Imaging of Cellular Dynamics by Two-Photon Excitation Microscopy

1995 ◽  
Vol 1 (1) ◽  
pp. 25-34 ◽  
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

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.

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 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.

Application of two-photon chromophore excitation to laser scanning microscopy

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 ◽  
Two 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.

Imaging Deep Optical Sections with Two-Photon Excitation Fluorescence Microscopy

1996 ◽  
Vol 54 ◽  
pp. 280-281
Author(s):  
J.G. White ◽  
V.F. Centonze ◽  
D.L. Wokosin
Keyword(s):  
Fluorescence Imaging ◽  
Photon Absorption ◽  
Laser Source ◽  
Excitation Wavelength ◽  
Scanning System ◽  
Focal Volume ◽  
Optical Sectioning ◽  
Two Photon ◽  
Photon Excitation ◽  
Two Photon Excitation

The technique of optical sectioning allows the visualization of a succession of images of parallel planes within a thick specimen with little or no out-of-focus interference. Ultimately, a limit is reached on the depth to which optical sections can be obtained from a given sample. This limit, up to the working distance of the objective, is largely determined by the degree of light scattering encountered by the incident excitation beam as well as the returning emission signal.Confocal imaging was one of the first optical sectioning techniques applied to fluorescence imaging. Two-photon excitation imaging is a recently developed alternative optical sectioning technique for fluorescence imaging where an excitation wavelength of around twice the excitation peak of the fluorophore is used in a laser-scanning microscope. This excitation wavelength produces very little fluorophore excitation in the bulk of the sample, but when the incident photons are confined in space and time sufficient two-photon absorption events can take place to obtain rapid imaging of fluorophores. With high peak powers—obtained with a sub-picosecond pulsed laser source focused by a lens—sufficient photon density can be obtained for easily detectable two-photon events. Thus fluorophore excitation occurs as two photons are absorbed essentially simultaneously, which act effectively as a single photon of twice the energy (half the wavelength). Two-photon events have a quadratic dependence on intensity, and, therefore, decrease rapidly away from the focal volume of the lens. In a raster scanning system, fluorophore excitation is confined to the optical section being viewed as fluorophore away from the lens focal volume is not excited by the long-wavelength illumination.

Crosstalk in Photoluminescence Readout of Three-Dimensional Memory in Vitreous Silica by One- and Two-Photon Excitation

2000 ◽  
Vol 39 (Part 1, No. 12A) ◽  
pp. 6763-6767 ◽  
Author(s):  
Mitsuru Watanabe ◽  
Saulius Juodkazis ◽  
Shigeki Matsuo ◽  
Junji Nishii ◽  
Hiroaki Misawa
Keyword(s):  
Three Dimensional ◽  
Vitreous Silica ◽  
Two Photon ◽  
Photon Excitation ◽  
Two Photon Excitation

High Speed Two Photon Excitation Microscopy in Live Cell Imaging using Image Correlation Spectroscopy (ICS)

2001 ◽  
Vol 7 (S2) ◽  
pp. 22-23
Author(s):  
P. W. Wiseman ◽  
J. C. Bouwer ◽  
S. Peltier ◽  
M. H. Ellisman
Keyword(s):  
Live Cell Imaging ◽  
High Speed ◽  
Cell Imaging ◽  
Live Cell ◽  
Image Correlation ◽  
Scanning System ◽  
Optical Sectioning ◽  
Two Photon ◽  
Photon Excitation ◽  
Two Photon Excitation

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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