Signal enhancement in multiphoton TIRF microscopy by shaping of broadband femtosecond pulses

Lane, Richard S. K.; Macpherson, Alisdair N.; Magennis, Steven W.

doi:10.1364/oe.20.025948

Cited by 11 publications

(9 citation statements)

References 44 publications

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“…In fact, since we started this work, a number of theoretical studies of the 1P photophysics of fluorescent nucleobases have appeared, including some of the molecules described herein. [9] New probe designs that incorporate structure-photophysical property relationships, and developments in optical instrumentation, such as the use of new ultrafast lasers for multiphoton excitation, [10] could facilitate the realization of fluorescent nucleobase labels that can probe the structure and dynamics of nucleic acids at the single-molecule level.…”

Section: Discussionmentioning

confidence: 99%

Two‐Photon‐Induced Fluorescence of Isomorphic Nucleobase Analogs

et al. 2014

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Five isomorphic fluorescent uridine mimics have been subjected to two-photon (2P) excitation analysis to investigate their potential applicability as non-perturbing probes for the single-molecule detection of nucleic acids. We find that small structural differences can cause major changes in the two-photon excitation probability, with the 2P cross sections varying by over one order of magnitude. Two of the probes, both furan-modified uridine analogs, have the highest 2P cross sections (3.8 GM and 7.6 GM) reported for nucleobase analogs, using a conventional Ti:sapphire laser for excitation at 690 nm; they also have the lowest emission quantum yields. In contrast, the analogs with the highest reported quantum yields have the lowest 2P cross sections. The structure-photophysical property relationship presented here is a first step towards the rational design of emissive nucleobase analogs with controlled 2P characteristics. The results demonstrate the potential for major improvements through judicious structural modifications.

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Section: Discussionmentioning

confidence: 99%

Two‐Photon‐Induced Fluorescence of Isomorphic Nucleobase Analogs

et al. 2014

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“…37,38 This approach can also be extended to wide-field excitation and detection. 39 For molecules with broad absorption spectra, it is possible to tailor the spectral phase [40][41][42] or amplitude 38 in a desired frequency range for selective excitation of fluorophores.…”

Section: Pulse-shaped 2p Microscopy Of Pa-containing Dnamentioning

confidence: 99%

Pulse-shaped two-photon excitation of a fluorescent base analogue approaches single-molecule sensitivity

Fisher

Nobis

Füchtbauer

et al. 2018

Phys. Chem. Chem. Phys.

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“…Analogous to scanning-type two-photon excitation fluorescence (2PEF) microscopy, femtosecond (fs)-pulsed EW excitation reduces nonevanescent background excitation because scattered photons are too dilute to generate appreciable fluorescence (21,(29)(30)(31). Although effective, 2PEF TIR has, in fact, rarely been used.…”

Section: Objective Glare and Aberrations Of Peripheral Beams Limit Exmentioning

confidence: 99%

Eliminating Unwanted Far-Field Excitation in Objective-Type TIRF. Part I. Identifying Sources of Nonevanescent Excitation Light

Brunstein

Térémetz

Hérault

et al. 2014

Biophysical Journal

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Total internal reflection fluorescence microscopy (TIRFM) achieves subdiffraction axial sectioning by confining fluorophore excitation to a thin layer close to the cell/substrate boundary. However, it is often unknown how thin this light sheet actually is. Particularly in objective-type TIRFM, large deviations from the exponential intensity decay expected for pure evanescence have been reported. Nonevanescent excitation light diminishes the optical sectioning effect, reduces contrast, and renders TIRFM-image quantification uncertain. To identify the sources of this unwanted fluorescence excitation in deeper sample layers, we here combine azimuthal and polar beam scanning (spinning TIRF), atomic force microscopy, and wavefront analysis of beams passing through the objective periphery. Using a variety of intracellular fluorescent labels as well as negative staining experiments to measure cell-induced scattering, we find that azimuthal beam spinning produces TIRFM images that more accurately portray the real fluorophore distribution, but these images are still hampered by far-field excitation. Furthermore, although clearly measureable, cell-induced scattering is not the dominant source of far-field excitation light in objective-type TIRF, at least for most types of weakly scattering cells. It is the microscope illumination optical path that produces a large cell- and beam-angle invariant stray excitation that is insensitive to beam scanning. This instrument-induced glare is produced far from the sample plane, inside the microscope illumination optical path. We identify stray reflections and high-numerical aperture aberrations of the TIRF objective as one important source. This work is accompanied by a companion paper (Pt.2/2).

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Signal enhancement in multiphoton TIRF microscopy by shaping of broadband femtosecond pulses

Cited by 11 publications

References 44 publications

Two‐Photon‐Induced Fluorescence of Isomorphic Nucleobase Analogs

Two‐Photon‐Induced Fluorescence of Isomorphic Nucleobase Analogs

Pulse-shaped two-photon excitation of a fluorescent base analogue approaches single-molecule sensitivity

Eliminating Unwanted Far-Field Excitation in Objective-Type TIRF. Part I. Identifying Sources of Nonevanescent Excitation Light

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