Photobleaching in two-photon excitation microscopy.

  title={Photobleaching in two-photon excitation microscopy.},
  author={George H. Patterson and David W. Piston},
  journal={Biophysical journal},
  volume={78 4},
The intensity-squared dependence of two-photon excitation in laser scanning microscopy restricts excitation to the focal plane and leads to decreased photobleaching in thick samples. However, the high photon flux used in these experiments can potentially lead to higher-order photon interactions within the focal volume. The excitation power dependence of the fluorescence intensity and the photobleaching rate of thin fluorescence samples ( approximately 1 microm) were examined under one- and two… Expand
High-order photobleaching in two-photon excitation fluorescence microscopy inside live cell
The two-photon excitation (TPE) microscopy has become an important tool of noninvasive imaging due to the better penetration and relative harmlessness of the longer wavelength. However, the highExpand
Photobleaching in two-photon scanning fluorescence correlation spectroscopy.
  • Z. Petrasek, P. Schwille
  • Chemistry, Medicine
  • Chemphyschem : a European journal of chemical physics and physical chemistry
  • 2008
Theoretical calculations assuming a nonuniform excitation intensity profile, and using the concept of generalized volume contrast, provide an explanation for the photobleaching effects commonly observed in two-photon FCS at high excitation intensities, without having to assume optical saturation. Expand
High-order photobleaching of green fluorescent protein inside live cells in two-photon excitation microscopy.
The results suggest that in applications where two-photon imaging of GFP is used to study dynamic molecular process, photobleaching may ruin the Imaging results and attention should be paid in interpreting the imaging results. Expand
Molecular photobleaching kinetics of Rhodamine 6G by one- and two-photon induced confocal fluorescence microscopy.
It is shown that fluorescence excitation is in all cases limited by photolysis from higher-excited electronic states, and comparison with experimental data and an exact theoretical model show that only minor deviations between the different theoretical approaches can be observed for high-pulsed excitation irradiances. Expand
Mechanisms of high-order photobleaching and its relationship to intracellular ablation
This work studied the photobleaching kinetics of an intrinsic and an extrinsic fluorophore in a cellular environment in two-photon microscopy and found that a low-density plasma is formed in both cases with a smooth transition in between. Expand
Mechanisms of high-order photobleaching and its relationship to intracellular ablation.
This work studied the photobleaching kinetics of an intrinsic and an extrinsic fluorophore in a cellular environment in two-photon microscopy and found that a low-density plasma is formed in both cases with a smooth transition in between. Expand
Michael Rubart Two-Photon Microscopy of Cells and Tissue
Two-photon excitation fluorescence imaging provides thin optical sections from deep within thick, scattering specimens by way of restricting fluorophore excitation (and thus emission) to the focalExpand
Reduction of higher-order photobleaching in two-photon excitation microscopy.
  • P. Mondal, A. Diaspro
  • Medicine, Physics
  • Physical review. E, Statistical, nonlinear, and soft matter physics
  • 2007
A theoretical microscopic technique is proposed that may reduce multiphoton interaction in the excitation volume of a two-photon microscope and higher-order processes can be minimized owing to their small molecular cross section. Expand
Two-photon microscopy of cells and tissue.
  • M. Rubart
  • Materials Science, Medicine
  • Circulation research
  • 2004
Its capability to resolve differences in calcium dynamics between individual cardiomyocytes deep within intact, buffer-perfused hearts is demonstrated and potential applications of two-photon laser scanning microscopy as applied to integrative cardiac physiology are pointed out. Expand
Two-photon-like microscopy with orders-of-magnitude lower illumination intensity via two-step fluorescence
Two-step fluorescence microscopy is described, a new approach to non-linear imaging based on positive reversible photoswitchable fluorescent probes that improves resolution and reduces unwanted out-of-focus excitation, and is compatible with structured illumination microscopy. Expand


Two-photon laser scanning fluorescence microscopy.
The fluorescence emission increased quadratically with the excitation intensity so that fluorescence and photo-bleaching were confined to the vicinity of the focal plane as expected for cooperative two-photon excitation. Expand
Mechanisms of photobleaching investigated by fluorescence correlation spectroscopy
Fluorescence correlation spectroscopy (FCS) can be used to investigate the photobleaching properties of fluorophores in solution. The advantage with this method is that in addition to theExpand
Multiphoton excitation provides optical sections from deeper within scattering specimens than confocal imaging.
It is demonstrated that this technique provides yet further improvements in the capability of multiphoton excitation imaging to produce good quality images from deeper within tissue relative to confocal imaging. Expand
Room-Temperature Fluorescence Imaging and Spectroscopy of Single Molecules by Two-Photon Excitation
We report fluorescence imaging of single dye molecules on a glass substrate by two-photon excitation with femtosecond pulses from a mode-locked Ti:sapphire laser. The single-molecule images exhibit aExpand
Photobleaching kinetics of fluorescein in quantitative fluorescence microscopy.
Both the theoretical simulation and experimental data show that photobleaching of fluorescein in microscopy is, in general, not a single-exponential process. Expand
Multiphoton excitation cross‐sections of molecular fluorophores
Nonlinear excitation of fluorophores through molecular absorption of two or three near-infra-red photons from the tightly focused femtosecond pulses of a mode-locked laser offers the cellularExpand
Influence of the triplet excited state on the photobleaching kinetics of fluorescein in microscopy.
Experiments demonstrated that a thiol-containing reducing agent, mercaptoethylamine (MEA or cysteamine), was the most effective, among other commonly known radical quenchers or singlet oxygen scavengers, in suppressing photobleaching of fluorescein while not reducing the fluorescence quantum yield. Expand
Quantitative imaging of metabolism by two-photon excitation microscopy.
The chapter describes the use of the naturally occurring reduced nicotinamide adenine dinucleotide (phosphate) [NAD(P)H] as a monitor of cellular metabolism and discusses the two-photon excitation microscopy methods used to image its activity. Expand
Handbook of Biological Confocal Microscopy
Foundations of Confocal Scanned Imaging in Light Microscopy -- Fundamental Limits in Confocal Microscopy -- Special Optical Elements -- Points, Pixels, and Gray Levels: Digitizing Image Data -- LaserExpand
Use of the green fluorescent protein and its mutants in quantitative fluorescence microscopy.
No single GFP variant is ideal for every application, but each one offers advantages and disadvantages for quantitative imaging in living cells. Expand