Widefield Two-Photon Excitation without Scanning: Live Cell Microscopy with High Time Resolution and Low Photo-Bleaching

For live-cell imaging, two-photon excitation microscopy (TPEM) is proving to be a significant technological advancement. The unique features offered by TPEM are the ability to image thick sections, excellent optical sectioning capabilities, low damage to living cells, and less out of focus fluorescence and out of focus photobleaching. of these features, the most useful for the biological microscopist, is optical sectioning. Optical sectioning is an intrinsic property of the two-photon process, whereby, two infrared (IR) photons are absorbed quickly to excite a single UV/blue transition. The probability for exciting a two photon transition is proportional to the instantaneous excitation intensity squared. Therefore, for a focused laser beam, only light at the focal point of the excitation beam excites a fluorescent transition. Thus, the need for confocal apertures and time consuming deconvolution algorithms are, for the most part, eliminated.We have continued to develop and enhance our ability to perform high-speed, two-photon excitation fluorescence microscopy. in 1998, we successfully deployed a prototype, video-rate twophoton laser scanning system (30 frames/sec or faster at reduced scan width) developed with support from Nikon Corporation. That system was built upon a Nikon RCM 8000 confocal microscope.

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Visible-wavelength two-photon excitation microscopy with multifocus scanning for volumetric live-cell imaging

Journal of Biomedical Optics ◽

10.1117/1.jbo.25.1.014502 ◽

2019 ◽

Vol 25 (01) ◽

pp. 1 ◽

Cited By ~ 1

Author(s):

Ryosuke Oketani ◽

Haruka Suda ◽

Kumiko Uegaki ◽

Toshiki Kubo ◽

Tomoki Matsuda ◽

...

Keyword(s):

Live Cell Imaging ◽

Cell Imaging ◽

Live Cell ◽

Two Photon ◽

Visible Wavelength ◽

Photon Excitation ◽

Two Photon Excitation

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Live-Cell Superresolution Imaging by Pulsed STED Two-Photon Excitation Microscopy

Biophysical Journal ◽

10.1016/j.bpj.2012.12.053 ◽

2013 ◽

Vol 104 (4) ◽

pp. 770-777 ◽

Cited By ~ 99

Author(s):

Kevin T. Takasaki ◽

Jun B. Ding ◽

Bernardo L. Sabatini

Keyword(s):

Live Cell ◽

Superresolution Imaging ◽

Two Photon ◽

Photon Excitation ◽

Two Photon Excitation

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Solar Corona Investigation by the Coronal Line Photometer at Lomnický Peak

International Astronomical Union Colloquium ◽

10.1017/s0252921100025744 ◽

1994 ◽

Vol 144 ◽

pp. 431-434

Author(s):

M. Minarovjech ◽

M. Rybanský

Keyword(s):

Solar Corona ◽

Time Resolution ◽

Active Regions ◽

High Time ◽

High Time Resolution ◽

List Type ◽

Type Number ◽

Coronal Line ◽

Global Cycle

AbstractThis paper deals with a possibility to use the ground-based method of observation in order to solve basic problems connected with the solar corona research. Namely:1.heating of the solar corona2.course of the global cycle in the corona3.rotation of the solar corona and development of active regions.There is stressed a possibility of high-time resolution of the coronal line photometer at Lomnický Peak coronal station, and use of the latter to obtain crucial observations.

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Two-photon excitation microscopy in cellular biophysics

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100136684 ◽

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.

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Two-photon excitation fluorescence microscopy in living systems

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100146618 ◽

1993 ◽

Vol 51 ◽

pp. 154-155 ◽

Cited By ~ 1

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.

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