scholarly journals Scanless two-photon excitation with temporal focusing

2020 ◽  
Vol 17 (6) ◽  
pp. 571-581 ◽  
Author(s):  
Eirini Papagiakoumou ◽  
Emiliano Ronzitti ◽  
Valentina Emiliani
Keyword(s):  
Two Photon ◽  
Photon Excitation ◽  
Temporal Focusing ◽  
Two Photon Excitation

scholarly journals Two-photon patterned photostimulation with low-power, high-efficiency and reliable single-cell optogenetic control

2022 ◽  
Author(s):  
Yifan Wang ◽  
Yao Zheng ◽  
Yongxian Xu ◽  
Rongrong Li ◽  
Yameng Zheng ◽  
...  
Keyword(s):  
Low Power ◽  
Single Cell ◽  
Laser Power ◽  
High Efficiency ◽  
Excitation Density ◽  
Two Photon ◽  
Photon Excitation ◽  
Temporal Focusing ◽  
Patterned Illumination ◽  
Two Photon Excitation

Two-photon optogenetics enables selectively stimulating individual cells for manipulating neuronal ensembles. As the general photostimulation strategy, the patterned two-photon excitation has enabled millisecond-timescale activation for single or multiple neurons, but its activation efficiency is suffered from high laser power due to low beam-modulation efficiency. Here, we develop a high-efficiency beam-shaping method based on the Gerchberg-Saxton (GS) algorithm with spherical-distribution initial phase (GSSIP) to reduce the patterned two-photon excitation speckles and intensity. It can well control the phase of shaped beams to attain speckle-free accurate patterned illumination with an improvement of 44.21% in the modulation efficiency compared with that of the traditional GS algorithm. A combination of temporal focusing and the GSSIP algorithm (TF-GSSIP) achieves patterned focusing through 500-μm-thickness mouse brain slices, which is 2.5 times deeper than the penetration depth of TF-GS with the same signal-to-noise ratio (SNR). With our method, the laser power can be reduced to only 55.56% of that with traditional method (the temporal focusing with GS, TF-GS) to reliably evoke GCaMP6s response in C1V1-expressing cultured neurons with single-cell resolution. Besides, the photostimulation efficiency is remarkably increased by 80.19% at the same excitation density of 0.27 mW/μm2. This two-photon stimulation method with low-power, reliable and patterned illumination may pave the way for analyzing neural circuits and neural coding and decoding mechanism.

scholarly journals 3D-resolved fluorescence and phosphorescence lifetime imaging using temporal focusing wide-field two-photon excitation

2012 ◽  
Vol 20 (24) ◽  
pp. 26219 ◽  
Author(s):  
Heejin Choi ◽  
Dimitrios S. Tzeranis ◽  
Jae Won Cha ◽  
Philippe Clémenceau ◽  
Sander J. G. de Jong ◽  
...  
Keyword(s):  
Wide Field ◽  
Phosphorescence Lifetime ◽  
Two Photon ◽  
Lifetime Imaging ◽  
Photon Excitation ◽  
Temporal Focusing ◽  
Phosphorescence Lifetime Imaging ◽  
Fluorescence And Phosphorescence ◽  
Two Photon Excitation

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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