A custom-made two-photon microscope and deconvolution system

Majewska, Ania K.; Yiu, Glenn; Yuste, Rafael

doi:10.1007/s004240000435

Cited by 162 publications

(99 citation statements)

References 18 publications

(23 reference statements)

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“…A custom-made two-photon laser scanning microscope (Majewska et al, 2000b) was used for in vivo imaging. The microscope consists of a modified Fluoview confocal scan head (Olympus Optical) and a titanium/sulphur laser providing 100 fs pulses at 80 MHz at a wavelength of 920 nm (Tsunami; Spectra-Physics, Menlo Park, CA) pumped by a 10 W solid-state source (Millenia; Spectra-Physics).…”

Section: Surgical Preparation For Rewiring Visual Input To the Auditomentioning

confidence: 99%

Remodeling of Synaptic Structure in Sensory Cortical AreasIn Vivo

Majewska

Newton²,

Sur³

2006

J. Neurosci.

223

178

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Although plastic changes are known to occur in developing and adult cortex, it remains unclear whether these changes require remodeling of cortical circuitry whereby synapses are formed and eliminated or whether they rely on changes in the strength of existing synapses. To determine the structural stability of dendritic spines and axon terminals in vivo, we chose two approaches. First, we performed time-lapse two-photon imaging of dendritic spine motility of layer 5 pyramidal neurons in juvenile [postnatal day 28 (P28)] mice in visual, auditory, and somatosensory cortices. We found that there were differences in basal rates of dendritic spine motility of the same neuron type in different cortices, with visual cortex exhibiting the least structural dynamics. Rewiring visual input into the auditory cortex at birth, however, failed to alter dendritic spine motility, suggesting that structural plasticity rates might be intrinsic to the cortical region. Second, we investigated the persistence of both the presynaptic (axon terminals) and postsynaptic (dendritic spine) structures in young adult mice (P40 -P61), using chronic in vivo two-photon imaging in different sensory areas. Both terminals and spines were relatively stable, with Ͼ80% persisting over a 3 week period in all sensory regions. Axon terminals were more stable than dendritic spines. These data suggest that changes in network function during adult learning and memory might occur through changes in the strength and efficacy of existing synapses as well as some remodeling of connectivity through the loss and gain of synapses.

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Section: Surgical Preparation For Rewiring Visual Input To the Auditomentioning

confidence: 99%

Remodeling of Synaptic Structure in Sensory Cortical AreasIn Vivo

Majewska

Newton²,

Sur³

2006

J. Neurosci.

223

178

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“…It consists of a modified upright Olympus microscope and a modified confocal scan head fitted with a ϫ60 water immersion objective (NA ϭ 0.9) and a Spectra-Physics Tsunami femtosecond laser tuned to 780 nm (26). A nondescanned photomultiplier tube mounted off the epifluorescence filter turret was used to detect fluorescence emission of Ͻ700 nm (12).…”

Section: Two-photon Microscopymentioning

confidence: 99%

Requirements for T Lymphocyte Migration in Explanted Lymph Nodes

Huang

Cárdenas-Navia

Caldwell

et al. 2007

The Journal of Immunology

117

127

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Although the requirements for T lymphocyte homing to lymph nodes (LNs) are well studied, much less is known about the requirements for T lymphocyte locomotion within LNs. Imaging of murine T lymphocyte migration in explanted LNs using two-photon laser-scanning fluorescence microscopy provides an opportunity to systematically study these requirements. We have developed a closed system for imaging an intact LN with controlled temperature, oxygenation, and perfusion rate. Naive T lymphocyte locomotion in the deep paracortex of the LN required a perfusion rate of >13 μm/s and a partial pressure of O2 (pO2) of >7.4%. Naive T lymphocyte locomotion in the subcapsular region was 38% slower and had higher turning angles and arrest coefficients than naive T lymphocytes in the deep paracortex. T lymphocyte activation decreased the requirement for pO2, but also decreased the speed of locomotion in the deep paracortex. Although CCR7−/− naive T cells displayed a small reduction in locomotion, systemic treatment with pertussis toxin reduced naive T lymphocyte speed by 59%, indicating a contribution of Gαi-mediated signaling, but involvement of other G protein-coupled receptors besides CCR7. Receptor knockouts or pharmacological inhibition in the adenosine, PG/lipoxygenase, lysophosphatidylcholine, and sphingosine-1-phosphate pathways did not individually alter naive T cell migration. These data implicate pO2, tissue architecture, and G-protein coupled receptor signaling in regulation of naive T lymphocyte migration in explanted LNs.

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“…Two-photon microscopes suitable for imaging dendritic spines can be custom-built (e.g., Mainen et al 1999;Majewska et al 2000) or obtained from a commercial vendor (e.g., Olympus, Prairie Technologies, Sutter Instruments, Zeiss). A typical custom imaging setup for two-photon imaging of dendritic spines is shown in Figure 1.…”

Section: Equipmentmentioning

confidence: 99%

Imaging Synaptic Protein Dynamics Using Photoactivatable Green Fluorescent Protein

Weimer¹,

Hill²,

Hamilton³

et al. 2012

Cold Spring Harb Protoc

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Considerable evidence has accumulated that structural changes in dendritic spines and their synapses are associated with adaptive functional changes in cortical circuits, such as during circuit refinement in young animals and in learning and memory in adults. Understanding the mechanisms of circuit plasticity requires detailed investigation of the structural dynamics of dendritic spines and how they are regulated by neural activity and sensory experience. Studying the dynamic localization of synaptic proteins in dendritic spines and how their stabilization and exchange rates influence spine structural plasticity is also important. This protocol describes imaging approaches to study synaptic protein dynamics in dendritic spines of the rodent cerebral cortex. It gives a strategy for generating photoactivatable green fluorescent protein (PA-GFP)-tagged synaptic proteins and in vitro and in vivo transfection methods for coexpression of these proteins with a spectrally separable cell-filling marker (DsRed-Express). Methods for tracking synaptic protein localization using photoactivation and time-lapse imaging of PA-GFP in spiny pyramidal neuron dendrites are given. A discussion of imaging hardware and software preferences is also included. The methods described here can be used to study the dynamic processes underlying spine synapse development during the formation and plasticity of neural circuits in the mammalian brain. Alternatively, use pregnant female mice if in vivo imaging will be performed.Reagents for testing PA-GFP-tagged synaptic proteins for their ability to retain native protein function (see Step 2)

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A custom-made two-photon microscope and deconvolution system

Cited by 162 publications

References 18 publications

Remodeling of Synaptic Structure in Sensory Cortical AreasIn Vivo

Remodeling of Synaptic Structure in Sensory Cortical AreasIn Vivo

Requirements for T Lymphocyte Migration in Explanted Lymph Nodes

Imaging Synaptic Protein Dynamics Using Photoactivatable Green Fluorescent Protein

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