scholarly journals In vivo two-photon excited fluorescence microscopy reveals cardiac- and respiration-dependent pulsatile blood flow in cortical blood vessels in mice

2012 ◽  
Vol 302 (7) ◽  
pp. H1367-H1377 ◽  
Author(s):  
Thom P. Santisakultarm ◽  
Nathan R. Cornelius ◽  
Nozomi Nishimura ◽  
Andrew I. Schafer ◽  
Richard T. Silver ◽  
...  
Keyword(s):  
Blood Flow ◽  
Fluorescence Microscopy ◽  
Capillary Tube ◽  
Three Dimensional ◽  
Vessel Diameter ◽  
Vascular Network ◽  
Maximum Speed ◽  
Flow Profile ◽  
Two Photon ◽  
Surface Vessels

Subtle alterations in cerebral blood flow can impact the health and function of brain cells and are linked to cognitive decline and dementia. To understand hemodynamics in the three-dimensional vascular network of the cerebral cortex, we applied two-photon excited fluorescence microscopy to measure the motion of red blood cells (RBCs) in individual microvessels throughout the vascular hierarchy in anesthetized mice. To resolve heartbeat- and respiration-dependent flow dynamics, we simultaneously recorded the electrocardiogram and respiratory waveform. We found that centerline RBC speed decreased with decreasing vessel diameter in arterioles, slowed further through the capillary bed, and then increased with increasing vessel diameter in venules. RBC flow was pulsatile in nearly all cortical vessels, including capillaries and venules. Heartbeat-induced speed modulation decreased through the vascular network, while the delay between heartbeat and the time of maximum speed increased. Capillary tube hematocrit was 0.21 and did not vary with centerline RBC speed or topological position. Spatial RBC flow profiles in surface vessels were blunted compared with a parabola and could be measured at vascular junctions. Finally, we observed a transient decrease in RBC speed in surface vessels before inspiration. In conclusion, we developed an approach to study detailed characteristics of RBC flow in the three-dimensional cortical vasculature, including quantification of fluctuations in centerline RBC speed due to cardiac and respiratory rhythms and flow profile measurements. These methods and the quantitative data on basal cerebral hemodynamics open the door to studies of the normal and diseased-state cerebral microcirculation.

scholarly journals High-speed volumetric two-photon fluorescence imaging of neurovascular dynamics

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Jiang Lan Fan ◽  
Jose A. Rivera ◽  
Wei Sun ◽  
John Peterson ◽  
Henry Haeberle ◽  
...  
Keyword(s):  
Blood Flow ◽  
High Speed ◽  
Laser Scanning ◽  
Three Dimensional ◽  
Laser Scanning Microscopy ◽  
Fluorescence Signal ◽  
Imaging Method ◽  
Spatiotemporal Resolution ◽  
Two Photon

AbstractUnderstanding the structure and function of vasculature in the brain requires us to monitor distributed hemodynamics at high spatial and temporal resolution in three-dimensional (3D) volumes in vivo. Currently, a volumetric vasculature imaging method with sub-capillary spatial resolution and blood flow-resolving speed is lacking. Here, using two-photon laser scanning microscopy (TPLSM) with an axially extended Bessel focus, we capture volumetric hemodynamics in the awake mouse brain at a spatiotemporal resolution sufficient for measuring capillary size and blood flow. With Bessel TPLSM, the fluorescence signal of a vessel becomes proportional to its size, which enables convenient intensity-based analysis of vessel dilation and constriction dynamics in large volumes. We observe entrainment of vasodilation and vasoconstriction with pupil diameter and measure 3D blood flow at 99 volumes/second. Demonstrating high-throughput monitoring of hemodynamics in the awake brain, we expect Bessel TPLSM to make broad impacts on neurovasculature research.

scholarly journals Network-enabled efficient image restoration for 3D microscopy of turbid biological specimens

2020 ◽  
Author(s):  
Le Xiao ◽  
Chunyu Fang ◽  
Yarong Wang ◽  
Tingting Yu ◽  
Yuxuan Zhao ◽  
...  
Keyword(s):  
Fluorescence Microscopy ◽  
Image Restoration ◽  
Signal To Noise Ratio ◽  
Three Dimensional ◽  
Science Research ◽  
Light Sheet ◽  
Cell Nuclei ◽  
Two Photon ◽  
Photon Excitation ◽  
3D Fluorescence Microscopy

AbstractThough three-dimensional (3D) fluorescence microscopy has been an essential tool for modern life science research, the light scattering by biological specimens fundamentally prevents its more widespread applications in live imaging. We hereby report a deep-learning approach, termed ScatNet, that enables reversion of 3D fluorescence microscopy from high-resolution targets to low-quality, light-scattered measurements, thereby allowing restoration for a single blurred and light-scattered 3D image of deep tissue, with achieving improved resolution and signal-to-noise ratio. Our approach can computationally extend the imaging depth for current 3D fluorescence microscopes, without the addition of complicated optics. Combining ScatNet approach with cutting-edge light-sheet fluorescence microscopy, we demonstrate that the image restoration of cell nuclei in the deep layer of live Drosophila melanogaster embryos at single-cell resolution. Applying our approach to two-photon excitation microscopy, we could improve the signal and resolution of neurons in mouse brain beyond the photon ballistic region.

Two-Photon Laser Scanning Confocal Microscopy

1997 ◽  
Vol 3 (S2) ◽  
pp. 847-848
Author(s):  
P.C. Cheng ◽  
S.J. Pan ◽  
A. Shih ◽  
W.S. Liou ◽  
M.S. Park ◽  
...  
Keyword(s):  
Fluorescence Microscopy ◽  
Laser Scanning ◽  
Three Dimensional ◽  
Laser Scanning Confocal Microscopy ◽  
Linear Absorption ◽  
Excitation Light ◽  
Excitation Beam ◽  
Scanning Confocal Microscopy ◽  
Two Photon ◽  
Photobleaching Recovery

Two-photon fluorescence microscopy has become an important research tool in both biological and material sciences. The technique uses long wavelength, typically in the near IR, as the excitation light to obtain shorter wavelength fluorescence (e.g. visible light). Because of the low linear absorption coefficient of most biological and polymeric specimens, this technique allows deeper penetration of the excitation beam, achieving optical sectioning to a depth of 250μm or more into the specimen. As a result of the quadratic dependency of the two-photon induced fluorescence to the excitation intensity, the fluorescent emission and photobleaching are limited to the vicinity of focal spot. This capability of addressing a specimen’s 3D space allows exciting possibilities in biological researches, such as 3D photobleaching recovery experiment.Two-photon confocal fluorescence microscopy is ideal for the study of thick biological and material specimen in 3D. For example, Figure 1 shows a three-dimensional isosurface rendered image of a vascular bundle from a maize stem.

Two-photon fluorescence microscopy of collateral blood flow following photothrombotic stroke in rat neocortex

2003 ◽  
Author(s):  
Chris B. Schaffer ◽  
Ford F. Ebner ◽  
Nozomi Nishimura ◽  
Beth Friedman ◽  
Philbert S. Tsai ◽  
...  
Keyword(s):  
Blood Flow ◽  
Fluorescence Microscopy ◽  
Collateral Blood Flow ◽  
Two Photon ◽  
Rat Neocortex ◽  
Photothrombotic Stroke ◽  
Two Photon Fluorescence ◽  
Photon Fluorescence

scholarly journals Precapillary sphincters and pericytes at first-order capillaries as key regulators for brain capillary perfusion

2021 ◽  
Vol 118 (26) ◽  
pp. e2023749118
Author(s):  
Stefan Andreas Zambach ◽  
Changsi Cai ◽  
Hans Christian Cederberg Helms ◽  
Bjørn Olav Hald ◽  
Yiqiu Dong ◽  
...  
Keyword(s):  
Blood Flow ◽  
Smooth Muscle Actin ◽  
Three Dimensional ◽  
Vessel Diameter ◽  
Synaptic Activity ◽  
Guanosine Monophosphate ◽  
Capillary Perfusion ◽  
First Order ◽  
The Impact

Rises in local neural activity trigger local increases of cerebral blood flow, which is essential to match local energy demands. However, the specific location of microvascular flow control is incompletely understood. Here, we used two-photon microscopy to observe brain microvasculature in vivo. Small spatial movement of a three-dimensional (3D) vasculature makes it challenging to precisely measure vessel diameter at a single x–y plane. To overcome this problem, we carried out four-dimensional (x–y–z–t) imaging of brain microvessels during exposure to vasoactive molecules in order to constrain the impact of brain movements on the recordings. We demonstrate that rises in synaptic activity, acetylcholine, nitric oxide, cyclic guanosine monophosphate, ATP-sensitive potassium channels, and endothelin-1 exert far greater effects on brain precapillary sphincters and first-order capillaries than on penetrating arterioles or downstream capillaries, but with similar kinetics. The high level of responsiveness at precapillary sphincters and first-order capillaries was matched by a higher level of α-smooth muscle actin in pericytes as compared to penetrating arterioles and downstream capillaries. Mathematical modeling based on 3D vasculature reconstruction showed that precapillary sphincters predominantly regulate capillary blood flow and pressure as compared to penetrating arterioles and downstream capillaries. Our results confirm a key role for precapillary sphincters and pericytes on first-order capillaries as sensors and effectors of endothelium- or brain-derived vascular signals.

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.

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