The methodology discussed here has been validated using various cell types, including fusion deficient Opa1-, Mfn1-, and Mfn2-depleted cells (Karbowski et al., 2004; Karbowski, Norris, Cleland, Jeong, & Youle, 2006; Lee, Jeong, Karbowski, Smith, & Youle, 2004; Fig. The resolution is a value that can be derived theoretically given the optical parameters of the instrument and the average wavelength of illumination. Resolution is directly related to the useful magnification of the microscope and the perception limit of specimen detail, though it is a somewhat subjective value in microscopy because at high magnification, an image may appear out of focus but still be resolved to the maximum ability of the objective and assisting optical components. Numerical Aperture (NA) = n • sin(α) In the above equation, ‘n’ is the refractive index of the medium between the cover glass and the front lens of the objective (for example; air, water or oil).
This hologram with its capability to focus laser light from all sides should become the decisive optical element of a laser scanning microscope. The point-to-point resolution at the specimen, d0, is listed in the table along with the magnified size of the image (D0) in the intermediate eyepiece plane (using green light of 550 nanometer wavelength).
Hence, the optical 3D resolution would be improved by a similar factor. Fast imaging (10+ fps) will require short exposure time and thus higher laser power. The superimposed profiles display two brightness maxima that are separated by a valley. The average focal power of the STED beam, which reduces the fluorescence by half, is on the order of 1 mW for YFP at a repetition rate of 80 MHz (measured without phase plate). While the diffusion rates of mito-PAGFP have not been measured, it is likely that they are similar to those of mito-GFP. Phase retrieval from a high-numerical-aperture intensity distribution by use of an aperture-array filter. An RNAi screen of mitochondrial genes in Caenorhabditis elegans indicated that nearly 80% of the knockdowns displayed altered mitochondrial morphology towards fragmentation and/or aggregation (Ichishita et al., 2008). With these, it is possible to activate and record several cells during each experiment. High-efficiency diffractive waveguide lenses by parametric optimization.
That is, the detector undersamples the image, and consequently, the image on the monitor or the image recorded digitally on the hard drive of a computer is limited by the pixel spacing of the camera, not the optical resolution of the objective. As mito-PAGFP is barely detectable before photoactivation (see Fig. This advantage makes it possible to develop assemblies of holograms as new focusing devices from all directions of the space with geometries and hence spatial light distributions which are impossible to realize with conventional high NA objective lenses. 4.4A). Contact your local ZEISS provider for more information. This is normally done with the auxiliary magnification wheel found on most modern microscopes or magnification in the phototube that holds the camera. Higher values of numerical aperture permit increasingly oblique rays to enter the objective front lens, which produces a more highly resolved image and allows smaller structures to be visualized with higher clarity. During the 3D acquisition, the SAC will dynamically move the correction lens relative to the z-position, therefore producing a 3D image that corrects SA at every plane in the series, as opposed to the correction collar that corrects for a single plane.
Due to the wave nature of light and the diffraction associated with these phenomena, the resolution of a microscope objective is determined by the angle of light waves that are able to enter the front lens and the instrument is therefore said to be diffraction limited. Note the increase in fluorescence intensity and decrease in the axial elongation of the diffraction-limited bead.
4.4B,C). Until recently, mitochondrial fusion in intact yeast and mammalian cells could only be assayed using cell fusion-based assays, like the polyethylene glycol (PEG) assay. It can never get any larger than 1 if air is the medium between the specimen and the lens. All parameters should be adjusted in a range satisfying characteristics of the camera used, typically 16 bit range. In this approach, cells stably expressing mitochondria-targeted GFP are co-plated with cells stably transfected with mitochondria-targeted red fluorescent protein (RFP). They also have a variable iris diaphragm (see next section). However, 63 × Plan-Apo objective lenses with a numerical aperture 1.4 are also suitable. Just like the correction collar, the SAC unit contains a motile lens doublet.
Appl Opt. When imaging single events of mitochondrial fusion, a time-lapse series with a maximum interval of 1 min can be collected. To fully capture the resolution provided by the objective, the camera should oversample the image so that the most closely spaced line pair imaged by the objective will be separated by at least one and preferably two rows of unstimulated detector pixels. By continuing you agree to the use of cookies. The illumination source (laser) together with exposure time should be adjusted according to the temporal resolution required and to minimize photobleaching. In order to enable two objectives to be compared and to obtain a quantitative handle on resolution, the numerical aperture, or the measure of the solid angle covered by an objective is defined as: 1993 Oct 1;32(28):5689-91. doi: 10.1364/AO.32.005689. This will lead to visualization of all mitochondria in an analyzed cell. Greenfield Sluder, Joshua J. Nordberg, in Methods in Cell Biology, 2013.
It is directly related to the angle of the cone which is formed between a point on the specimen and the front lens of the objective or condenser, determined by the equation NA = n sin ∝.
This hologram with its capability to focus laser light from all sides should become the decisive optical element of a laser scanning microscope. The point-to-point resolution at the specimen, d0, is listed in the table along with the magnified size of the image (D0) in the intermediate eyepiece plane (using green light of 550 nanometer wavelength).
Hence, the optical 3D resolution would be improved by a similar factor. Fast imaging (10+ fps) will require short exposure time and thus higher laser power. The superimposed profiles display two brightness maxima that are separated by a valley. The average focal power of the STED beam, which reduces the fluorescence by half, is on the order of 1 mW for YFP at a repetition rate of 80 MHz (measured without phase plate). While the diffusion rates of mito-PAGFP have not been measured, it is likely that they are similar to those of mito-GFP. Phase retrieval from a high-numerical-aperture intensity distribution by use of an aperture-array filter. An RNAi screen of mitochondrial genes in Caenorhabditis elegans indicated that nearly 80% of the knockdowns displayed altered mitochondrial morphology towards fragmentation and/or aggregation (Ichishita et al., 2008). With these, it is possible to activate and record several cells during each experiment. High-efficiency diffractive waveguide lenses by parametric optimization.
That is, the detector undersamples the image, and consequently, the image on the monitor or the image recorded digitally on the hard drive of a computer is limited by the pixel spacing of the camera, not the optical resolution of the objective. As mito-PAGFP is barely detectable before photoactivation (see Fig. This advantage makes it possible to develop assemblies of holograms as new focusing devices from all directions of the space with geometries and hence spatial light distributions which are impossible to realize with conventional high NA objective lenses. 4.4A). Contact your local ZEISS provider for more information. This is normally done with the auxiliary magnification wheel found on most modern microscopes or magnification in the phototube that holds the camera. Higher values of numerical aperture permit increasingly oblique rays to enter the objective front lens, which produces a more highly resolved image and allows smaller structures to be visualized with higher clarity. During the 3D acquisition, the SAC will dynamically move the correction lens relative to the z-position, therefore producing a 3D image that corrects SA at every plane in the series, as opposed to the correction collar that corrects for a single plane.
Due to the wave nature of light and the diffraction associated with these phenomena, the resolution of a microscope objective is determined by the angle of light waves that are able to enter the front lens and the instrument is therefore said to be diffraction limited. Note the increase in fluorescence intensity and decrease in the axial elongation of the diffraction-limited bead.
4.4B,C). Until recently, mitochondrial fusion in intact yeast and mammalian cells could only be assayed using cell fusion-based assays, like the polyethylene glycol (PEG) assay. It can never get any larger than 1 if air is the medium between the specimen and the lens. All parameters should be adjusted in a range satisfying characteristics of the camera used, typically 16 bit range. In this approach, cells stably expressing mitochondria-targeted GFP are co-plated with cells stably transfected with mitochondria-targeted red fluorescent protein (RFP). They also have a variable iris diaphragm (see next section). However, 63 × Plan-Apo objective lenses with a numerical aperture 1.4 are also suitable. Just like the correction collar, the SAC unit contains a motile lens doublet.
Appl Opt. When imaging single events of mitochondrial fusion, a time-lapse series with a maximum interval of 1 min can be collected. To fully capture the resolution provided by the objective, the camera should oversample the image so that the most closely spaced line pair imaged by the objective will be separated by at least one and preferably two rows of unstimulated detector pixels. By continuing you agree to the use of cookies. The illumination source (laser) together with exposure time should be adjusted according to the temporal resolution required and to minimize photobleaching. In order to enable two objectives to be compared and to obtain a quantitative handle on resolution, the numerical aperture, or the measure of the solid angle covered by an objective is defined as: 1993 Oct 1;32(28):5689-91. doi: 10.1364/AO.32.005689. This will lead to visualization of all mitochondria in an analyzed cell. Greenfield Sluder, Joshua J. Nordberg, in Methods in Cell Biology, 2013.
It is directly related to the angle of the cone which is formed between a point on the specimen and the front lens of the objective or condenser, determined by the equation NA = n sin ∝.