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The advantage of confocal microscopes over conventional wide-field microscopes is the ability to reject out-of-focus background light. This enables the ability to optically section thick cells or tissues, penetrate deep light-scattering tissues, and to generate three-dimensional views at high resolution. A confocal microscope optically sections by restricting the field of view of the objective lens and the excitation illumination to a single spot. This is most often accomplished by scanning the illumination source (usually a laser beam) over the specimen and collecting the emitted fluorescence through an exit pinhole placed before the photodetector. In this manner, light originating from out of focus regions of the specimen is excluded from detection. The optical section is then a function of the size of the pinhole in the intermediate image plane and the numerical aperture (N.A.) of the lens. Typically, with a high NA lens, in plane resolution can be around 0.2 microns while axial resolution is a little less than 1 micron. While this technique can provide crisp clear optical sectioning capability, there are sacrifices made over conventional wide–field fluorescence microscopy.

Two main disadvantages of conventional CLSM are a slow speed of acquisition and potential photodamage. Photodamage can arise from the high illumination intensities produced from focused laser light. Use of acoutic optical tunable filters (AOTF’s) is one method available on many commercial confocal systems for rapid attenuation of laser power over a broad range of intensities. Photodamage is also a danger during 3D-imaging. During 3D-image generation, although emmision light is captured from one image plane at a time as the plane of focus is moved through the specimen, excitation light is not restricted. Therefore, the cone of excitation light is exciting, and potentially photobleaching, all planes of focus regardless of which plane is being imaged at a time. Restricting the excitation light to the plane of focus using multi-photon microscopy can reduce this problem. The second drawback to conventional CLSM is the by-product of raster scanning. Raster scanning allows for the sectioning capabilities of CLSM but is much slower than wide field camera microscopy. Typically a 512 x 512 pixel 8 bit image will take 1-2 s to acquire compared to conventional WF microscopy were images can be acquired at video rates or higher.

Detailed information (including many references) about CLSM can be found at http://www.microscopyu.com/articles/confocal/confocalintrobasics.html, http://micro.magnet.fsu.edu/primer/virtual/confocal/index.html, and http://micro.magnet.fsu.edu/primer/resources/confocal.html.

Two-photon (2P) excitation microscopy is an alternative optical sectioning technique to confocal laser scanning microscopy (CLSM). The main advantage of two-photon microscopy is that excitation occurs predominantly in the focal plane of the objective. In CLSM excitation light is wasted above and below the focal plane in a cone even though emission information is restricted to light coming from the focal plane by the pinhole placed before the detector. Thus, in two-photon microscopy there is less photobleaching and photodamage in out of focus planes of the specimen. The second main advantage of 2P excitation is that long wavelength light penetrates much deeper into tissues. This provides for 2-3 times deeper penetration than confocal microscopy and allows for information to be gathered in much thicker cells or tissues.

Two-photon excitation occurs when two photons are simultaneously absorbed by a fluorophore. Two photons of twice the wavelength that would normally excite a fluorophore absorbed in a single quantitized event provide the same energy as single photon absorption at one-half the wavelength. For example, two red photons (~ 700 nm) absorbed at the same time would excite a fluorophore that would normally be excited by ultraviolet light (~ 350 nM). To initiate enough 2P absorption events to excite fluorophores effectively requires very high photon densities, typically much higher than are usually used for epi-fluorescent imaging {Denk, Piston, et al. 1995 ID: 765}{Denk & Svoboda ID: 766}. In 2P excitation microscopy high photon densities are achieved at the focal plane of the specimen both by temporal and spatial means. Temporal crowding is achieved using mode-locked (pulsed) lasers. Typically the lasers are pulsed on the femto- or pico-second timescale at high powers (milliwatt levels) and although the peak powers are high, the average powers are low due to the duration of the pulses. Spatial crowding is achieved by the optics of the microscope. The focusing of the laser beam through the optics results in crowding of the photons at the plane of focus. It is precisely this crowding that allows for the optical sectioning capabilities of multi-photon excitation. Above and below the focal plane the photon density is not high enough to elicit significant two-photon absorption events. In addition, no pinhole is necessary in the emission pathway due to the excitation occurring only in the focal plane. Therefore all the emission light is collected and is not limited by a pinhole in the intermediate image plane.

Detailed information (including many references) about two-photon microscopy can be found at http://micro.magnet.fsu.edu/primer/techniques/fluorescence/
multiphoton/multiphotonintro.ht ml.