Quantitative Imaging of Metabolism by Two-Photon Excitation Microscopy

1999 ◽  
Vol 5 (S2) ◽  
pp. 1048-1049
Author(s):  
David W. Piston ◽  
Susan Knobel ◽  
George Patterson
Keyword(s):  
Quantitative Imaging ◽  
Photon Absorption ◽  
Limiting Factors ◽  
Focal Volume ◽  
Time Resolved ◽  
Two Photon Absorption ◽  
Two Photon ◽  
Photon Excitation ◽  
Small Absorption ◽  
Two Photon Excitation

Two-photon excitation microscopy provides attractive advantages over confocal microscopy for three-dimensionally resolved fluorescence imaging. Because of the intensity-squared dependence of the two-photon absorption, the excitation is limited to the focal volume. This inherent localization minimizes photobleaching and photodamage - the ultimate limiting factors in fluorescence microscopy of living cells. One of the most powerful applications of two-photon excitation microscopy is imaging from the naturally occurring reduced pyridine nucleotides (NAD(P)H). NAD(P)H is a useful indicator of cellular metabolism, but it is not a “good“ fluorophore (it has a small absorption cross-section and a low quantum yield). Two-photon excitation of NAD(P)H yields minimal photodamage, thus allowing time-resolved threedimensional metabolic mapping of cellular redox state. We have used two-photon excitation microscopy to examine glucose metabolism in pancreatic and muscle cells. As glucose is metabolized by these cells, intermediate metabolism results in an increase in the reduced-tooxidized NAD(P)H/NAD(P)+ ratio, and a concomitant increase in autofluorescence.

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.

Two-photon excitation spectroscopy of photosynthetic light-harvesting complexes and pigments

2019 ◽  
Vol 216 ◽  
pp. 494-506 ◽  
Author(s):  
Alexander Betke ◽  
Heiko Lokstein
Keyword(s):  
Spectral Region ◽  
Protein Complexes ◽  
Photosynthetic Pigment ◽  
Photon Absorption ◽  
Light Harvesting Complexes ◽  
Two Photon Absorption ◽  
Two Photon ◽  
Photon Excitation ◽  
Pigment Protein Complexes ◽  
Two Photon Excitation

Two-photon excitation (TPE) profiles of LHCII samples containing different xanthophyll complements were measured in the presumed 11Ag− → 21Ag− (S0 → S1) transition region of xanthophylls. Additionally, TPE profiles of Chls a and b in solution and of WSCP, which does not contain carotenoids, were measured. The results indicate that direct two-photon absorption by Chls in the presumed S0 → S1 transition spectral region of carotenoids is dominant over that of carotenoids, with negligible contributions of the latter. These results suggest the re-evaluation of previously published TPE data obtained with photosynthetic pigment–protein complexes containing (B)Chls and carotenoids.

UV-excited fluorophore images obtained with IR excitation

1996 ◽  
Vol 54 ◽  
pp. 906-907
Author(s):  
D. Wokosin ◽  
V.F. Centonze ◽  
J.G. White
Keyword(s):  
Short Pulse ◽  
Argon Laser ◽  
Confocal Imaging ◽  
Photon Absorption ◽  
Focal Volume ◽  
Two Photon ◽  
Photon Excitation ◽  
Biological Studies ◽  
Short Pulse Lasers ◽  
Two Photon Excitation

The widespread use of two-photon excitation fluorescence imaging has been somewhat inhibited by the necessity to use large, expensive, high-power, short-pulse lasers. These ultra-short pulse lasers are used as an excitation source in a raster scanning configuration to provide sufficient peak power density in a lens focal volume to generate detectable two-photon absorption events for rapid imaging. Biological studies often benefit from multiple fluorescent labels and multi-labelled samples often require different excitation wavelengths for adequate excitation of the various colored fluorophores. This is achieved inexpensively with the three Krypton Argon laser lines in standard confocal imaging systems, but multiple two-photon excitation lasers--if available-- would be a very expensive system. The lasers commonly used for two-photon imaging are tuneable, but this is not a non-trivial and time consuming process. The tuning range on these lasers allows good access to the blue-emitting and green-emitting fluorophores via two-photon excitation; however, we have found that a fixed-wavelength, compact,

Two-Photon Excitation Microscopy in Cellular Biophysics

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

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 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.

scholarly journals Phenothiazin-N-yl-capped 1,4-diketo-3,6-diphenylpyrrolo[3,4-c]pyrrole exhibiting strong two-photon absorption and aggregation-enhanced one- and two-photon excitation red fluorescence

RSC Advances ◽  
2017 ◽  
Vol 7 (49) ◽  
pp. 30610-30617 ◽  
Author(s):  
Kai Zhang ◽  
Zhongwei Liu ◽  
Shian Ying ◽  
Mingshuai Chen ◽  
Shanfeng Xue ◽  
...  
Keyword(s):  
Cross Section ◽  
Absorption Cross Section ◽  
Photon Absorption ◽  
Absorption Cross ◽  
Two Photon Absorption ◽  
Red Fluorescence ◽  
Two Photon ◽  
Photon Excitation ◽  
Two Photon Excitation

A phenothiazin-capped simple DPP dye exhibits a large two-photon absorption cross section and aggregation-enhanced one- and two-photon excitation red fluorescence.

Gas Phase Two-Photon Spectroscopy of Polyazines: Pyrazine

1979 ◽  
Vol 34 (8) ◽  
pp. 979-982 ◽  
Author(s):  
I. Knoth ◽  
H. J. Neusser ◽  
E. W. Schlag
Keyword(s):  
Excited State ◽  
Gas Phase ◽  
Vibrational Frequencies ◽  
Photon Absorption ◽  
Excitation Spectra ◽  
Selection Rules ◽  
Two Photon Absorption ◽  
Two Photon ◽  
Photon Excitation ◽  
Two Photon Excitation

Abstract The two-photon excitation spectra of gas phase pyrazine-h4 and -d 4 in the region of the S1←S0 transition are presented. The two-photon absorption which is electronically forbidden is induced by-various vibrations of ungerade parity. Because of the different selection rules for two-photon transitions hitherto unknown vibrational frequencies in the excited state 1B3u have been determined.

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