MCI Pryer Endoscopic Objective: An Introduction
In recent years, fluorescence imaging techniques have become central to the study of live neurons. Unfortunately, high-resolution fluorescence imaging studies of neurons remain largely confined to in vitro preparations of cultured cells or tissue slices (Yuste et al. 2000), because of the experimental difficulties in accessing many neuron types in vivo. These difficulties mainly involve light scattering, which leads to an exponential attenuation of light intensity with distance traveled through solid tissue. In the mammalian brain, this limits fluorescence imaging depths to several hundred microns.
Another limitation of fluorescence microscopy is the requirement that samples to be studied need to be placed directly under the instrument. This experimental setup alters the sample for the simple reason that the actual observation is done outside the natural environment. A primary challenge for in vivo microscopy is miniaturization of the instrument to permit access to regions of interest.
To date, fluorescence microscopy studies of neuronal properties in live mammals have been limited to superficial or easily accessible tissues or to within ∼500 μm of the neocortical (Grutzendler et al. 2002; Helmchen et al. 1999; Stosiek et al. 2003; Svoboda et al. 1997, 1999; Trachtenberg et al. 2002) or olfactory bulb (Charpak et al. 2001) surface. However, most mammalian brain regions, which are not superficial and lie deeper than 500–1,000 μm within tissue, have largely remained inaccessible to cellular-level fluorescence imaging in vivo.
To address these challenges, we have designed and manufactured the first of its kind imaging modality that allows non- or minimally- invasive image acquisition in vivo and in real time with sub-cellular spatial resolution, and at sub-second sampling rates- the MCI Pryer Endoscopic Objective (abbreviated as MCI Pryer).
The MCI Pryer is a combined endoscopic objective and is compatible with current confocal and 2-photon microscopes from Olympus, Nikon and Zeiss. This state-of -the-art objective allows for deep tissue access and visualization in anesthetized animals, awake animals with head fixation and even chronic recordings with our custom-built Pryer adaptor. Non-invasive in vivo endoscopy could therefore accelerate diagnosis, by providing real-time information and would permit more sample sites, since the tissue is not removed. It also makes it possible to study tissues without artifacts that can be caused by fixation, sectioning, mounting, and staining.