scholarly journals Brain heating induced by near infrared lasers during multi-photon microscopy

2016 ◽  
Author(s):  
Kaspar Podgorski ◽  
Gayathri Ranganathan
Keyword(s):  
High Power ◽  
Heat Dissipation ◽  
Near Infrared ◽  
Total Power ◽  
Continuous Illumination ◽  
Two Photon Microscopy ◽  
Two Photon ◽  
Cranial Window ◽  
Shock Proteins ◽  
The Brain

AbstractTwo-photon imaging and optogenetic stimulation rely on high illumination powers, particularly for state-of-the-art applications that target deeper structures, achieve faster measurements, or probe larger brain areas. However, little information is available on heating and resulting damage induced by high-power illumination in the brain. Here we used thermocouple probes and quantum dot nanothermometers to measure temperature changes induced by two-photon microscopy in the neocortex of awake and anaesthetized mice. We characterized heating as a function of wavelength, exposure time, and distance from the center of illumination. Although total power is highest near the surface of the brain, heating was most severe hundreds of microns below the focal plane, due to heat dissipation through the cranial window. Continuous illumination of a 1mm2area produced a peak temperature increase of approximately 1.8°C/100mW. Continuous illumination with powers above 250 mW induced lasting damage, detected with immunohistochemistry against Iba1, GFAP, heat shock proteins, and activated Caspase-3. Higher powers were usable in experiments with limited duty ratios, suggesting an approach to mitigate damage in high-power microscopy experiments.

scholarly journals Brain heating induced by near-infrared lasers during multiphoton microscopy

2016 ◽  
Vol 116 (3) ◽  
pp. 1012-1023 ◽  
Author(s):  
Kaspar Podgorski ◽  
Gayathri Ranganathan
Keyword(s):  
High Power ◽  
Heat Dissipation ◽  
Near Infrared ◽  
Multiphoton Microscopy ◽  
Total Power ◽  
Continuous Illumination ◽  
Two Photon ◽  
Cranial Window ◽  
Shock Proteins ◽  
The Brain

Two-photon imaging and optogenetic stimulation rely on high illumination powers, particularly for state-of-the-art applications that target deeper structures, achieve faster measurements, or probe larger brain areas. However, little information is available on heating and resulting damage induced by high-power illumination in the brain. In the current study we used thermocouple probes and quantum dot nanothermometers to measure temperature changes induced by two-photon microscopy in the neocortex of awake and anaesthetized mice. We characterized heating as a function of wavelength, exposure time, and distance from the center of illumination. Although total power is highest near the surface of the brain, heating was most severe hundreds of micrometers below the focal plane, due to heat dissipation through the cranial window. Continuous illumination of a 1-mm2 area produced a peak temperature increase of ∼1.8°C/100 mW. Continuous illumination with powers above 250 mW induced lasting damage, detected with immunohistochemistry against Iba1, glial fibrillary acidic protein, heat shock proteins, and activated caspase-3. Higher powers were usable in experiments with limited duty ratios, suggesting an approach to mitigate damage in high-power microscopy experiments.

scholarly journals Two-photon imaging induces brain heating and calcium microdomain hyperactivity in cortical astrocytes

2018 ◽  
Author(s):  
Elke Schmidt ◽  
Martin Oheim
Keyword(s):  
Laser Power ◽  
Near Infrared ◽  
Continuous Wave ◽  
Average Power ◽  
Infrared Light ◽  
Near Infrared Light ◽  
Two Photon Microscopy ◽  
Two Photon ◽  
Cortical Astrocytes ◽  
The Brain

ABSTRACTUnraveling how neural networks process and represent sensory information and how this cellular dynamics instructs behavioral output is a main goal in current neuroscience. Two-photon activation of optogenetic actuators and fluorescence calcium (Ca2+) imaging with genetically encoded Ca2+ indicators allow, respectively, the all-optical stimulation and readout of activity from genetically identified cell populations. However, these techniques expose the brain to high near-infrared light doses raising the concern of light-induced adverse effects on the biological phenomena being studied. Combing Ca2+ imaging of GCaMP6f-expressing cortical astrocytes as a sensitive readout for photodamage and an unbiased machine-based event detection, we demonstrate the subtle build-up of aberrant microdomain Ca2+ signals in fine astroglial processes. Illumination conditions routinely being used in biological two-photon microscopy (920-nm excitation, 100-fs regime, ten mW average power) increased the frequency of microdomain Ca2+ events, but left their amplitude, area and duration rather unchanged. This increase in local Ca2+ activity was followed by Ca2+ transients in the otherwise silent soma. Ca2+ hyperactivity occurred without overt morphological damage. Surprisingly, at the same average power, continuous-wave 920-nm illumination was as damaging as fs pulses, indicating a linear, heating-mediated (rather than a highly non-linear) damage mechanism. In an astrocyte-specific IP3-receptor knock-out mouse (IP3R2-KO), Near-infrared light-induced Ca2+ microdomains signals persisted in the small processes, underpinning their resemblance to physiological IP3R2-independent Ca2+ signals, while somatic activity was abolished. Contrary to what has generally been believed in the field, shorter pulses and lower average power are advantageous to alleviate photodamage and allow for longer useful recording windows.SIGNIFICANCE STATEMENTImaging the fine structure and function of the brain has become possible with two-photon microscopy that uses ultrashort-pulsed infrared laser light for better tissue penetration. The high peak energy of these light pulses has raised concerns about photodamage resulting from multi-photon processes. Here, we show that the time-averaged rather than the peak laser power matters. At wavelengths and with laser powers now commonly used in neuroscience brain damage occurs as a consequence of direct infrared light absorption, i.e., heating. To counteract brain heating we explore a strategy that uses even shorter, more energetic pulses but a lower time-averaged laser power to produce the same image quality while making two-photon microscopy less invasive.

scholarly journals P02.08 Visualizing tumor cell - lymphocyte interactions in the brain metastatic cascade using in vivo two photon microscopy

2018 ◽  
Vol 20 (suppl_3) ◽  
pp. iii273-iii273
Author(s):  
M Piechutta ◽  
A S Berghoff ◽  
M A Karreman ◽  
K Gunkel ◽  
W Wick ◽  
...  
Keyword(s):  
Tumor Cell ◽  
Metastatic Cascade ◽  
Two Photon Microscopy ◽  
Two Photon ◽  
The Brain

scholarly journals IMMU-26. VISUALIZING TUMOR CELL - LYMPHOCYTE INTERACTIONS IN THE BRAIN METASTATIC CASCADE USING IN VIVO TWO PHOTON MICROSCOPY

2018 ◽  
Vol 20 (suppl_6) ◽  
pp. vi126-vi127
Author(s):  
Manuel Piechutta ◽  
Anna Berghoff ◽  
Matthia Karreman ◽  
Katharina Gunkel ◽  
Wolfgang Wick ◽  
...  
Keyword(s):  
Tumor Cell ◽  
Metastatic Cascade ◽  
Two Photon Microscopy ◽  
Two Photon ◽  
The Brain

scholarly journals Two-Photon Microscopy as a Tool to Study Blood Flow and Neurovascular Coupling in the Rodent Brain

2012 ◽  
Vol 32 (7) ◽  
pp. 1277-1309 ◽  
Author(s):  
Andy Y Shih ◽  
Jonathan D Driscoll ◽  
Patrick J Drew ◽  
Nozomi Nishimura ◽  
Chris B Schaffer ◽  
...  
Keyword(s):  
Blood Flow ◽  
Laser Scanning ◽  
Vascular System ◽  
Neurovascular Coupling ◽  
Laser Scanning Microscopy ◽  
Small Scale ◽  
Two Photon Microscopy ◽  
Two Photon ◽  
Rats And Mice ◽  
The Brain

The cerebral vascular system services the constant demand for energy during neuronal activity in the brain. Attempts to delineate the logic of neurovascular coupling have been greatly aided by the advent of two-photon laser scanning microscopy to image both blood flow and the activity of individual cells below the surface of the brain. Here we provide a technical guide to imaging cerebral blood flow in rodents. We describe in detail the surgical procedures required to generate cranial windows for optical access to the cortex of both rats and mice and the use of two-photon microscopy to accurately measure blood flow in individual cortical vessels concurrent with local cellular activity. We further provide examples on how these techniques can be applied to the study of local blood flow regulation and vascular pathologies such as small-scale stroke.

scholarly journals Post-capillary venules is the locus for transcytosis of therapeutic nanoparticles to the brain

2020 ◽  
Author(s):  
Krzysztof Kucharz ◽  
Kasper Kristensen ◽  
Kasper Bendix Johnsen ◽  
Mette Aagaard Lund ◽  
Micael Lønstrup ◽  
...  
Keyword(s):  
Drug Carrier ◽  
Basement Membranes ◽  
List Type ◽  
Biologic Drugs ◽  
Large Molecules ◽  
Two Photon Microscopy ◽  
Two Photon ◽  
Resolution Imaging ◽  
The Brain

SUMMARYTreatments of neurodegenerative diseases require biologic drugs to be actively transported across the blood-brain barrier (BBB). To answer outstanding questions regarding transport mechanisms, we determined how and where transcytosis occurs at the BBB. Using two-photon microscopy, we characterized the transport of therapeutic nanoparticles at all steps of delivery to the brain and at the nanoscale resolution in vivo. Transferrin receptor-targeted nanoparticles were taken up by endothelium at capillaries and venules, but not at arterioles. The nanoparticles moved unobstructed within endothelial cells, but transcytosis across the BBB occurred only at post-capillary venules, where endothelial and glial basement membranes form a perivascular space that can accommodate biologics. In comparison, transcytosis was absent in capillaries with closely apposed basement membranes. Thus, post-capillary venules, not capillaries, provide an entry point for transport of large molecules across the BBB, and targeting therapeutic agents to this locus may be an effective way for treating brain disorders.HIGHLIGHTSIntegration of drug carrier nanotechnology with two-photon microscopy in vivoReal-time nanoscale-resolution imaging of nanoparticle transcytosis to the brainDistinct trafficking pattern in the endothelium of cerebral venules and capillariesVenules, not capillaries, is the locus for brain uptake of therapeutic nanoparticles

scholarly journals Dual Near Infrared Two-Photon Microscopy for Deep-Tissue Dopamine Nanosensor Imaging

2017 ◽  
Author(s):  
Jackson T. Del Bonis-O’Donnell ◽  
Ralph H. Page ◽  
Abraham G. Beyene ◽  
Eric G. Tindall ◽  
Ian McFarlane ◽  
...  
Keyword(s):  
Near Infrared ◽  
Photon Absorption ◽  
Deep Tissue ◽  
Biologically Relevant ◽  
Two Photon Microscopy ◽  
Two Photon ◽  
Tissue Phantom ◽  
Photon Excitation ◽  
Photon Absorption And Scattering ◽  
Two Photon Excitation

A key limitation for achieving deep imaging in biological structures lies in photon absorption and scattering leading to attenuation of fluorescence. In particular, neurotransmitter imaging is challenging in the biologically-relevant context of the intact brain, for which photons must traverse the cranium, skin and bone. Thus, fluorescence imaging is limited to the surface cortical layers of the brain, only achievable with craniotomy. Herein, we describe optimal excitation and emission wavelengths for through-cranium imaging, and demonstrate that near-infrared emissive nanosensors can be photoexcited using a two-photon 1560 nm excitation source. Dopamine-sensitive nanosensors can undergo two-photon excitation, and provide chirality-dependent responses selective for dopamine with fluorescent turn-on responses varying between 20% and 350%. We further calculate the two-photon absorption cross-section and quantum yield of dopamine nanosensors, and confirm a two-photon power law relationship for the nanosensor excitation process. Finally, we show improved image quality of the nanosensors embedded 2 mm deep into a brain-mimetic tissue phantom, whereby one-photon excitation yields 42% scattering, in contrast to 4% scattering when the same object is imaged under two-photon excitation. Our approach overcomes traditional limitations in deep-tissue fluorescence microscopy, and can enable neurotransmitter imaging in the biologically-relevant milieu of the intact and living brain.

scholarly journals Lanthanide nano-drums: a new class of molecular nanoparticles for potential biomedical applications

2014 ◽  
Vol 175 ◽  
pp. 241-255 ◽  
Author(s):  
Richard A. Jones ◽  
Annie J. Gnanam ◽  
Jonathan F. Arambula ◽  
Jessica N. Jones ◽  
Jagannath Swaminathan ◽  
...  
Keyword(s):  
Total Internal Reflection ◽  
Biomedical Applications ◽  
Near Infrared ◽  
Photophysical Properties ◽  
Diagnostic Systems ◽  
Two Photon Microscopy ◽  
New Class ◽  
Two Photon ◽  
Self Assembling ◽  
Molecular Nanoparticles

We are developing a new class of lanthanide-based self-assembling molecular nanoparticles as potential reporter molecules for imaging, and as multi-functional nanoprobes or nanosensors in diagnostic systems. These lanthanide “nano-drums” are homogeneous 4d–4f clusters approximately 25 to 30 Å in diameter that can emit from the visible to near-infrared (NIR) wavelengths. Here, we present syntheses, crystal structures, photophysical properties, and comparative cytotoxicity data for six nano-drums containing either Eu, Tb, Lu, Er, Yb or Ho. Imaging capabilities of these nano-drums are demonstrated using epifluorescence, total internal reflection fluorescence (TIRF), and two-photon microscopy. We discuss how these molecular nanoparticles can to be adapted for a range of assays, particularly by taking advantage of functionalization strategies with chemical moieties to enable conjugation to protein or nucleic acids.

scholarly journals Role of Pial Microvasospasms and Leukocyte Plugging for Parenchymal Perfusion after Subarachnoid Hemorrhage Assessed by In Vivo Multi-Photon Microscopy

2021 ◽  
Vol 22 (16) ◽  
pp. 8444
Author(s):  
Julian Schwarting ◽  
Kathrin Nehrkorn ◽  
Hanhan Liu ◽  
Nikolaus Plesnila ◽  
Nicole Angela Terpolilli
Keyword(s):  
Subarachnoid Hemorrhage ◽  
Cerebral Ischemia ◽  
Fluorescent Dyes ◽  
Delayed Cerebral Ischemia ◽  
Tissue Imaging ◽  
Two Photon Microscopy ◽  
Two Photon ◽  
Cranial Window ◽  
Pial Arterioles

Subarachnoid hemorrhage (SAH) is associated with acute and delayed cerebral ischemia. We suggested spasms of pial arterioles as a possible mechanism; however, it remained unclear whether and how pial microvasospasms (MVSs) induce cerebral ischemia. Therefore, we used in vivo deep tissue imaging by two-photon microscopy to investigate MVSs together with the intraparenchymal microcirculation in a clinically relevant murine SAH model. Male C57BL/6 mice received a cranial window. Cerebral vessels and leukocytes were labelled with fluorescent dyes and imaged by in vivo two-photon microscopy before and three hours after SAH induced by filament perforation. After SAH, a large clot formed around the perforation site at the skull base, and blood distributed along the perivascular space of the middle cerebral artery up to the cerebral cortex. Comparing the cerebral microvasculature before and after SAH, we identified three different patterns of constrictions: pearl string, global, and bottleneck. At the same time, the volume of perfused intraparenchymal vessels and blood flow velocity in individual arterioles were significantly reduced by more than 60%. Plugging of capillaries by leukocytes was observed but infrequent. The current study demonstrates that perivascular blood is associated with spasms of pial arterioles and that these spasms result in a significant reduction in cortical perfusion after SAH. Thus, the pial microvasospasm seems to be an important mechanism by which blood in the subarachnoid space triggers cerebral ischemia after SAH. Identifying the mechanisms of pial vasospasm may therefore result in novel therapeutic options for SAH patients.

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