytometry ◽  
Green Fluorescent Protein ◽  
Fluorescence Microscopy ◽  
Gene Fusion ◽  
Fluorescent Protein ◽  
Fusion System ◽  
Chromosomal Dna ◽  
Brucella Suis ◽  
Green Fluorescent ◽  
Inducible Genes

ABSTRACT A gene fusion system based on plasmid pBBR1MCS and the expression of green fluorescent protein was developed for Brucella suis, allowing isolation of constitutive and inducible genes. Bacteria containing promoter fusions of chromosomal DNA togfp were visualized by fluorescence microscopy and examined by flow cytometry. Twelve clones containing gene fragments induced inside J774 murine macrophages were isolated and further characterized.

Use of Green Fluorescent Protein To Detect Expression of an Endopolygalacturonase Gene of Colletotrichum lindemuthianum during Bean Infection

1999 ◽  
Vol 65 (4) ◽  
pp. 1769-1771 ◽  
Author(s):  
Bernard Dumas ◽  
Sylvie Centis ◽  
Nathalie Sarrazin ◽  
Marie-Thérèse Esquerré-Tugayé
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescence Microscopy ◽  
Fluorescent Protein ◽  
Colletotrichum Lindemuthianum ◽  
Fungal Genome ◽  
Appressorium Formation ◽  
Gene Encoding ◽  
Green Fluorescent ◽  
Microscopy Examination

ABSTRACT The 5′ noncoding region of clpg2, an endopolygalacturonase gene of the bean pathogenColletotrichum lindemuthianum, was fused to the coding sequence of a gene encoding a green fluorescent protein (GFP), and the construct was introduced into the fungal genome. Detection of GFP accumulation by fluorescence microscopy examination revealed thatclpg2 was expressed at the early stages of germination of the conidia and during appressorium formation both in vitro and on the host plant.

scholarly journals Dual‐color‐emitting green fluorescent protein from the sea cactus Cavernularia obesa and its use as a pH indicator for fluorescence microscopy

Luminescence ◽  
2013 ◽  
Vol 28 (4) ◽  
pp. 582-591 ◽  
Author(s):  
Katsunori Ogoh ◽  
Takashi Kinebuchi ◽  
Mariko Murai ◽  
Takeo Takahashi ◽  
Yoshihiro Ohmiya ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescence Microscopy ◽  
Fluorescent Protein ◽  
Ph Indicator ◽  
Dual Color ◽  
Green Fluorescent
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