scholarly journals Deep tissue scattering compensation with three-photon F-SHARP

2021 ◽  
Author(s):  
Caroline Berlage ◽  
Malinda L. S. Tantirigama ◽  
Mathias Babot ◽  
Diego Di Battista ◽  
Clarissa Whitmire ◽  
...  
Keyword(s):  
Penetration Depth ◽  
Dendritic Spines ◽  
Imaging Techniques ◽  
Biological Research ◽  
Biological Applications ◽  
Deep Tissue ◽  
Two Photon ◽  
Tissue Scattering ◽  
Compensation Technique ◽  
Wavefront Shaping

Optical imaging techniques are widely used in biological research, but their penetration depth is limited by tissue scattering. Wavefront shaping techniques are able to overcome this problem in principle, but are often slow and their performance depends on the sample. This greatly reduces their practicability for biological applications. Here we present a scattering compensation technique based on three-photon (3P) excitation, which converges faster than comparable two-photon (2P) techniques and works reliably even on densely labeled samples, where 2P approaches fail. To demonstrate its usability and advantages for biomedical imaging we apply it to the imaging of dendritic spines on GFP-labeled layer 5 neurons in an anesthetized mouse.

scholarly journals Automated remote focusing, drift correction, and photostimulation to evaluate structural plasticity in dendritic spines

2016 ◽  
Author(s):  
Michael S Smirnov ◽  
Paul R Evans ◽  
Tavita R Garrett ◽  
Long Yan ◽  
Ryohei Yasuda
Keyword(s):  
Dendritic Spines ◽  
High Throughput Screening ◽  
Imaging Techniques ◽  
Structural Plasticity ◽  
Fold Increase ◽  
Signaling Proteins ◽  
Correction Algorithm ◽  
Drift Correction ◽  
Two Photon

AbstractLong-term structural plasticity of dendritic spines plays a key role in synaptic plasticity, the cellular basis for learning and memory. The biochemical step is mediated by a complex network of signaling proteins in spines. Two-photon imaging techniques combined with two-photon glutamate uncaging allows researchers to induce and quantify structural plasticity in single dendritic spines. However, this method is laborious and slow, making it unsuitable for high throughput screening of factors necessary for structural plasticity. Here we introduce a MATLAB-based module built for Scanimage to automatically track, image, and stimulate multiple dendritic spines. We implemented an electrically tunable lens in combination with a drift correction algorithm to rapidly and continuously track targeted spines and correct sample movements. With a straightforward user interface to design custom multi-position experiments, we were able to adequately image and produce targeted plasticity in multiple dendritic spines using glutamate uncaging. Our methods are inexpensive, open source, and provides up to a five-fold increase in throughput for quantifying structural plasticity of dendritic spines.

scholarly journals Dual GRIN lens two-photon endoscopy for high-speed volumetric and deep brain imaging

2020 ◽  
Author(s):  
Yu-Feng Chien ◽  
Jyun-Yi Lin ◽  
Po-Ting Yeh ◽  
Kuo-Jen Hsu ◽  
Yu-Hsuan Tsai ◽  
...  
Keyword(s):  
Penetration Depth ◽  
High Speed ◽  
Gradient Index ◽  
Spatiotemporal Resolution ◽  
Deep Tissue ◽  
Volumetric Imaging ◽  
Two Photon ◽  
Brain Functions ◽  
Grin Lens ◽  
Deep Brain

AbstractStudying neural connections and activities in vivo is fundamental to understanding brain functions. Given the cm-size brain and three-dimensional neural circuit dynamics, deep-tissue, high-speed volumetric imaging is highly desirable for brain study. With sub-micrometer spatial resolution, intrinsic optical sectioning, and deep-tissue penetration capability, two-photon microscopy (2PM) has found a niche in neuroscience. However, current 2PM typically relies on slow axial scan for volumetric imaging, and the maximal penetration depth is only about 1 mm. Here, we demonstrate that by integrating two gradient-index (GRIN) lenses into 2PM, both penetration depth and volume-imaging rate can be significantly improved. Specifically, an 8-mm long GRIN lens allows imaging relay through a whole mouse brain, while a tunable acoustic gradient-index (TAG) lens provides sub-second volume rate via 100 kHz ∼ 1 MHz axial scan. This technique enables the study of calcium dynamics in cm-deep brain regions with sub-cellular and sub-second spatiotemporal resolution, paving the way for interrogating deep-brain functional connectome.

scholarly journals Universal intracellular biomolecule delivery with precise dosage control

2018 ◽  
Vol 4 (10) ◽  
pp. eaat8131 ◽  
Author(s):  
Y. Cao ◽  
H. Chen ◽  
R. Qiu ◽  
M. Hanna ◽  
E. Ma ◽  
...  
Keyword(s):  
Cell Membrane ◽  
Electric Fields ◽  
Low Voltage ◽  
Intracellular Delivery ◽  
Cell Types ◽  
Controlled Delivery ◽  
Biological Research ◽  
Biological Applications ◽  
Primary Cells ◽  
Cell Densities

Intracellular delivery of mRNA, DNA, and other large macromolecules into cells plays an essential role in an array of biological research and clinical therapies. However, current methods yield a wide variation in the amount of material delivered, as well as limitations on the cell types and cargoes possible. Here, we demonstrate quantitatively controlled delivery into a range of primary cells and cell lines with a tight dosage distribution using a nanostraw-electroporation system (NES). In NES, cells are cultured onto track-etched membranes with protruding nanostraws that connect to the fluidic environment beneath the membrane. The tight cell-nanostraw interface focuses applied electric fields to the cell membrane, enabling low-voltage and nondamaging local poration of the cell membrane. Concurrently, the field electrophoretically injects biomolecular cargoes through the nanostraws and into the cell at the same location. We show that the amount of material delivered is precisely controlled by the applied voltage, delivery duration, and reagent concentration. NES is highly effective even for primary cell types or different cell densities, is largely cargo agnostic, and can simultaneously deliver specific ratios of different molecules. Using a simple cell culture well format, the NES delivers into >100,000 cells within 20 s with >95% cell viability, enabling facile, dosage-controlled intracellular delivery for a wide variety of biological applications.

scholarly journals The Power of Single and Multibeam Two-Photon Microscopy for High-Resolution and High-Speed Deep Tissue and Intravital Imaging

2007 ◽  
Vol 93 (7) ◽  
pp. 2519-2529 ◽  
Author(s):  
Raluca Niesner ◽  
Volker Andresen ◽  
Jens Neumann ◽  
Heinrich Spiecker ◽  
Matthias Gunzer
Keyword(s):  
High Resolution ◽  
High Speed ◽  
Intravital Imaging ◽  
Deep Tissue ◽  
Two Photon Microscopy ◽  
Two Photon

Two-Photon Glutamate Uncaging to Study Structural and Functional Plasticity of Dendritic Spines

2019 ◽  
pp. 65-85
Author(s):  
Ivar S. Stein ◽  
Travis C. Hill ◽  
Won Chan Oh ◽  
Laxmi K. Parajuli ◽  
Karen Zito
Keyword(s):  
Dendritic Spines ◽  
Functional Plasticity ◽  
Two Photon

scholarly journals Multiphoton Microscopy for Ophthalmic Imaging

2011 ◽  
Vol 2011 ◽  
pp. 1-11 ◽  
Author(s):  
Emily A. Gibson ◽  
Omid Masihzadeh ◽  
Tim C. Lei ◽  
David A. Ammar ◽  
Malik Y. Kahook
Keyword(s):  
Harmonic Generation ◽  
Second Harmonic ◽  
Imaging Techniques ◽  
Multiphoton Microscopy ◽  
Tissue Imaging ◽  
Third Harmonic ◽  
Two Photon ◽  
Disease Mechanisms ◽  
Ophthalmic Imaging ◽  
Anti Stokes

We review multiphoton microscopy (MPM) including two-photon autofluorescence (2PAF), second harmonic generation (SHG), third harmonic generation (THG), fluorescence lifetime (FLIM), and coherent anti-Stokes Raman Scattering (CARS) with relevance to clinical applications in ophthalmology. The different imaging modalities are discussed highlighting the particular strength that each has for functional tissue imaging. MPM is compared with current clinical ophthalmological imaging techniques such as reflectance confocal microscopy, optical coherence tomography, and fluorescence imaging. In addition, we discuss the future prospects for MPM in disease detection and clinical monitoring of disease progression, understanding fundamental disease mechanisms, and real-time monitoring of drug delivery.

scholarly journals Microglia motility depends on neuronal activity and promotes structural plasticity in the hippocampus

2019 ◽  
Author(s):  
Felix C. Nebeling ◽  
Stefanie Poll ◽  
Lena C. Schmid ◽  
Manuel Mittag ◽  
Julia Steffen ◽  
...  
Keyword(s):  
Neuronal Activity ◽  
Dendritic Spines ◽  
Synapse Formation ◽  
Brain Parenchyma ◽  
Two Photon ◽  
Contact Rates ◽  
Health And Disease ◽  
The Impact ◽  
The Brain

AbstractMicroglia, the resident immune cells of the brain, play a complex role in health and disease. They actively survey the brain parenchyma by physically interacting with other cells and structurally shaping the brain. Yet, the mechanisms underlying microglia motility and their significance for synapse stability, especially during adulthood, remain widely unresolved. Here we investigated the impact of neuronal activity on microglia motility and its implication for synapse formation and survival. We used repetitive two-photon in vivo imaging in the hippocampus of awake mice to simultaneously study microglia motility and their interaction with synapses. We found that microglia process motility depended on neuronal activity. Simultaneously, more dendritic spines emerged in awake compared to anesthetized mice. Interestingly, microglia contact rates with individual dendritic spines were associated with their stability. These results suggest that microglia are not only sensing neuronal activity, but participate in synaptic rewiring of the hippocampus during adulthood, which has profound relevance for learning and memory processes.

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