Optogenetics

Optogenetics

Optogenetics

Neuroscience is the multidisciplinary branch of biology in which the fundamental properties of neurons, the organization of neural networks, and their behavioral and sensory functional products are studied. Because the functions of neural networks are inextricably linked to their spatial organization, neuroscience research is heavily dependent on a variety of imaging techniques. For cellular and tissue level investigations, fluorescence microscopy is the most instructive and widely applicable of these techniques. Optogenetic techniques provides researchers with ways of externally modulating neuronal network function using light as an intermediary.

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Optogenetics

Transmitted Light Microscopy

Transmitted Light Microscopy

Transmitted light microscopy is a technique of light microscopy where the light passes from the source to the opposite side of specimens from the objective. This method is employed to distinguish the morphological characteristics and optic properties of the observed sample. Transmitted light microscopy generally has lower spatial resolution and less sensitivity than fluorescence microscopy.

Techniques the require transmitted light are bright-field, dark-field, phase contrast, polarization, and differential interference contrast (DIC) optics.

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Optogenetics

Live-Cell Imaging

Live-Cell Imaging

Live-cell imaging is a non-invasive imaging technique that allows for scientists to follow biological processes in living cells. This is an umbrella term used to describe all techniques where live cells are observed with microscopes encompassing techniques such as optogenetics, confocal microscopy, and fluorescence microscopy.

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Optogenetics

Fluorescence Microscopy

Fluorescence Microscopy

Fluorescence microscopy is a type of light microscopy in which light for image formation is generated indirectly from the fluorescence of specialized molecules (known as fluorophores or dyes) that are embedded in the specimen. Unlike transmitted light microscopy, there is no background from light passing directly from the illumination source to the detector. For biologist, this means that much smaller objects in smaller numbers can be detected and observed. However, in order to make those observations, light sources capable of generating much higher intensity outputs than those used for transmitted light microscopy are required.

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Optogenetics

Super-Resolution

Super-Resolution

Super-resolution is a term used to refer to methods that allow spatial discrimination of objects that are smaller than about 200 nm (0.2 microns). Two basic methods are used to achieve super-resolution. (1) Methods that constrain the illumination field used for fluorescence excitation or (2) methods that constrain the population of detectable fluorophores in the specimen. Notwithstanding these advances, light microscopy cannot yet deliver sub-nanometer spatial resolution comparable to electron microscopy. Techniques for correlative light and electron microscopy (CLEM) are therefore important for combining information obtained from these two contrast modalities.

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Optogenetics

Confocal Microscopy

Confocal Microscopy

The optical imaging technique of Laser Confocal Scanning Microscopy (LCSM) is used to increase the optical resolution and contrast of an image. Although laser scanning is most commonly used, the scanning process is relatively slow and requires high light levels that may produce photo-damage, particularly in living specimens. Spinning disk confocal microscopy uses a wide illumination field that is projected through microscopic apertures in a rotating disk to generate a scan pattern. This parallelizes the scanning process, making it faster. Confocal imaging is compatible with many spectral applications including multi-fluorescence imaging, time-lapse imaging, FLIM, FRAP, and FCS measurements.

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