Two‐photon fluorescence absorption and emission spectra of dyes relevant for cell imaging

Bestvater, Felix; Spieß, Eberhard; Stobrawa, G.; Häcker, Martin; Feurer, Thomas; Porwol, T.; Berchner‐Pfannschmidt, Utta; Wotzlaw, Christoph; Acker, H.

doi:10.1046/j.1365-2818.2002.01074.x

Cited by 202 publications

(140 citation statements)

References 21 publications

(32 reference statements)

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“…The mice were perfusion fixed, followed by perfusion with a silicone rubber compound called Microfil (FlowTech Inc., Carver, MA, USA) according to a method we developed previously (Pathak et al, 2008b). In brief, mice were deeply anesthetized with isoflurane and then perfused through the left ventricle-first with heparinized phosphate-buffered saline, and then with 10% buffered formalin (Bestvater et al, 2002) for fixation, and finally with a 1:2 mixture of Microfil and diluent added with 5% curing agent by volume. The Microfil was allowed to cure at room temperature for 90 minutes, and then the heads were fixed in cold formalin for 2 days.…”

Section: Whole-brain Sample Preparationmentioning

confidence: 99%

Vascular phenotyping of brain tumors using magnetic resonance microscopy (μMRI)

Kim

Zhang

Hong

et al. 2011

J Cereb Blood Flow Metab

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Abnormal vascular phenotypes have been implicated in neuropathologies ranging from Alzheimer's disease to brain tumors. The development of transgenic mouse models of such diseases has created a crucial need for characterizing the murine neurovasculature. Although histologic techniques are excellent for imaging the microvasculature at submicron resolutions, they offer only limited coverage. It is also challenging to reconstruct the three-dimensional (3D) vasculature and other structures, such as white matter tracts, after tissue sectioning. Here, we describe a novel method for 3D whole-brain mapping of the murine vasculature using magnetic resonance microscopy (lMRI), and its application to a preclinical brain tumor model. The 3D vascular architecture was characterized by six morphologic parameters: vessel length, vessel radius, microvessel density, length per unit volume, fractional blood volume, and tortuosity. Region-of-interest analysis showed significant differences in the vascular phenotype between the tumor and the contralateral brain, as well as between postinoculation day 12 and day 17 tumors. These results unequivocally show the feasibility of using lMRI to characterize the vascular phenotype of brain tumors. Finally, we show that combining these vascular data with coregistered images acquired with diffusion-weighted MRI provides a new tool for investigating the relationship between angiogenesis and concomitant changes in the brain tumor microenvironment.

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Section: Whole-brain Sample Preparationmentioning

confidence: 99%

Vascular phenotyping of brain tumors using magnetic resonance microscopy (μMRI)

Kim

Zhang

Hong

et al. 2011

J Cereb Blood Flow Metab

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“…We used a custom two-photon laser-scanning microscope with a 60ϫ, 1.1 numerical aperture (NA) objective (Olympus Optical, Tokyo, Japan) and a Ti:sapphire laser (Mira; Coherent, Santa Clara, CA). A single excitation wavelength of 810 nm was effective for simultaneous excitation of both green and red fluorophores (Bestvater et al, 2002;Yasuda et al, 2006). Fluorescence signals were collected in the epifluorescence and transfluorescence pathways (using a 1.4 NA oil immersion condenser).…”

Section: Methodsmentioning

confidence: 99%

Reliability and Heterogeneity of Calcium Signaling at Single Presynaptic Boutons of Cerebellar Granule Cells

Brenowitz¹,

Regehr²

2007

J. Neurosci.

106

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res transients while loading boutons to a steady-state indicator concentration. These experiments revealed that, in the absence of exogenous calcium buffers, a single action potential evokes transients of Ca res that vary widely in different boutons both in amplitude (400 -900 nM) and time course (25-55 ms). Variation in calcium influx density, endogenous buffer capacity, and calcium extrusion density contribute to differences in Ca res among boutons. Heterogeneity in Ca res within different boutons suggests that plasticity can be regulated independently at different synapses arising from an individual granule cell. In a given bouton, Ca res signals were highly reproducible from trial to trial and failures of calcium influx were not observed. We find that a factor contributing to this reliability is that an action potential opens a large number of calcium channels (20 -125) in a bouton. Presynaptic calcium signals were also used to assess the ability of granule cell axons to convey somatically generated action potentials to distant synapses. In response to pairs of action potentials or trains, granule cell boutons showed a remarkable ability to respond reliably at frequencies up to 500 Hz. Thus, individual boutons appear specialized for reliable calcium signaling during bursts of high-frequency activation such as those that are observed in vivo.

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“…During imaging, the brain slice was mechanically stabilized by a "harp" ( Figure 1B) and perfused by the carbogenated ACSF at 37 8C. Hoechst-33342 dye (H-342) was used to label nuclear DNA of mouse brain cells [11,14,53]. H-342 is a cell-permeable nuclear counterstain for live and fixed cells and tissue sections.…”

Section: Sample Preparation and Image Acquisitionmentioning

confidence: 99%

Quantitative comparison of 3D third harmonic generation and fluorescence microscopy images

Zhang

Kuzmin

Groot

et al. 2017

Journal of Biophotonics

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Third harmonic generation (THG) microscopy is a label-free imaging technique that shows great potential for rapid pathology of brain tissue during brain tumor surgery. However, the interpretation of THG brain images should be quantitatively linked to images of more standard imaging techniques, which so far has been done qualitatively only. We establish here such a quantitative link between THG images of mouse brain tissue and all-nuclei-highlighted fluorescence images, acquired simultaneously from the same tissue area. For quantitative comparison of a substantial pair of images, we present here a segmentation workflow that is applicable for both THG and fluorescence images, with a precision of 91.3 % and 95.8 % achieved respectively. We find that the correspondence between the main features of the two imaging modalities amounts to 88.9 %, providing quantitative evidence of the interpretation of dark holes as brain cells. Moreover, 80 % bright objects in THG images overlap with nuclei highlighted in the fluorescence images, and they are 2 times smaller than the dark holes, showing that cells of different morphologies can be recognized in THG images. We expect that the described quantitative comparison is applicable to other types of brain tissue and with more specific staining experiments for cell type identification.

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Two‐photon fluorescence absorption and emission spectra of dyes relevant for cell imaging

Cited by 202 publications

References 21 publications

Vascular phenotyping of brain tumors using magnetic resonance microscopy (μMRI)

Vascular phenotyping of brain tumors using magnetic resonance microscopy (μMRI)

Reliability and Heterogeneity of Calcium Signaling at Single Presynaptic Boutons of Cerebellar Granule Cells

Quantitative comparison of 3D third harmonic generation and fluorescence microscopy images

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