Depth-targeted intracortical microstroke by two-photon photothrombosis in rodent brain

Significance: Photothrombosis is a widely used model of ischemic stroke in rodent experiments. In the photothromboris model, the photosensitizer Rose Bengal is systemically introduced to the blood stream and activated by green light to induce aggregation of platelets that eventually cause vessel occlusion. Since the activation of Rose Bengal is a one-photon phenomenon and the molecules in the illuminated area (light path) are subject to excitation, targeting of thrombosis is unspecific especially in the depth dimension. We have developed a photothrombosis protocol that can target a single vessel in the cortical parenchyma by two-photon excitation. Aim: We aim to induce a thrombotic stroke in the cortical parenchyma by two-photon activation of Rose Bengal so that we confine photothrombosis within a vessel of a target depth. Approach: FITC-dextran is injected into the blood stream to visualize the cerebral blood flow in anesthetized adult mice with a cranial window. After a target vessel is chosen by two-photon imaging (950 nm), Rose Bengal is injected into the blood stream. The scanning wavelength is changed to 720 nm and photothrombosis was induced by scanning the target vessel. Results: Two-photon depth-targeted single vessel photothrombosis was achieved with a success rate of 84.9+/-1.7% within 80 s. Attempts without Rose Bengal (i.e., only with FITC) did not result in photothrombosis at the excitation wavelength of 720 nm. Conclusions: We described a protocol that achieves depth-targeted single vessel photothrombosis by two-photon excitation. Simultaneous imaging of blood flow in the targeted vessel using FITC dextran enabled the confirmation of vessel occlusion and prevention of excess irradiation that possibly induces unintended photodamage.

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Two-Photon Imaging of Cortical Microvascular Blood Flow in Response to Single Vessel Occlusion

Frontiers in Optics ◽

10.1364/fio.2005.jmb2 ◽

2005 ◽

Author(s):

N. Nishimura ◽

C. B. Schaffer ◽

B. Friedman ◽

P. S. Tsai ◽

P. D. Lyden ◽

...

Keyword(s):

Blood Flow ◽

Microvascular Blood Flow ◽

Vessel Occlusion ◽

Two Photon ◽

Single Vessel ◽

Photon Imaging ◽

Two Photon Imaging

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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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Two‐photon excitation fluorescence microscopy using a semiconductor laser

Bioimaging ◽

10.1002/1361-6374(199506)3:2<70::aid-bio3>3.3.co;2-n ◽

1995 ◽

Vol 3 (2) ◽

pp. 70-75 ◽

Cited By ~ 15

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

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Two-Photon Excitation Imaging of Glucose Metabolism in Living Tissue

Microscopy and Microanalysis ◽

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

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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