Extending the Reach of Immunoassays to Optically Dense Specimens by Using Two-Photon Excited Fluorescence Polarization

2000 ◽  
Vol 72 (22) ◽  
pp. 5748-5752 ◽  
Author(s):  
Gary A. Baker ◽  
Siddharth Pandey ◽  
Frank V. Bright
Keyword(s):  
Fluorescence Polarization ◽  
Two Photon

Two-Photon Fluorescence Polarization Anisotropy Decay on Highly Diluted Solutions by Phase Fluorometry

2001 ◽  
Vol 55 (3) ◽  
pp. 311-317 ◽  
Author(s):  
Francesca Olivini ◽  
Sabrina Beretta ◽  
Giuseppe Chirico
Keyword(s):  
Fluorescence Polarization ◽  
Light Polarization ◽  
Correlation Spectroscopy ◽  
Polarization Anisotropy ◽  
Two Photon ◽  
Protein Size ◽  
High Dilutions ◽  
Fluorescence Polarization Anisotropy ◽  
Two Photon Fluorescence ◽  
Photon Fluorescence

We report here the phase-sensitive measurements of two-photon fluorescence polarization anisotropy (FPA) on highly diluted solutions. We first describe the characterization of the response of the two-photon microscope to the light polarization and its test by means of measurements of the FPA on rhodamine 6G as a function of the viscosity. Further, we report the study of the FPA at high dilutions on a globular protein, the beta-lactoglobulin B (BLG) labeled with Alexa 532. The number of molecules (from 0.4 to 17 molecules per excitation volume) is measured by means of fluorescence correlation spectroscopy (FCS). The average rotational and translational diffusion coefficients measured with the FPA and FCS methods are in good agreement with the protein size and do not show a substantial dependence on the protein concentration, despite the very low signal (≌3 times the background) observed for highly diluted solutions.

Maximum degree of fluorescence polarization for two-photon-excited complex molecules

1972 ◽  
Vol 17 (3) ◽  
pp. 1138-1140
Author(s):  
E. S. Voropai ◽  
I. I. Zholnerevich ◽  
A. M. Sarzhevskii
Keyword(s):  
Fluorescence Polarization ◽  
Maximum Degree ◽  
Complex Molecules ◽  
Two Photon ◽  
Excited Complex

scholarly journals Two-Photon Excitation in Fluorescence Polarization Receptor-Ligand Binding Assay

2005 ◽  
Vol 10 (4) ◽  
pp. 314-319 ◽  
Author(s):  
Marko E. Tirri ◽  
Roope J. Huttunen ◽  
Juha Toivonen ◽  
Pirkko L. Härkönen ◽  
Juhani T. Soini ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Fluorescence Polarization ◽  
Binding Assay ◽  
Ligand Binding Assay ◽  
Receptor Ligand ◽  
Lead Compounds ◽  
Two Photon ◽  
Photon Excitation ◽  
Homogeneous Assay ◽  
Estrogen Receptor Ligand

Fluorescence polarization is one of the most commonly used homogeneous assay principles in drug discovery for screening of potential lead compounds. In this article, the fluorescence polarization technique is combined with 2-photon excitation of fluorescence. Theoretically, the use of 2-photon excitation of fluorescence increases the volumetric sensitivity and polarization contrast of fluorescence polarization assays. The work in this report demonstrates these predictions for an estrogen receptor ligand binding assay.

Two-photon excitation microscopy in cellular biophysics

1995 ◽  
Vol 53 ◽  
pp. 62-63
Author(s):  
David W. Piston ◽  
Brian D. Bennett ◽  
Robert G. Summers
Keyword(s):  
Confocal Microscopy ◽  
Laser Beam ◽  
Duty Cycle ◽  
Excited Electronic State ◽  
Input Power ◽  
Two Photon ◽  
Simultaneous Absorption ◽  
Photon Excitation ◽  
Very High ◽  
Two Photon Excitation

Two-photon excitation microscopy (TPEM) provides attractive advantages over confocal microscopy for three-dimensionally resolved fluorescence imaging and photochemistry. Two-photon excitation arises from the simultaneous absorption of two photons in a single quantitized event whose probability is proportional to the square of the instantaneous intensity. For example, two red photons can cause the transition to an excited electronic state normally reached by absorption in the ultraviolet. In practice, two-photon excitation is made possible by the very high local instantaneous intensity provided by a combination of diffraction-limited focusing of a single laser beam in the microscope and the temporal concentration of 100 femtosecond pulses generated by a mode-locked laser. Resultant peak excitation intensities are 106 times greater than the CW intensities used in confocal microscopy, but the pulse duty cycle of 10-5 maintains the average input power on the order of 10 mW, only slightly greater than the power normally used in confocal microscopy.

Two-photon excitation fluorescence microscopy in living systems

1993 ◽  
Vol 51 ◽  
pp. 154-155 ◽  
Author(s):  
David W. Piston
Keyword(s):  
Confocal Microscopy ◽  
Fluorescence Microscopy ◽  
Singlet State ◽  
Excited Electronic State ◽  
Input Power ◽  
Two Photon ◽  
Simultaneous Absorption ◽  
Photon Excitation ◽  
Very High ◽  
Two Photon Excitation

Two-photon excitation fluorescence microscopy provides attractive advantages over confocal microscopy for three-dimensionally resolved fluorescence imaging. Two-photon excitation arises from the simultaneous absorption of two photons in a single quantitized event whose probability is proportional to the square of the instantaneous intensity. For example, two red photons can cause the transition to an excited electronic state normally reached by absorption in the ultraviolet. In our fluorescence experiments, the final excited state is the same singlet state that is populated during a conventional fluorescence experiment. Thus, the fluorophore exhibits the same emission properties (e.g. wavelength shifts, environmental sensitivity) used in typical biological microscopy studies. In practice, two-photon excitation is made possible by the very high local instantaneous intensity provided by a combination of diffraction-limited focusing of a single laser beam in the microscope and the temporal concentration of 100 femtosecond pulses generated by a mode-locked laser. Resultant peak excitation intensities are 106 times greater than the CW intensities used in confocal microscopy, but the pulse duty cycle of 10−5 maintains the average input power on the order of 10 mW, only slightly greater than the power normally used in confocal microscopy.

Second order interference in two photon absorption

1996 ◽  
Vol 43 (9) ◽  
pp. 1765-1771 ◽  
Author(s):  
M. W. HAMILTON and D. S. ELLIOTT
Keyword(s):  
Second Order ◽  
Photon Absorption ◽  
Two Photon Absorption ◽  
Two Photon
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