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Journal ArticleDOI

Two-photon imaging to a depth of 1000 µm microm in living brains by use of a Ti:Al2O3 regenerative amplifier

15 Jun 2003-Optics Letters (Optical Society of America)-Vol. 28, Iss: 12, pp 1022-1024
TL;DR: It is shown that two-photon fluorescence images can be obtained throughout almost the entire gray matter of the mouse neocortex by using optically amplified femtosecond pulses.
Abstract: It is shown that two-photon fluorescence images can be obtained throughout almost the entire gray matter of the mouse neocortex by using optically amplified femtosecond pulses The achieved imaging depth approaches the theoretical limit set by excitation of out-of-focus fluorescence
Citations
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Journal ArticleDOI
TL;DR: Fundamental concepts of nonlinear microscopy are reviewed and conditions relevant for achieving large imaging depths in intact tissue are discussed.
Abstract: With few exceptions biological tissues strongly scatter light, making high-resolution deep imaging impossible for traditional⎯including confocal⎯fluorescence microscopy. Nonlinear optical microscopy, in particular two photon–excited fluorescence microscopy, has overcome this limitation, providing large depth penetration mainly because even multiply scattered signal photons can be assigned to their origin as the result of localized nonlinear signal generation. Two-photon microscopy thus allows cellular imaging several hundred microns deep in various organs of living animals. Here we review fundamental concepts of nonlinear microscopy and discuss conditions relevant for achieving large imaging depths in intact tissue.

3,781 citations


Cites background from "Two-photon imaging to a depth of 10..."

  • ...can now be examined in detail at depths of up to one millimete...

    [...]

Journal ArticleDOI
TL;DR: Multiphoton microscopy has found a niche in the world of biological imaging as the best noninvasive means of fluorescence microscopy in tissue explants and living animals and its use is now increasing exponentially.
Abstract: Multiphoton microscopy (MPM) has found a niche in the world of biological imaging as the best noninvasive means of fluorescence microscopy in tissue explants and living animals. Coupled with transgenic mouse models of disease and 'smart' genetically encoded fluorescent indicators, its use is now increasing exponentially. Properly applied, it is capable of measuring calcium transients 500 microm deep in a mouse brain, or quantifying blood flow by imaging shadows of blood cells as they race through capillaries. With the multitude of possibilities afforded by variations of nonlinear optics and localized photochemistry, it is possible to image collagen fibrils directly within tissue through nonlinear scattering, or release caged compounds in sub-femtoliter volumes.

3,738 citations

Journal ArticleDOI
TL;DR: Non-invasive, high-resolution, in vivo imaging of subcortical structures (the external capsule and hippocampus) within an intact mouse brain is demonstrated using three-photon fluorescence microscopy at the new spectral window of 1700 nm.
Abstract: Two-photon fluorescence microscopy (2PM)1 enables scientists in various fields including neuroscience2,3, embryology4, and oncology5 to visualize in vivo and ex vivo tissue morphology and physiology at a cellular level deep within scattering tissue. However, tissue scattering limits the maximum imaging depth of 2PM within the mouse brain to the cortical layer, and imaging subcortical structures currently requires the removal of overlying brain tissue3 or the insertion of optical probes6,7. Here we demonstrate non-invasive, high resolution, in vivo imaging of subcortical structures within an intact mouse brain using three-photon fluorescence microscopy (3PM) at a spectral excitation window of 1,700 nm. Vascular structures as well as red fluorescent protein (RFP)-labeled neurons within the mouse hippocampus are imaged. The combination of the long excitation wavelength and the higher order nonlinear excitation overcomes the limitations of 2PM, enabling biological investigations to take place at greater depth within tissue.

1,182 citations


Additional excerpts

  • ...< FF(tt)2PP >= 8CC2nn0 <PP>2 ππππ ee −2zz llee , (1) while that of 3PM is(3) < FF(tt)3PP >= 3....

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Journal ArticleDOI
08 Mar 2012-Neuron
TL;DR: This Primer briefly reviews the general mechanisms of neuronal calcium signaling, and introduces the calcium imaging devices, including confocal and two-photon microscopy as well as miniaturized devices used in freely moving animals.

1,113 citations


Cites background from "Two-photon imaging to a depth of 10..."

  • ...Even when using two-photon microscopy combined with improved depth penetration, imaging depth is ultimately limited (Andresen et al., 2009; Theer et al., 2003)....

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References
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Journal ArticleDOI
06 Apr 1990-Science
TL;DR: The fluorescence emission increased quadratically with the excitation intensity so that fluorescence and photo-bleaching were confined to the vicinity of the focal plane as expected for cooperative two-photon excitation.
Abstract: Molecular excitation by the simultaneous absorption of two photons provides intrinsic three-dimensional resolution in laser scanning fluorescence microscopy. The excitation of fluorophores having single-photon absorption in the ultraviolet with a stream of strongly focused subpicosecond pulses of red laser light has made possible fluorescence images of living cells and other microscopic objects. The fluorescence emission increased quadratically with the excitation intensity so that fluorescence and photo-bleaching were confined to the vicinity of the focal plane as expected for cooperative two-photon excitation. This technique also provides unprecedented capabilities for three-dimensional, spatially resolved photochemistry, particularly photolytic release of caged effector molecules.

8,905 citations

Journal ArticleDOI
TL;DR: In this article, the authors used two-photon laser scanning microscopy to image the motion of red blood cells (RBCs) in individual capillaries that lie as far as 600 μm below the pia mater of primary somatosensory cortex in rat.
Abstract: Cortical blood flow at the level of individual capillaries and the coupling of neuronal activity to flow in capillaries are fundamental aspects of homeostasis in the normal and the diseased brain. To probe the dynamics of blood flow at this level, we used two-photon laser scanning microscopy to image the motion of red blood cells (RBCs) in individual capillaries that lie as far as 600 μm below the pia mater of primary somatosensory cortex in rat; this depth encompassed the cortical layers with the highest density of neurons and capillaries. We observed that the flow was quite variable and exhibited temporal fluctuations around 0.1 Hz, as well as prolonged stalls and occasional reversals of direction. On average, the speed and flux (cells per unit time) of RBCs covaried linearly at low values of flux, with a linear density of ≈70 cells per mm, followed by a tendency for the speed to plateau at high values of flux. Thus, both the average velocity and density of RBCs are greater at high values of flux than at low values. Time-locked changes in flow, localized to the appropriate anatomical region of somatosensory cortex, were observed in response to stimulation of either multiple vibrissae or the hindlimb. Although we were able to detect stimulus-induced changes in the flux and speed of RBCs in some single trials, the amplitude of the stimulus-evoked changes in flow were largely masked by basal fluctuations. On average, the flux and the speed of RBCs increased transiently on stimulation, although the linear density of RBCs decreased slightly. These findings are consistent with a stimulus-induced decrease in capillary resistance to flow.

779 citations

Journal Article
TL;DR: Two-photon laser scanning microscopy is used to image the motion of red blood cells in individual capillaries that lie as far as 600 micrometers below the pia mater of primary somatosensory cortex in rat, finding both the average velocity and density of RBCs are greater at high values of flux than at low values.
Abstract: Cortical blood flow at the level of individual capillaries and the coupling of neuronal activity to flow in capillaries are fundamental aspects of homeostasis in the normal and the diseased brain. To probe the dynamics of blood flow at this level, we used two-photon laser scanning microscopy to image the motion of red blood cells (RBCs) in individual capillaries that lie as far as 600 μm below the pia mater of primary somatosensory cortex in rat; this depth encompassed the cortical layers with the highest density of neurons and capillaries. We observed that the flow was quite variable and exhibited temporal fluctuations around 0.1 Hz, as well as prolonged stalls and occasional reversals of direction. On average, the speed and flux (cells per unit time) of RBCs covaried linearly at low values of flux, with a linear density of ≈70 cells per mm, followed by a tendency for the speed to plateau at high values of flux. Thus, both the average velocity and density of RBCs are greater at high values of flux than at low values. Time-locked changes in flow, localized to the appropriate anatomical region of somatosensory cortex, were observed in response to stimulation of either multiple vibrissae or the hindlimb. Although we were able to detect stimulus-induced changes in the flux and speed of RBCs in some single trials, the amplitude of the stimulus-evoked changes in flow were largely masked by basal fluctuations. On average, the flux and the speed of RBCs increased transiently on stimulation, although the linear density of RBCs decreased slightly. These findings are consistent with a stimulus-induced decrease in capillary resistance to flow.

698 citations

Journal ArticleDOI
TL;DR: This study studied this kind of damage in bovine adrenal chromaffin cells, using two different indicators of damage: changes in resting [Ca(2+)] level and the degranulation reaction, and found that damage is proportional to the integral (over space and time) of light intensity raised to a power approximately 2.5.

454 citations

Journal ArticleDOI
TL;DR: A range of imaging modes based on 2-photon laser scanning microscopy (TPLSM) are shown as applicable to problems in neuroscience, showing how the distribution of neurotransmitter receptors on the surface of specific cells can be mapped.

431 citations