Two-Photon Microscopy in Highly Scattering Tissue

Wallace, Vincent P.; Dunn, Andrew K.; Coleno, Mariah L.; Tromberg, Bruce J.

doi:10.1007/978-1-4614-7513-2_11

Cited by 14 publications

(11 citation statements)

References 23 publications

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“…Moreover, resolution in the Z-direction is sufficient to obtain high density XYZ-stacks sufficient for high quality 3-dimensional reconstructions and animated rotations. This is consistent with the view that multiphoton confocal microscopy does not enhance optical resolution, especially in the Z-direction (for discussion, see symposium paper by Tauer in this issue; and also Dickinson & Fraser, 2001;Wallace et al 2001). In addition, we obtain most of our images using < 2-3 mW power from the 488 nm laser line.…”

Section: Figuresupporting

confidence: 88%

Dynamic Confocal Imaging in Acute Brain Slices and Organotypic Slice Cultures Using a Spectral Confocal Microscope with Single Photon Excitation

Kasparov

Teschemacher

Paton

2002

Experimental Physiology

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Confocal imaging in living brain slices allows the resolution of submicrometre structures of nerve cells, glia and brain vessels. Imaging living brain slices is in many respects different from conventional fixed histological preparations for which confocal microscopes were designed originally. Several problems (i.e. mechanical and thermal drift, and autofluorescence) resulting from the optical and structural properties of brain slices are discussed. Fluorescent indicators may be used to monitor numerous intracellular parameters such as pH and Ca2+ concentration, but not all of them are equally suitable for this type of work. Genetically engineered fluorescent proteins can be used to visualise the fine dendritic structure of neurones or track particular intracellular structures and proteins. They have also been used to generate indicators for Ca2+, cAMP and other molecules. While conventional chemical indicators can be either loaded into neurones via patch pipettes or as membrane‐permeable esters, protein indicators can be expressed in various types of cells using adenoviral vectors. Adenoviral transgenesis can be performed in vitro in both acute slices and organotypic slice cultures. Organotypic slice cultures give excellent optical access to neurones loaded with either conventional fluorescent indicators or transfected with adenovirus to express fluorescent proteins. They are most suitable for experiments where both conventional and genetically engineered indicators are combined. Single photon imaging in brain slices is limited to the superficial layers (∼≤50 μm), while multiphoton excitation has a much greater depth of penetration. However, the overall optical resolution achievable in single photon mode is at least as good as when using multiphoton excitation.

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Section: Figuresupporting

confidence: 88%

Dynamic Confocal Imaging in Acute Brain Slices and Organotypic Slice Cultures Using a Spectral Confocal Microscope with Single Photon Excitation

Kasparov

Teschemacher

Paton

2002

Experimental Physiology

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“…In comparison to nuclear morphology techniques, this assay has been shown to have a higher degree of accuracy for the detection of apoptosis [38] and, as such, has been used to quantify programmed cell death in a number of brain tumor cell lines [39,40]. The fluorescein label was detected by two-photon fluorescence microscopy [41]. The fluorescein was excited at a wavelength of 400 nm, and the resultant fluorescence images were collected using a long-pass (530 nm cut-off) filter (CVI, Albuquerque, NM).…”

Section: Tunel Assaymentioning

confidence: 99%

Enhanced cytotoxic effects of 5-aminolevulinic acid-mediated photodynamic therapy by concurrent hyperthermia in glioma spheroids

et al. 2004

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“…In addition, light collection efficiency in MPEM is also a function of sample optical properties heavily weighted to the transport characteristics of the emitted light (i.e. tissue light scattering and absorption) (Wallace et al ., 2001).…”

Section: Introductionmentioning

confidence: 99%

Optimization of multiphoton excitation microscopy by total emission detection using a parabolic light reflector

Combs¹,

Смирнов

Riley³

et al. 2007

Journal of Microscopy

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SummaryWe have constructed a device that maximizes the probability of collecting all of the scattered and ballistic light isotropically generated at the focal spot of multiphoton excited emissions (MPE) to optimize the signal-to-noise ratio (SNR) for microimaging. This was accomplished by optically coupling a parabolic reflector (that surrounds the sample and top of the objective) to a pair of collimating lenses (above the sample) that redirects emitted light to a separate detector. These additional optics, combined with the objective, allow the total emission detection (TED) condition to be approached. Numerical simulations suggest an approximately 10-fold improvement in SNR with TED. Comparisons between the objective detection and TED reveal an enhancement of 8.9 in SNR (77% of predicted) for GFP-labelled brain slices and similar results for fluorescent beads. This increase in SNR can be used to improve time resolution, reduce laser power requirements/photodynamic damage, and, in certain cases, detection depth, for MPE imaging techniques.

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Two-Photon Microscopy in Highly Scattering Tissue

Cited by 14 publications

References 23 publications

Dynamic Confocal Imaging in Acute Brain Slices and Organotypic Slice Cultures Using a Spectral Confocal Microscope with Single Photon Excitation

Dynamic Confocal Imaging in Acute Brain Slices and Organotypic Slice Cultures Using a Spectral Confocal Microscope with Single Photon Excitation

Enhanced cytotoxic effects of 5-aminolevulinic acid-mediated photodynamic therapy by concurrent hyperthermia in glioma spheroids

Optimization of multiphoton excitation microscopy by total emission detection using a parabolic light reflector

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