Non-degenerate two-photon excitation for increasing the fluorescence photon yield and maximum microscopy imaging depth (Conference Presentation)

2017 ◽  
Author(s):  
Mu-Han Yang ◽  
Christopher G. L. Ferri ◽  
Payam A. Saisan ◽  
Maxim Abashin ◽  
Peifang Tian ◽  
...  
Keyword(s):  
Photon Yield ◽  
Microscopy Imaging ◽  
Two Photon ◽  
Fluorescence Photon ◽  
Photon Excitation ◽  
Imaging Depth ◽  
Two Photon Excitation

scholarly journals Two-photon Excitation Fluorescent Spectral and Decay Properties of Retrograde Neuronal Tracer Fluoro-Gold

2021 ◽  
Author(s):  
Matthew Q. Miller ◽  
Iván Coto Hernández ◽  
Jenu V. Chacko ◽  
Steven Minderler ◽  
Nate Jowett
Keyword(s):  
Single Photon ◽  
Fluorescence Decay ◽  
Two Photon ◽  
Temporal Properties ◽  
Decay Properties ◽  
Photon Excitation ◽  
Deep Imaging ◽  
Refractive Index Matching ◽  
Imaging Depth ◽  
Two Photon Excitation

Abstract Fluoro-Gold is a fluorescent neuronal tracer suitable for targeted deep imaging of the nervous system. Widefield fluorescence microscopy enables visualization of fluoro-gold, but lacks depth discrimination. Though scanning laser confocal microscopy yields volumetric data, imaging depth is limited, and optimal single-photon excitation of fluoro-gold requires an unconventional ultraviolet excitation line. Two-photon excitation microscopy employs ultrafast pulsed infrared lasers to image fluorophores at high-resolution at unparalleled depths in opaque tissue. Deep imaging of fluoro-gold-labeled neurons carries potential to advance understanding of the central and peripheral nervous systems, yet its two-photon spectral and temporal properties remain uncharacterized. Herein, we report the relative two-photon excitation spectrum of fluoro-gold between 720 nm and 1100 nm, and its fluorescence decay rate in aqueous solution and murine brainstem tissue. We demonstrate unprecedented imaging depth of whole-mounted murine brainstem via two-photon excitation microscopy of fluoro-gold-labeled facial motor nuclei. Optimal two-photon excitation of fluoro-gold occurred at 730 nm, while maximum lifetime contrast was observed at 760 nm with mean fluorescence lifetime of 1.4 ns. Whole-mount brainstem explants were readily imaged to depths in excess of 450 µm via immersion in refractive-index matching solution.

scholarly journals Two-photon excitation fluorescent spectral and decay properties of retrograde neuronal tracer Fluoro-Gold

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Matthew Q. Miller ◽  
Iván Coto Hernández ◽  
Jenu V. Chacko ◽  
Steven Minderler ◽  
Nate Jowett
Keyword(s):  
Single Photon ◽  
Fluorescence Decay ◽  
Two Photon ◽  
Temporal Properties ◽  
Decay Properties ◽  
Photon Excitation ◽  
Deep Imaging ◽  
Refractive Index Matching ◽  
Imaging Depth ◽  
Two Photon Excitation

AbstractFluoro-Gold is a fluorescent neuronal tracer suitable for targeted deep imaging of the nervous system. Widefield fluorescence microscopy enables visualization of Fluoro-Gold, but lacks depth discrimination. Though scanning laser confocal microscopy yields volumetric data, imaging depth is limited, and optimal single-photon excitation of Fluoro-Gold requires an unconventional ultraviolet excitation line. Two-photon excitation microscopy employs ultrafast pulsed infrared lasers to image fluorophores at high-resolution at unparalleled depths in opaque tissue. Deep imaging of Fluoro-Gold-labeled neurons carries potential to advance understanding of the central and peripheral nervous systems, yet its two-photon spectral and temporal properties remain uncharacterized. Herein, we report the two-photon excitation spectrum of Fluoro-Gold between 720 and 990 nm, and its fluorescence decay rate in aqueous solution and murine brainstem tissue. We demonstrate unprecedented imaging depth of whole-mounted murine brainstem via two-photon excitation microscopy of Fluoro-Gold labeled facial motor nuclei. Optimal two-photon excitation of Fluoro-Gold within microscope tuning range occurred at 720 nm, while maximum lifetime contrast was observed at 760 nm with mean fluorescence lifetime of 1.4 ns. Whole-mount brainstem explants were readily imaged to depths in excess of 450 µm via immersion in refractive-index matching solution.

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.

Two‐photon excitation fluorescence microscopy using a semiconductor laser

Bioimaging ◽  
1995 ◽  
Vol 3 (2) ◽  
pp. 70-75 ◽  
Author(s):  
Pekka E Hänninen ◽  
Martin Schrader ◽  
Erkki Soini ◽  
Stefan W Hell
Keyword(s):  
Fluorescence Microscopy ◽  
Semiconductor Laser ◽  
Two Photon ◽  
Photon Excitation ◽  
Two Photon Excitation

Two-Photon Excitation Imaging of Glucose Metabolism in Living Tissue

1997 ◽  
Vol 3 (S2) ◽  
pp. 305-306
Author(s):  
David W. Piston
Keyword(s):  
Confocal Microscopy ◽  
Collection Efficiency ◽  
Three Dimensional ◽  
Spatial Filter ◽  
Input Power ◽  
Photon Absorption ◽  
Focal Volume ◽  
Two Photon ◽  
Photon Excitation ◽  
Two Photon Excitation

Two-photon excitation microscopy (TPEM) provides attractive advantages over confocal microscopy for three-dimensionally resolved fluorescence imaging and photochemistry. It provides three-dimensional resolution and eliminates background equivalent to an ideal confocal microscope without requiring a confocal spatial filter, whose absence enhances fluorescence collection efficiency. This results in inherent submicron optical sectioning by excitation alone. In practice, TPEM 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 limits the average input power to less than 10 mW, only slightly greater than the power normally used in confocal microscopy. Because of the intensity-squared dependence of the two-photon absorption, the excitation is limited to the focal volume.

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