Multiphoton excitation provides optical sections from deeper within scattering specimens than confocal imaging.

  title={Multiphoton excitation provides optical sections from deeper within scattering specimens than confocal imaging.},
  author={Victoria E. Centonze and J. G. White},
  journal={Biophysical journal},
  volume={75 4},
Multiphoton excitation fluorescence imaging generates an optical section of sample by restricting fluorophore excitation to the plane of focus. High photon densities, achieved only in the focal volume of the objective, are sufficient to excite the fluorescent probe molecules by density-dependent, multiphoton excitation processes. We present comparisons of confocal with multiphoton excitation imaging of identical optical sections within a sample. These side-by-side comparisons of imaging modes… 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
Detection sensitivity enhancements for fluorescence imaging with multi-photon excitation microscopy
  • D. Wokosin, W. Amos, John White
  • Materials Science
  • Proceedings of the 20th Annual International Conference of the IEEE Engineering in Medicine and Biology Society. Vol.20 Biomedical Engineering Towards the Year 2000 and Beyond (Cat. No.98CH36286)
  • 1998
Multiphoton excitation microscopy (MPEM) offers several distinct advantages over laser scanning confocal microscopy (LSCM). One is that the resolution and the thickness of the optical section areExpand
The effects of spherical aberration on multiphoton fluorescence excitation microscopy
It is demonstrated that signal levels and resolution both rapidly decline with depth into refractive index mismatched samples, demonstrating that spherical aberration is an important limiting factor in multiphoton fluorescence excitation microscopy of biological samples. Expand
Improving Signal Levels in Intravital Multiphoton Microscopy using an Objective Correction Collar.
It is found that spherical aberration, as evaluated from axial asymmetry in the point spread function, can be corrected by adjustment of the correction collar of a water immersion objective lens. 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
Principles of Multiphoton Microscopy
Improvements in multiphoton fluorescence microscopy will involve optimizing objectives for the unique characteristics of multiphotons, improving the speed at which images may be collected and extending the depth to which imaging may be conducted. Expand
Applications of two-photon fluorescence microscopy in deep-tissue imaging
Based on the non-linear excitation of fluorescence molecules, two-photon fluorescence microscopy has become a significant new tool for biological imaging. The point-like excitation characteristic ofExpand
Multifocal multiphoton microscopy with adaptive optical correction
Fluorescence lifetime imaging microscopy (FLIM) is a well established approach for measuring dynamic signalling events inside living cells, including detection of protein-protein interactions. TheExpand
Multiphoton versus confocal high resolution z‐sectioning of enhanced green fluorescent microtubules: increased multiphoton photobleaching within the focal plane can be compensated using a Pockels cell and dual widefield detectors
It is shown quantitatively that for any particular bleach rate multiphoton excitation produces significantly more signal than one photon excitation confocal microscopy in high resolution Z‐axis sectioning of thin samples. Expand
Multiphoton Imaging of Neurons in Living Tissue: Acquisition and Analysis of Time-Lapse Morphological Data
Object-Image, an extension of the popular public-domain NIH Image software, together with a set of custom macros for time-lapse morphometric analysis, is used to analyze neuronal branch growth and complexity in three dimensions over time. Expand


All-solid-state ultrafast lasers facilitate multiphoton excitation fluorescence imaging
Improvements in ultrafast laser technology have enabled a new excitation mode for optical sectioning fluorescence microscopy: multiphoton excitation fluorescence imaging. The primary advantages ofExpand
Optimization of the design of a multiple-photon excitation laser scanning fluorescence imaging system
Multi-photon (two or more photon) excitation imaging offers three significant advantages compared to laser-scanning confocal fluorescence microscopy for 3-D and 4-D fluorescence microscopy:Expand
Multiple-photon excitation imaging with an all-solid-state laser
Two-photon excitation imaging is a recently described optical sectioning technique where fluorophore excitation is confined to--and therefore defines--the optical section being observed. ThisExpand
An evaluation of confocal versus conventional imaging of biological structures by fluorescence light microscopy
It is found that confocal imaging gives greatly enhanced images of biological structures viewed with epifluorescence, and the improvements are such that it is possible to optically section thick specimens with little degradation in the image quality of interior sections. 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
Intravital imaging of green fluorescent protein using two-photon laser-scanning microscopy.
It is shown that TPLSM provides greater sensitivity, better resolution and less photo-bleaching, as compared to confocal laser-scanning microscopy, which allows a safer and higher-resolution means of imaging living cells labeled with a variety of fluorophores, including green fluorescent protein. Expand
Two‐photon molecular excitation provides intrinsic 3‐dimensional resolution for laser‐based microscopy and microphotochemistry
  • R. Williams, D. Piston, W. Webb
  • Materials Science, Medicine
  • FASEB journal : official publication of the Federation of American Societies for Experimental Biology
  • 1994
The working principles of new, nonlinear laser microscopies based on two‐photon molecular excitation are described, specifically, Imaging of vital DNA stains in developing cells and embryos, imaging of cellular metabolic activity from NADH autofluorescence, spatially resolved measurements of cytoplasmic calcium ion activity, and optically induced micropharmacology using caged bioeffector molecules. Expand
Anatomical and functional imaging of neurons using 2-photon laser scanning microscopy
A range of imaging modes based on 2-photon laser scanning microscopy (TPLSM) are shown as applicable to problems in neuroscience, showing how the distribution of neurotransmitter receptors on the surface of specific cells can be mapped. Expand
It is shown that two-photon absorption confines the illumination volume and present quantitative evidence that an additional confocal arrangement of the detector further improves the resolution byExpand
Confocal microscopy in turbid media.
The major conclusion of the paper is that the trade-off between signal level and background scattered-light rejection places a fundamental limit on the sectioning capability of the microscope. Expand