![]() 1 A and S2).Ī dual-color 3D single-molecule localization microscopy approach. ![]() The same dichroic can also be used to introduce an axial separation between the focal planes of the two channels to implement biplane 3D imaging ( Figs. The assignment of this localization pair to either dye is based on the ratiometric fluorescence intensity. Both channels exhibit cross-talk in either direction, and an emitting fluorophore produces a localization on each side of the camera chip. Their partially overlapping emission is spectrally separated by a dichroic beamsplitter and imaged onto two separate parts of the camera chip. ![]() Here we used the far-red dyes AlexaFluor647 (AF647) and CF680, which are both highly performing in terms of brightness and duty-cycle in the same conventional blinking buffer ( Fig. S1 in the Supporting Material). In spectral-demixing, a single laser is used to excite two spectrally close fluorophores. Existing setups can be readily extended to two-color imaging capability with only minor changes. In this Letter we provide a simple and robust approach to two-color SMLM imaging in three dimensions by combining an established dual-color scheme based on spectral-demixing ( 13–16) with biplane imaging. Some of these methods provide isotropic or close-to-isotropic 3D resolution, but require complex optical setups as a tradeoff. Other methods employ interferometric detection, such as iPALM ( 11) and 4PI-SMS ( 12). The z position of a single molecule can be obtained from the shape of its point-spread function (PSF) by employing two focal planes as in biplane imaging ( 7) or after shaping the PSF by using an astigmatic ( 8), double-helical ( 9), or self-bending PSF ( 10). Several optical methods have been developed to access the axial dimension for subdiffraction 3D resolution. Methods to estimate and correct for this exist ( 6), but rely on additional experiments and the extent of cross-talk will vary with the employed activator/reporter conjugated reagent. ![]() Although being completely free from chromatic aberrations and requirement for image registration, the activator/reporter method can be prone to color cross-talk, owing to spontaneous or nonspecific activation. ![]() In the latter approach, the same reporter dye can be switched by spectrally distinct activator dyes in close proximity. In the former approach, the compound multicolor resolution will also depend on the precision with which the two images can be registered, and accuracies of <10 nm involve very elaborate and sophisticated calibration experiments, even more so in three dimensions ( 5). Regarding multicolor imaging, the two most widely used strategies include either excitation of spectrally well-separated fluorophores with multiple laser lines and detection through a dichroic beamsplitter or different emission filters ( 3), or the use of an activator/reporter dye system ( 4). Major challenges in technical development now lie in multicolor and three-dimensional (3D) imaging. Such techniques allow the resolution of important cellular structures in unprecedented detail. Single-molecule localization microscopy (SMLM) techniques ( 1,2) rely on stochastic activation and precise localization of individual fluorophores. ![]()
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