scholarly journals Accuracy and precision in quantitative fluorescence microscopy

2009 ◽  
Vol 185 (7) ◽  
pp. 1135-1148 ◽  
Author(s):  
Jennifer C. Waters
Keyword(s):  
Fluorescence Microscopy ◽  
Cell Biology ◽  
Imaging System ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Digital Cameras ◽  
Accuracy And Precision ◽  
Fluorescent Molecules ◽  
Quantitative Fluorescence ◽  
Biology Research

The light microscope has long been used to document the localization of fluorescent molecules in cell biology research. With advances in digital cameras and the discovery and development of genetically encoded fluorophores, there has been a huge increase in the use of fluorescence microscopy to quantify spatial and temporal measurements of fluorescent molecules in biological specimens. Whether simply comparing the relative intensities of two fluorescent specimens, or using advanced techniques like Förster resonance energy transfer (FRET) or fluorescence recovery after photobleaching (FRAP), quantitation of fluorescence requires a thorough understanding of the limitations of and proper use of the different components of the imaging system. Here, I focus on the parameters of digital image acquisition that affect the accuracy and precision of quantitative fluorescence microscopy measurements.

scholarly journals Directed evolution of a monomeric, bright and photostable version of Clavularia cyan fluorescent protein: structural characterization and applications in fluorescence imaging

2006 ◽  
Vol 400 (3) ◽  
pp. 531-540 ◽  
Author(s):  
Hui-wang Ai ◽  
J. Nathan Henderson ◽  
S. James Remington ◽  
Robert E. Campbell
Keyword(s):  
Directed Evolution ◽  
Cell Biology ◽  
Fluorescent Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Selection For ◽  
Monomeric Structure ◽  
Fluorescence Lifetime Decay ◽  
Lifetime Decay ◽  
Biology Research

The arsenal of engineered variants of the GFP [green FP (fluorescent protein)] from Aequorea jellyfish provides researchers with a powerful set of tools for use in biochemical and cell biology research. The recent discovery of diverse FPs in Anthozoa coral species has provided protein engineers with an abundance of alternative progenitor FPs from which improved variants that complement or supersede existing Aequorea GFP variants could be derived. Here, we report the engineering of the first monomeric version of the tetrameric CFP (cyan FP) cFP484 from Clavularia coral. Starting from a designed synthetic gene library with mammalian codon preferences, we identified dimeric cFP484 variants with fluorescent brightness significantly greater than the wild-type protein. Following incorporation of dimer-breaking mutations and extensive directed evolution with selection for blue-shifted emission, high fluorescent brightness and photostability, we arrived at an optimized variant that we have named mTFP1 [monomeric TFP1 (teal FP 1)]. The new mTFP1 is one of the brightest and most photostable FPs reported to date. In addition, the fluorescence is insensitive to physiologically relevant pH changes and the fluorescence lifetime decay is best fitted as a single exponential. The 1.19 Å crystal structure (1 Å=0.1 nm) of mTFP1 confirms the monomeric structure and reveals an unusually distorted chromophore conformation. As we experimentally demonstrate, the high quantum yield of mTFP1 (0.85) makes it particularly suitable as a replacement for ECFP (enhanced CFP) or Cerulean as a FRET (fluorescence resonance energy transfer) donor to either a yellow or orange FP acceptor.

A fluorescence enhancement assay of cell fusion

1977 ◽  
Vol 28 (1) ◽  
pp. 167-177
Author(s):  
P.M. Keller ◽  
S. Person ◽  
W. Snipes
Keyword(s):  
Absorption Spectrum ◽  
Energy Transfer ◽  
Emission Spectrum ◽  
Cell Fusion ◽  
Saturated Hydrocarbon ◽  
Photon Emission ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Hydrocarbon Chains ◽  
Fluorescent Molecules

Two probes were synthesized which consist of fluorescent molecules conjugated to saturated hydrocarbon chains, 18 carbons long, to ensure their localization into cellular membranes. There is an overlap between the emission spectrum of one probe (donor) and the absorption spectrum of the other probe (acceptor). By the use of appropriate wavelengths it is possible to specifically excite the donor probe and record the fluorescence of the acceptor probe. Two cell populations, each labelled with one of the probes, were infected with a virus that causes cell fusion, mixed in equal proportions, and the fluorescence of the acceptor probe measured as a function of time after infection. An increase in fluorescence was observed beginning at the time of onset of cell fusion indicating a mixing of the fluorescent membrane molecules. An investigation of the distance dependence indicated that the increase in fluorescence was mainly due to resonance energy transfer and not to photon emission and reabsorption. Resonance energy transfer requires that the 2 probes be close together and that there be an overlap of the emission spectrum of the donor probe and the absorption spectrum of the acceptor probe. The possible application of this assay to other types of membrane fusion is noted.

Super-resolution FRET measurements

Nanoscale ◽  
2021 ◽  
Author(s):  
Fernando D Stefani ◽  
Alan M. M. Szalai ◽  
Cecilia Zaza
Keyword(s):  
Energy Transfer ◽  
Fluorescence Microscopy ◽  
Biological Systems ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Super Resolution ◽  
Forster Resonance Energy Transfer ◽  
Förster Resonance Energy Transfer ◽  
Dynamic Architecture ◽  
Förster Resonance

Super-resolution fluorescence microscopy and Förster Resonance Energy Transfer (FRET) form a well-established family of techniques that has provided unique tools to study the dynamic architecture and functionality of biological systems,...

scholarly journals MipTec Proceedings: Transcriptional Assay Screens Using Renilla Luciferase as an Internal Control

1998 ◽  
Vol 3 (4) ◽  
pp. 41-44
Author(s):  
Rita R. Hannah ◽  
Martha L. Jennens-Clough ◽  
Keith V. Wood
Keyword(s):  
Internal Control ◽  
Cell Biology ◽  
Mammalian Cells ◽  
Imaging System ◽  
Test Sample ◽  
Firefly Luciferase ◽  
Renilla Luciferase ◽  
Cell Lysate ◽  
Biology Research ◽  
Assay Conditions

In cell biology research and pharmaceutical discovery, physiological responses of mammalian cells are commonly screened using transcriptional assays. Although firefly luciferase is widely used because of its rapid and simple assay, greater precision can be achieved using a second reporter as an internal control. Renilla luciferase serves as an efficient internal control because it can be measured as easily and rapidly using the same instrument. The Dual-Luciferase™ Reporter (DLR™) Assay developed at Promega measures both reporters sequentially within each sample, which eliminates the need to separate the test sample into aliquots for each assay. The expression of each reporter is independently quantitated using selective assay conditions based on their distinctive chemical characteristics. The firefly luciferase is initiated first by the addition of LAR II to the sample or cell lysate. Following measurement of the luminescent output, the firefly reaction is rapidly quenched and the Renilla reaction is simultaneously activated by addition of Stop & Glo™ Reagent, and the luminescence is measured a second time. The DLR™ Assay allows quantitation of both reporters within 4 seconds per well using a 96-well luminometer equipped with two reagent injectors. Multi-well plates may be processed even more rapidly using a CCD-based imaging system.

scholarly journals Genetically encoded sensors of protein hydrodynamics and molecular proximity

2015 ◽  
Vol 112 (20) ◽  
pp. E2569-E2574 ◽  
Author(s):  
Alexander C. Hoepker ◽  
Ariel Wang ◽  
Alix Le Marois ◽  
Klaus Suhling ◽  
Yuling Yan ◽  
...  
Keyword(s):  
Fluorescence Lifetime ◽  
Bound States ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Small Mass ◽  
Photobacterium Leiognathi ◽  
Fret Efficiency ◽  
Quantitative Fluorescence ◽  
Control Protein ◽  
Cell Division Control

The specialized light organ of the ponyfish supports the growth of the bioluminescent symbiont Photobacterium leiognathi. The bioluminescence of P. leiognathi is generated within a heteromeric protein complex composed of the bacterial luciferase and a 20-kDa lumazine binding protein (LUMP), which serves as a Förster resonance energy transfer (FRET) acceptor protein, emitting a cyan-colored fluorescence with an unusually long excited state lifetime of 13.6 ns. The long fluorescence lifetime and small mass of LUMP are exploited for the design of highly optimized encoded sensors for quantitative fluorescence anisotropy (FA) measurements of protein hydrodynamics. In particular, large differences in the FA values of the free and target-bound states of LUMP fusions appended with capture sequences of up to 20 kDa are used in quantitative FA imaging and analysis of target proteins. For example, a fusion protein composed of LUMP and a 5-kDa G protein binding domain is used as an FA sensor to quantify the binding of the GTP-bound cell division control protein 42 homolog (Cdc42) (21 kDa) in solution and within Escherichia coli. Additionally, the long fluorescence lifetime and the surface-bound fluorescent cofactor 6,7-dimethyl-8- (1′-dimethyl-ribityl) lumazine in LUMP are utilized in the design of highly optimized FRET probes that use Venus as an acceptor probe. The efficiency of FRET in a zero-length LUMP-Venus fusion is 62% compared to ∼31% in a related CFP-Venus fusion. The improved FRET efficiency obtained by using LUMP as a donor probe is used in the design of a FRET-optimized genetically encoded LUMP-Venus substrate for thrombin.

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