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

Abstract

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.

Spectral Unmixing: Analysis of Performance in the Olfactory Bulb In Vivo

“…In contrast to confocal microscopy, which limits detection of fluorescence to few tens microns in depth in the brain [27], [39], our approach allowed us to greatly increase this distance in the anesthetized animal and detect odor-evoked FRET transients up to 180–250 µm in depth in the main olfactory bulb. To our knowledge this is the first demonstration of in vivo spectral imaging and unmixing of FRET signals at such depth.…”

Targeting specific neuronal populations using adeno‐ and lentiviral vectors: applications for imaging and studies of cell function

“…In addition, water immersion lenses also allow a clearer view of the upper surface of the slice, where fluorescent cells can be targeted by patch pipettes. Altogether, this set‐up provides superior conditions for imaging such that ∼0.5‐μm ‐wide objects, close to the theoretical limit of optical microscopy, may be visualized in three‐dimensional (3‐D) space (Kasparov et al 2002) (see Fig. 2).…”

“…Owing to the good optical properties of the organotypic cell culture membranes (Millipore) and the flattening of the tissue, which occurs over approximately 2 weeks in culture, very high‐resolution confocal imaging of fluorescent neurones in slice cultures can be achieved (Kasparov et al 2002; Teschemacher et al 2005). For imaging, explants are excised together with the surrounding culture membrane and transferred to a glass‐bottomed tissue chamber mounted on the microscope stage.…”

Nanobiolistic delivery of indicators to the living mouse retina

“…A Plan-Apochromat 25× objective was used to generate a single image for each sample. All samples were processed under the same settings (Kasparov et al, 2002;Nonet, 2003).…”