In vitro and In vivo Imaging

In vitro and in vivo imaging Prairie’s imaging systems are used for a broad range of imaging applications, from single molecule imaging, immunofluorescense, to cell motility and long-term imaging of neuronal development.

In confocal imaging systems, a single or multipoint of excitation light is scanned across the specimen. In the case of single point light is detected by a PMT and in the case of the multipoint, light is detected via a high speed camera. Fluorescent probes serve as markers for labeling specific structures or proteins in a cell or tissue. Different cellular structures can be studied simultaneously when several proteins are labeled separately. Moreover, cells and tissues can express a number of different fluorescent proteins.

Time-lapse Imaging
The ability for the scientist to match the optical recording with the temporal biological fluorescence response has great promise for cell and developmental biology studies where live dynamic events must be captured quickly in high resolution.

Two-photon excitation microscopy is anticipated to have an ample impact in areas such as physiology, neurobiology, embryology and tissue engineering, for which imaging of highly scattering tissue is required. Highly opaque tissues such as human skin have been visualized with cellular detail³. Clinically, two-photon microscopy may find an application in noninvasive optical biopsy, for which high–speed imaging is required. This need has been addressed by video rate two-photon microscopy). In cell biology, the most promising applications are those that rely on two-photon excitation to produce localized chemical reactions, such as in 3D resolved uncaging and photobleaching recovery studies⁴.

Calcium Dynamics
Two-photon laser scanning microscopy was first employed to measure calcium dynamics in subcellular compartments of single neurons with unprecedented spatial resolution^5-8. These experimental results bear on theories that associate activity dependent, long-lasting cognitive processes such as learning and memory with possible physiological and anatomical changes in brain cells⁹. It has been proposed that in certain categories of neurons, subcellular appendages present on neuronal extensions, called dendritic spines, play a central role in the integration of input signals coming from other neurons. The geometry and size of dendritic spines could, in principle, allow local retention of calcium, an ion known to be critical for many long-term intracellular events. However, basic physiological properties of dendritic spines could not be measured prior to the advent of two-photon excitation (2PE) microscopy, since the volume of dendritic spines is in the order of one cubic micrometer or less⁹. These experiments on dendritic spines were performed using in vitro brain slicing^5,6.

2PE microscopy is especially well suited for imaging in the retina. Since the retina’s photoreceptors are exquisitely sensitive to visible light, they are rapidly bleached by conventional fluorescence microscopy methods. In contrast, the IR light used for 2PE microscopy is not absorbed by photopigments and therefore allows simultaneous functional imaging of retinal neurons and visual stimulation of photoreceptors. 2PE microscopy has been exploited to dissect the mechanisms underlying direction-selectivity in the dendrites of retinal interneurons^10,11. It also has been used to image the pancreatic islets; N. Takahashi et al.¹⁶ assessed various fluorescent indicators for investigating intact islets of Langerhans using two-photon excitation imaging.

Developmental Biology - Long-term fluorescence imaging
Living embryos are among those biological samples that are most sensitive to damage and require the imaging of whole specimens. 2PE microscopy has been used to study sea urchin embryogenesis¹². The dynamics of mitochondrial distribution in hamster embryos at frequent intervals over 24 hours using two-photon microscopy while maintaining blastocyst, and even fetal developmental competence, have been monitored¹³. Various aspects of C. elegans development are beginning to be studied with 2PE as well¹⁴.

Imaging Neuronal Populations
Populations of neurons can be loaded with membrane-permeable Ca2+ indicators both in vitro5 and in vivo¹⁵. Using this loading technique, it has been possible to image the dynamics of populations of individual neurons both in vivo and in vitro.

In vivo Cellular Applications of 2PE Microscopy
The ability to of 2PE microscopy to image deep inside neural tissue allows investigators to link neuronal activity and morphology at higher levels of organization in the brain. Experimental evidence of activity-dependent remodeling of neuronal wiring in response to sensory manipulation was provided by 2PE imaging in the cerebral cortex of developing rats^17-19. Svoboda et al.²¹ have used 2PE to measure in vivo calcium dynamics elicited by sensory stimulation in neocortical between pyramidal cells. Calcium-sensitive fluorescent dyes⁹ are currently the indicators of choice because their fluorescence varies robustly with changes in concentration of intracellular calcium that, in turn, is strongly correlated with neuronal activity. However, a recurrent problem with the in vivo experiments is the ability to label potentially thousands of cells of interest with intracellular dyes in a volume that corresponds to the extent of the network of interest. A method that involves intracerebral perfusion of dyes is described by Stosiek et al.¹⁵. Using this approach, Ohki et al. managed to accurately map domains of common neuronal responses involved in vision in the adult rat cerebral cortex²⁰.

2PE microscopy has also revealed rich dynamism of non-neuronal structures in vivo. GFP-labeled microglia, the main immune cells of the brain, show pronounced structural dynamics over tens of seconds and rapidly migrate to sites of brain injury^22,23. 2PE microscopy of blood flow²⁴ has been combined with nonlinear techniques for tissue ablation to study the hemodynamics in response to vascular.

Targeted intracellular recording in vivo
2PE microscopy is particularly useful as a tool to target specific neurons for intracellular recording in the brain of mammals in vivo, a technique that was not possible until recently. Margrie et al.²⁶ specifically targeted cortical inhibitory interneurons. Targeting intracellular electrodes in specific classes of mammalian neurons in vivo will enable future experiments to reach a level of sophistication that was once achieved exclusively in invertebrate preparations and opens the possibility for many future variations of this method⁹.

Cancer Research
Using a Prairie Ultima IV system, A. Leimgruber et al.²⁷ assessed in vivo the behavior of tumor-associated macrophages, using a functionalized nanoparticle.

 

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