Supraresolution Imaging in Brain Slices using Stimulated-Emission Depletion Two-Photon Laser Scanning Microscopy

Ding, Jun; Takasaki, Kevin T.; Sabatini, Bernardo L.

doi:10.1016/j.neuron.2009.07.011

Cited by 162 publications

(133 citation statements)

References 34 publications

Supporting

Mentioning

129

Contrasting

Unclassified

Order By: Relevance

“…Indeed, with adaptive optics we can now image to >400 μm deep into the brain with resolution similar to that normally seen at the superficial depth in brain slices. As a result, adaptive optics would play an important role in the application of many physiological techniques in vivo, such as two-photon uncaging (19), two-photon imaging of dendrites, spines, and axons (20), multiphoton ablation of neuronal structures (21), optogenetic stimulation (22,23), and superresolution imaging (24).…”

Section: Resultsmentioning

confidence: 99%

Characterization and adaptive optical correction of aberrations during in vivo imaging in the mouse cortex

Satō

Betzig

2011

Proc. Natl. Acad. Sci. U.S.A.

178

158

View full text Add to dashboard Cite

The signal and resolution during in vivo imaging of the mouse brain is limited by sample-induced optical aberrations. We find that, although the optical aberrations can vary across the sample and increase in magnitude with depth, they remain stable for hours. As a result, two-photon adaptive optics can recover diffraction-limited performance to depths of 450 μm and improve imaging quality over fields of view of hundreds of microns. Adaptive optical correction yielded fivefold signal enhancement for small neuronal structures and a threefold increase in axial resolution. The corrections allowed us to detect smaller neuronal structures at greater contrast and also improve the signal-to-noise ratio during functional Ca 2 imaging in single neurons.T he ability to visualize biological systems in vivo has been a major attraction of optical microscopy, because studying biological systems as they evolve in their natural, physiological state provides relevant information that in vitro preparations often do not allow (1). However, for conventional optical microscopes to achieve their optimal, diffraction-limited resolution, the specimen needs to have identical optical properties to those of the immersion media for which the microscope objective is designed. For example, one of the most widely applied microscopy techniques for in vivo imaging, two-photon fluorescence microscopy, often uses water-dipping objectives. Because biological samples are comprised of structures (i.e., proteins, nuclear acids, and lipids) with refractive indices different from that of water, they induce optical aberrations to the incoming excitation wave and result in an enlarged focal spot within the sample and a concomitant deterioration of signal and resolution (2, 3). As a result, the resolution and contrast of optical microscopes is compromised in vivo, especially deep in tissue.Many questions related to how the brain processes information on both the neuronal circuit level and the cell biological level can be addressed by observing the morphology and activity of neurons inside a living and, preferably, awake and behaving mouse (1). In a typical experiment, an area of the skull is surgically removed and replaced with a cover glass to provide optical access to the underlying structure of interest (4). For imaging during behavior, the cover glass is often attached to an optically transparent plug embedded in the skull to improve mechanical stability and to prevent the skull from growing back and blocking the optical access (5, 6) (Fig. 1A). Before the excitation light of a two-photon microscope reaches the desired focal plane inside the brain, it has to traverse first the cranial window and then the brain tissue, both with optical properties different from water. Thus, they both impart optical aberrations on the excitation light, which leads to a distorted focus, even at the surface of the brain.These sample-induced aberrations can be corrected with adaptive optics (AO) to recover diffraction-limited resolution. In AO, a wavefront-shaping device m...

show abstract

Section: Resultsmentioning

confidence: 99%

Characterization and adaptive optical correction of aberrations during in vivo imaging in the mouse cortex

Satō

Betzig

2011

Proc. Natl. Acad. Sci. U.S.A.

178

158

View full text Add to dashboard Cite

show abstract

“…The STED methodology has successfully been used to monitor the fine structure of dendritic spines (Bethge et al, 2013; Ding et al, 2009; Tonnesen et al, 2014, 2011), and it has recently been applied to image astroglia (Panatier et al, 2014) (Fig. 5A).…”

Section: Monitoring Of Astroglia On the Nanoscale: Emerging Techniquesmentioning

confidence: 99%

Morphological plasticity of astroglia: Understanding synaptic microenvironment

Heller

Rusakov

2015

Glia

130

137

View full text Add to dashboard Cite

Memory formation in the brain is thought to rely on the remodeling of synaptic connections which eventually results in neural network rewiring. This remodeling is likely to involve ultrathin astroglial protrusions which often occur in the immediate vicinity of excitatory synapses. The phenomenology, cellular mechanisms, and causal relationships of such astroglial restructuring remain, however, poorly understood. This is in large part because monitoring and probing of the underpinning molecular machinery on the scale of nanoscopic astroglial compartments remains a challenge. Here we briefly summarize the current knowledge regarding the cellular organisation of astroglia in the synaptic microenvironment and discuss molecular mechanisms potentially involved in use‐dependent astroglial morphogenesis. We also discuss recent observations concerning morphological astroglial plasticity, the respective monitoring methods, and some of the newly emerging techniques that might help with conceptual advances in the area. GLIA 2015;63:2133–2151

show abstract

“…For this reason, several groups have extended STED microscopy to two-photon excitation using different optical systems and modalities, allowing imaging deeper into e.g. brain tissue slices [15,16,17,18].…”

Section: Major Technological Progress Since 2007mentioning

confidence: 99%

STED microscopy—towards broadened use and scope of applications

Blom

Widengren

2014

Current Opinion in Chemical Biology

View full text Add to dashboard Cite

PostprintThis is the accepted version of a paper published in Current opinion in chemical biology. This paper has been peer-reviewed but does not include the final publisher proof-corrections or journal pagination.Citation for the original published paper (version of record):Blom, H., Widengren, J. (2014) STED microscopy -towards broadened use and scope of applications. Current opinion in chemical biology AbstractStimulated Emission Depletion (STED) microscopy has been convincingly demonstrated for fundamental studies in cells, living tissue and organisms. Today, a major trend in the STED technique development is to make the instruments simpler and more user-friendly, without compromising performance. This has become possible by new low-cost, turnkey laser technology and by implementing specifically designed phase plates and birefringent segmented polarization elements. With these simpler and cheaper realizations of STED now becoming more broadly available, and with a parallel development of sample preparation and fluorophore reporter molecules ultimately setting the limit of the image quality, contrast and resolution, we can expect a significant increase in the use of STED, in science as well as for clinical and drug development purposes. Highlights-STED imaging of cells, living tissue and organisms has been convincingly demonstrated -The STED technology has in the last 6-7 years become far easier to realize -This largely relies on new laser technology and customized optical elements -New fluorescent markers and sample preparation procedures support the development -By increased accessibility, a strong increase in the use of STED can be expected

show abstract

Supraresolution Imaging in Brain Slices using Stimulated-Emission Depletion Two-Photon Laser Scanning Microscopy

Cited by 162 publications

References 34 publications

Characterization and adaptive optical correction of aberrations during in vivo imaging in the mouse cortex

Characterization and adaptive optical correction of aberrations during in vivo imaging in the mouse cortex

Morphological plasticity of astroglia: Understanding synaptic microenvironment

STED microscopy—towards broadened use and scope of applications

Contact Info

Product

Resources

About