Fluorescence Lifetime Imaging Microscopy

Herman, Brian; Wang, X. F.; Wodnicki, Pawel; Perisamy, A.; Mahajan, Nupam P.; Berry, Gail; Gordon, Gerald W.

doi:10.1007/978-3-642-59903-3_20

Cited by 12 publications

(11 citation statements)

References 30 publications

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“…The spatial confinement of excitation and the ability to excite molecules with half of the energy required for linear absorption can be employed in three-dimensional microfabrication [2,3], three-dimensional optical storage [4,5], two-photon fluorescence imaging [6][7][8], two-photon photodynamic cancer therapy [9,10] and optical power limiting [11,12]. Motivated by these applications, efforts to understand the relationship between molecular structure and 2PA, aiming to design compounds with larger 2PA cross-section, have been carried out, leading to design strategies for novel organic materials [13][14][15][16][17].…”

Section: Introductionmentioning

confidence: 99%

Two-photon absorption spectrum in diazoaromatic compounds

Boni¹,

Andrade²,

Yamaki

et al. 2008

Chemical Physics Letters

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Section: Introductionmentioning

confidence: 99%

Two-photon absorption spectrum in diazoaromatic compounds

Boni¹,

Andrade²,

Yamaki

et al. 2008

Chemical Physics Letters

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“…Fluorescence lifetime has been shown to be sensitive to local environmental factors such as pH, calcium ion concentration or viscosity (Dowling et al ., 1998; Valeur, 2002) and is directly dependent upon excited‐state reactions but is independent of fluorophore concentration and light‐path length (Herman et al ., 1999). Thus, FLIM can provide a robust means to obtain information about molecular interactions or the biophysical environment beyond mere localization of a labelled protein.…”

Section: Introductionmentioning

confidence: 99%

Imaging fluorescence lifetime heterogeneity applied to GFP‐tagged MHC protein at an immunological synapse*

Treanor¹,

Lanigan

Suhling

et al. 2005

Journal of Microscopy

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Key words. Fluorescence lifetime imaging, green fluorescent protein, immunological synapse, natural killer cells, refractive index, TCSPC. SummaryFluorescence imaging of green fluorescent protein (GFP) may be used to locate proteins in live cells and fluorescence lifetime imaging (FLIM) may be employed to probe the local microenvironment of proteins. Here we apply FLIM to GFP-tagged proteins at the cell surface and at an inhibitory natural killer (NK) cell immunological synapse (IS). We present a novel quantitative analysis of fluorescence lifetime images that we believe is useful to determine whether apparent FLIM heterogeneity is statistically significant. We observe that, although the variation of observed fluorescence lifetime of GFP-tagged proteins at the cell surface is close to the expected statistical range, the lifetime of GFP-tagged proteins in cells is shorter than recombinant GFP in solution. Furthermore the lifetime of GFP-tagged major histocompatibility complex class I protein is shortened at the inhibitory NK cell IS compared with the unconjugated membrane. Following our previous work demonstrating the ability of FLIM to report the local refractive index of GFP in solution, we speculate that these lifetime variations may indicate local refractive index changes. This application of our method for detecting small but significant differences in fluorescence lifetimes shows how FLIM could be broadly useful in imaging discrete membrane environments for a given protein.

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“…26 These values are presented in Figure 3a (9). ν 01 (T) was extracted from the peak of the absorption spectra shown in Figure 2.…”

mentioning

confidence: 99%

Two-Photon Absorption Dependence on the Temperature for Azoaromatic Compounds: Effect of Molecular Conformation

et al. 2007

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This work reports on the effect of temperature on the two-photon absorption cross section of azoaromatic chromophores. A linear decrease in the two-photon absorption cross section with the temperature was observed for several azochromophores. This process was characterized by introducing a two-photon absorption thermal coefficient (ddelta/dT), whose typical values are approximately 2GM/degrees C for all the azochromophores studied here. Such an effect was attributed to thermal induced molecular conformation changes, described by the sum-over-states model and semiempirical calculations, which affect the molecular dipole moments. The characterization of the phenomenon reported here for other nonlinear materials can help in the design of specific applications using two-photon absorption.

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Fluorescence Lifetime Imaging Microscopy

Cited by 12 publications

References 30 publications

Two-photon absorption spectrum in diazoaromatic compounds

Two-photon absorption spectrum in diazoaromatic compounds

Imaging fluorescence lifetime heterogeneity applied to GFP‐tagged MHC protein at an immunological synapse*

Two-Photon Absorption Dependence on the Temperature for Azoaromatic Compounds: Effect of Molecular Conformation

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