Two-photon fluorescence excitation spectra of aromatic amino acids

Rehms, Aden A.; Callis, Patrik R.

doi:10.1016/0009-2614(93)89075-s

Cited by 80 publications

(44 citation statements)

References 27 publications

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“…Furthermore, we believe that the current study is only the first step in exploring the potential utility of 4CN-Trp in biological research; we expect that future studies will investigate its applicability in single-molecule spectroscopy and multiphoton microscopy. In comparison with Trp, which poses several challenges in these types of applications (30)(31)(32), 4CN-Trp has the advantages of being more photostable and having an excitation spectrum that is readily accessible by a typical femtosecond Ti:Sapphire laser (700-1,000 nm). Moreover, besides its attractive photophysical properties, the small size of 4CN-Trp, which only differs from its naturally occurring counterpart by two atoms, will make this BFAA especially useful in applications where a minimally perturbing fluorescence probe is required.…”

Section: Discussionmentioning

confidence: 99%

Blue fluorescent amino acid for biological spectroscopy and microscopy

Hilaire

Ahmed

Lin

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

116

119

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Many fluorescent proteins are currently available for biological spectroscopy and imaging measurements, allowing a wide range of biochemical and biophysical processes and interactions to be studied at various length scales. However, in applications where a small fluorescence reporter is required or desirable, the choice of fluorophores is rather limited. As such, continued effort has been devoted to the development of amino acid-based fluorophores that do not require a specific environment and additional time to mature and have a large fluorescence quantum yield, long fluorescence lifetime, good photostability, and an emission spectrum in the visible region. Herein, we show that a tryptophan analog, 4-cyanotryptophan, which differs from tryptophan by only two atoms, is the smallest fluorescent amino acid that meets these requirements and has great potential to enable in vitro and in vivo spectroscopic and microscopic measurements of proteins.fluorescence protein | fluorescence probe | unnatural amino acid | imaging O f the amino acids that are inherently responsible for the fluorescence of proteins, tyrosine (Tyr), tryptophan (Trp), and phenylalanine (Phe), Trp is the most widely used fluorescence reporter of protein structure, function, and dynamics, as its fluorescence quantum yield (QY) is comparatively large and is also sensitive to environment (1-3). However, Trp absorbs/emits in the UV wavelength region and has low photostability. Combined, these factors render this naturally occurring fluorescent amino acid hardly useful as a fluorophore for single-molecule measurements (4) and imaging applications, especially under in vivo conditions. For this reason, significant efforts have been devoted to identifying small unnatural fluorophores (5-11) that could overcome this limitation. So far, only naphthalene-based fluorophores, such as prodan (8, 9) and aladan (11) have been shown to be useful in this regard. However, these fluorophores are not structurally based on any naturally occurring amino acids and are larger in size than Trp, making them less attractive in applications where there are stringent requirements for the fluorophore size and structure. Therefore, the development of a smaller, ideally amino acid-based, fluorophore that does not require additional time to mature, have a large fluorescence QY, long fluorescence lifetime, good photostability, and an emission spectrum in the visible range, would enhance biological research. Herein, we show that a Trp analog, 4-cyanotryptophan (4CN-Trp), meets these requirements and has great potential to expand biological fluorescence spectroscopy and microscopy into additional territory.Many past studies have focused on Trp-based unnatural amino acids, including azatryptophans (5, 6, 12) and various indole-ring substituted analogs (13-16), aiming to identify useful biological fluorophores. Whereas some Trp analogs indeed exhibit improved fluorescent properties over Trp, none of them has found broad applications due to certain photophysical limitations. Recently, Ta...

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Section: Discussionmentioning

confidence: 99%

Blue fluorescent amino acid for biological spectroscopy and microscopy

Hilaire

Ahmed

Lin

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

116

119

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“…This technique promises to selectively excite transitions in the UV by visible pulsed laser sources without the broad unspecific background of conventional, one-photon excitation (OPE) in the UV. Compared with fluorescent dyes (33) that were studied in the first two-photon experiments (32), Trp has a rather low two-photon absorption cross section (34). This disadvantage seems to be partly compensated by the large number of Trps in a single Hc.…”

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confidence: 99%

Two-photon excitation microscopy of tryptophan-containing proteins

Lippitz

Erker²,

Decker³

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

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We have examined the feasibility of observing single protein molecules by means of their intrinsic tryptophan emission after two-photon excitation. A respiratory protein from spiders, the 24-meric hemocyanin, containing 148 tryptophans, was studied in its native state under almost in vivo conditions. In this specific case, the intensity of the tryptophan emission signals the oxygen load, allowing one to investigate molecular cooperativity. As a system with even higher tryptophan content, we also investigated latex spheres covered with the protein avidin, resulting in 340 tryptophans per sphere. The ratio of the fluorescence quantum efficiency to the bleaching efficiency was found to vary between 2 and 180 after two-photon excitation for tryptophan free in buffer solution, in hemocyanin, and in avidin-coated spheres. In the case of hemocyanin, this ratio leads to about four photons detected before photobleaching. Although this number is quite small, the diffusion of individual protein molecules could be detected by fluorescence correlation spectroscopy. In avidin-coated spheres, the tryptophans exhibit a higher photostability, so that even imaging of single spheres becomes possible. As an unexpected result of the measurements, it was discovered that the population of the oxygenated state of hemocyanin can be changed by means of a one-photon process with the same laser source that monitors this population in a two-photon process.

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“…Only corrections for the transmission of the microscope objective at the excitation wavelength and laser power at each excitation wavelength are required. A two-photon excitation spectrum for tryptophan has been reported by Rehms and Callis [22] and σ 2 of 1.9 GM units (10 −50 cm 4 s photon −1 ) calculated for 3-methyl indole at 552 nm. Their data suggests σ 2r of 0.21 GM for tryptophan two-photon excitation at 590 nm, and this was used as one of the standard values.…”

Section: Resultsmentioning

confidence: 86%

Fluorescence Lifetime Imaging of Propranolol Uptake in Living Glial C6 Cells

Bisby

Botchway²,

Crisostomo³

et al. 2012

Journal of Spectroscopy

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Abstract. Uptake of the β-blocker drug propranolol by living glial C6 cells has been observed using fluorescence lifetime imaging with two-photon excitation at 630 nm. Both uptake and release of propranolol occur within minutes and are temperature dependent, being about 5 times faster at 37 • C than at 20 • C. The intracellular fluorescence lifetime of propranolol is generally shorter than the value of 9.8 ns determined in dilute neutral aqueous solution, and the difference is ascribed to concentration quenching. Within the cells, propranolol is accumulated within intracellular acidic vesicles and the cytoplasm but is excluded from the cell nucleus. On incubation of cells in medium containing 100 μM propranolol, the drug is accumulated to reach intracellular concentrations up to 10 mM in a process that is believed to be driven by protonation within acidic cellular compartments.

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Two-photon fluorescence excitation spectra of aromatic amino acids

Cited by 80 publications

References 27 publications

Blue fluorescent amino acid for biological spectroscopy and microscopy

Blue fluorescent amino acid for biological spectroscopy and microscopy

Two-photon excitation microscopy of tryptophan-containing proteins

Fluorescence Lifetime Imaging of Propranolol Uptake in Living Glial C6 Cells

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