Two-photon excitation microscopy in cellular biophysics

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 Imaging of Glucose Metabolism in Living Tissue

10.1017/s1431927600008412 ◽

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 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.

Two-Photon Excitation Microscopy in Cellular Biophysics

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100163848 ◽

1996 ◽

Vol 54 ◽

pp. 276-277

Author(s):

David W. Piston

Keyword(s):

Confocal Microscopy ◽

Living Cells ◽

Input Power ◽

Photon Absorption ◽

Infrared Light ◽

Focal Volume ◽

Excitation Light ◽

Two Photon ◽

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 limits the average input power to less than 10 mW, only slightly greater than the power normally used in confocal microscopy.Three properties TPEM give this method a tremendous advantage over conventional optical sectioning microscopies for the study of thick samples: 1) The excitation is limited to the focal volume because of the intensity-squared dependence of the two-photon absorption. This inherent localization 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. Confinement of excitation to the focal volume also minimizes photobleaching and photo damage - the ultimate limiting factors in fluorescence microscopy of living cells and tissues. 2) The two-photon technique allows imaging of UV fluorophores with conventional visible light optics in both the scanning and imaging systems, because both the red excitation light (~700 nm) and the blue fluorescence (>400 nm) are within the visible spectrum. 3) Red or infrared light is far less damaging to most living cells and tissues than bluer light because fewer biological molecules absorb at the higher wavelengths. Longer wavelength excitation also reduces scattering of the incident light by the specimen, thus allowing more of the input power to reach the focal plane. This relative transparency of biological specimens to 700 nm light permits deeper sectioning, since both absorbance and scattering are reduced.

Imaging of Cellular Dynamics by Two-Photon Excitation Microscopy

10.1017/s1431927695110259 ◽

1995 ◽

Vol 1 (1) ◽

pp. 25-34 ◽

Cited By ~ 2

Author(s):

David W. Piston ◽

Brian D. Bennett ◽

Guangtao Ying

Keyword(s):

Uv Light ◽

Excited Electronic State ◽

Red Light ◽

Three Dimensional ◽

Focal Volume ◽

Optical Sectioning ◽

Two Photon ◽

Simultaneous Absorption ◽

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 (∼700 nm) can cause the transition to an excited electronic state normally reached by absorption in the ultraviolet (∼350 nm). In the fluorescence experiments described here, 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. Three properties of two-photon excitation give this method its advantage over conventional optical sectioning microscopies: (1) the excitation is limited to the focal volume, thus providing inherent three-dimensional resolution and minimizing photobleaching and photodamage; (2) the two-photon technique allows imaging of UV fluorophores with only conventional visible light optics; (3) red light is far less damaging to most living cells and tissues than UV light and permits deeper sectioning, because both absorbance and scattering are reduced. Many cell biological applications of two-photon excitation microscopy have been successfully realized, demonstrating the wide ranging power of this technique.

Video-Rate Scanning Two-Photon Excitation Fluorescence Microscopy

10.1017/s1431927600022248 ◽

1998 ◽

Vol 4 (S2) ◽

pp. 424-425

Author(s):

G.Y. Fan ◽

H. Fujisaki ◽

R.-K. Tsay ◽

R.Y. Tsien ◽

Mark H. Ellisman

Keyword(s):

Laser Beam ◽

Group Velocity Dispersion ◽

Laser System ◽

Average Power ◽

Laser Pulses ◽

Green Laser ◽

Two Photon ◽

Photon Excitation ◽

Schematic Layout ◽

A video-rate scanning two-photon excitation microscope (TPEM) has been successfully constructed and tested. The TPEM, based on a Nikon RCM-8000, incorporates a femtosecond pulsed laser, a pre-chirper, and a non-confocal detection box for ratio imaging. Fig. 1 shows the schematic layout of the main components of the instrument, each of which is briefly discussed below.Laser System: A Tsunami Ti: Sapphire laser (from Spectra-Physics) is optically pumped by a 5 W green laser (Millennia from Spectra-Physics) and is capable of generating 100 fs pulses at a repetition rate of 82 MHz and an average power of 0.8 W. The output wavelength is tunable from 690 to 1050 nm with three optical sets, each covering part of the spectrum with some overlapping.Pre-chirper: After leaving the Tsunami, the laser beam enters an optic unit known as a pre-chirper which pre-chirps laser pulses to compensate for the group velocity dispersion which will result when the laser beam goes through the microscope optics.

Application of two-photon chromophore excitation to laser scanning microscopy

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100086325 ◽

1991 ◽

Vol 49 ◽

pp. 404-405

Author(s):

David W. Piston ◽

James H. Strickler ◽

Watt W. Webb

Keyword(s):

Laser Scanning ◽

Uv Light ◽

Excited Electronic State ◽

Laser Scanning Confocal Microscopy ◽

Photochemical Reactions ◽

Laser Scanning Microscopy ◽

Scanning Confocal Microscopy ◽

Two Photon ◽

Photon Excitation ◽

The non-linear optical technique of two-photon excitation of fluorescence and photochemical reactions makes possible new applications that are not possible using linear one-photon excitation in laser scanning confocal microscopy. The two-photon excitation effect arises from the simultaneous absorption of two red photons, which causes the transition to an excited electronic state with its normal absorption in the ultraviolet. In our fluorescence experiments, this excited state is the same singlet state, S1, that is populated during a conventional fluorescence experiment, and thus exhibits the same emission properties (e.g. wavelength shifts, environmental sensitivity) that are observed in typical biological microscopy studies. Likewise, photochemical reactions such as light induced polymerization and photolytic uncaging that are normally catalyzed by UV light can be generated using two-photon excitation. In practice, twophoton excitation is made possible by the very high local instantaneous intensity that is provided by a combination of the diffraction limited focusing in the microscope and the temporal concentration of a subpicosecond mode-locked laser.

Noninvasive redox and back-scattered light imaging of keratocyte cells in the cornea: two-photon excitation and scanning slit confocal microscopy

10.1117/12.206831 ◽

1995 ◽

Author(s):

Barry R. Masters

Keyword(s):

Confocal Microscopy ◽

Scattered Light ◽

Two Photon ◽

Photon Excitation ◽

Two-photon controlled sol–gel condensation for the microfabrication of silica based microstructures. The role of photoacids and photobases

RSC Advances ◽

10.1039/c7ra08608c ◽

2017 ◽

Vol 7 (74) ◽

pp. 46615-46620 ◽

Cited By ~ 4

Author(s):

J. Kustra ◽

E. Martin ◽

D. Chateau ◽

F. Lerouge ◽

C. Monnereau ◽

...

Keyword(s):

Laser Beam ◽

Focal Point ◽

Sol Gel ◽

Ph Changes ◽

Two Photon ◽

Photon Excitation ◽

Sol Gel Process ◽

And Control ◽

Two-photon excitation of photobases is used to induce pH changes and control the condensation step of the sol–gel process at the focal point of a laser beam in a confocal configuration.

Real-Time Polarization-Resolved Imaging of Living Tissues Based on Two-Photon Excitation Spinning-Disk Confocal Microscopy

Frontiers in Physics ◽

10.3389/fphy.2019.00056 ◽

2019 ◽

Vol 7 ◽

Cited By ~ 2

Author(s):

Ai Goto ◽

Kohei Otomo ◽

Tomomi Nemoto

Keyword(s):

Confocal Microscopy ◽

Real Time ◽

Two Photon ◽

Spinning Disk ◽

Photon Excitation ◽

Living Tissues ◽

Multi-Photon Excitation Microscopy: An Old Idea in Quantum Theory Applied to Modern Scientific Problems

10.1017/s1431927600038393 ◽

2000 ◽

Vol 6 (S2) ◽

pp. 1180-1181

Author(s):

David W. Piston

Keyword(s):

Physical Principle ◽

Excitation Intensity ◽

Appreciable Amount ◽

Photon Density ◽

Two Photon ◽

Simultaneous Absorption ◽

Photon Excitation ◽

Dramatic Difference ◽

Basic Physical Principle ◽

Multi-photon excitation microscopy provides attractive advantages over confocal microscopy for three-dimensionalry resolved fluorescence imaging and photochemistry. The most commonly used type of multi-photon excitation is two-photon excitation where simultaneous absorption of two photons leads to a single quantitized event. The powerful advantages of using two-photon excitation microscopy arise from the basic physical principle that the absorption depends on the square of the excitation intensity. In practice, two-photon excitation is generated by focusing a single pulsed laser through the microscope. As the laser beam is focused, the photons become more crowded, but the only place at which they are crowded enough to generate an appreciable amount of two-photon excitation is at the focus. Above and below the focus, the photon density is not high enough for two of them to interact with a single fluorophore at the same time. This dramatic difference between confocal and two-photon excitation microscopy is shown in Fig. 1.