Localization-Based Super-Resolution Light Microscopy
Multicolor
imaging.
Figure 2: Experimental geometry for two-color bi-plane FPALM. (Left) A set of lasers is aligned into a collinear path to allow a choice of one activation and one readout illumination wavelength. These two beams are directed through a series of mirrors into a focusing lens positioned at one focal length from the back aperture of a microscope objective within a fluorescence microscope (here shown as inverted). (Right) The sample is illuminated on the microscope stage, and emitted fluorescence from single molecules is separated from illumination by a dichroic (DM1) and sent through the tube lens and out the microscope side port. Next, the fluorescence image is magnified by a telescope and then split by a second dichroic (DM2) into two detection channels. Each detection channel can then be passed through a 50:50 beamsplitter to form separate images on each of two EMCCD cameras. Note that the two longer arms of the red and green paths should have the same distance between sample and camera, as should the two shorter arms of the red and green paths. For illustrative purposes, the figure does not show the optics arranged in this way. Also shown are an optional spectrometer, for confirmation of molecular species, and a pulsed laser for multi-photon activation and/or readout.
Activation laser intensity is regulated, either by pulsing with progressively higher frequency or gradually increasing intensity as the pool of inactive molecules is depleted, to maintain a small number of activated molecules within the field of view. Readout laser intensity is controlled such that the active fluorescent molecules are clearly visible above the background. Typically, hundreds to thousands of frames are acquired and then analyzed to localize visible molecules. Images are rendered by plotting each molecule as a Gaussian spot with a weight and size proportional to the number of photons detected and the localization precision, respectively. An example of single-color imaging of dendritic spines is shown in Figure 3. Te first published example of localization microscopy
in living cells used single-color FPALM of the viral surface protein hemagglutinin (HA) to distinguish between several competing models of membrane organization [26]. A wide variety of biological applications have been presented [27–30]; localization microscopy is applicable to virtually any subcellular structure, as long as the structure can be labeled and the molecular millieu is compatible with imaging and localizing single molecules. In addition to molecular coordinates, a direct count of the
numbers of localized molecules (and thus, with careful analysis, the densities of molecules) is obtained, and sub-populations of molecules can be identified based on brightness, or other spectroscopic properties, which would otherwise be averaged out in ensemble imaging methods. Correlation of localization microscopy results with other methods, such as confocal or widefield fluorescence, atomic-force microcsopy, or electron microscopy, is extremely valuable as a way to provide context for the molecular positions.
14
Multicolor super-resolution imaging is invaluable for the direct visualization of molecular interactions at the nanoscale level [8, 18–20]. Multicolor imaging and analysis has been performed as an extension of standard FPALM using a detection scheme similar to that reported previously for multicolor single molecule imaging [19]. With a detection path divided by a dichroic into two wavelength ranges [19], camera frames con- taining two spatially distinct (but simultaneous) images representing the two emission wavelength ranges are ob- tained. Te two images are correlated and superimposed for localization, while the intensity ratio of one channel over the sum of both channels is used to distinguish the multiple molecular species.
Both PALM and STORM have also reported multicolor
super-resolution images obtained using combinations of cyanine dyes or PA-FPs [18, 20]. Advantages of PA-FPs for localization microscopy include the ability to perform live-cell imaging (that is, without thiol or oxygen-scavenging reagents),
Figure 3: FPALM image of dendrites of fixed rat hippocampal neurons in a culture labeled with Dendra2-PSD95. Green: convention fluorescence. Orange: FPALM image. Credit: D. Santucci, T. Gould, T. Newpher, A. Mabb, M. Ehlers, J. Lisman, and S. Hess.
www.microscopy-today.com • 2011 July
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