Localization based super-resolution microscopy techniques require precise drift correction methods because the achieved spatial resolution is close to both the mechanical and optical performance limits of modern light microscopes. Multi-color imaging methods require corrections in addition to those dealing with drift due to the static, but spatially-dependent, chromatic offset between images. We present computer simulations to quantify this effect, which is primarily caused by the high-NA objectives used in super-resolution microscopy. Although the chromatic offset in well corrected systems is only a fraction of an optical wavelength in magnitude (<50 nm) and thus negligible in traditional diffraction limited imaging, we show that object colocalization by multi-color super-resolution methods is impossible without appropriate image correction. The simulated data are in excellent agreement with experiments using fluorescent beads excited and localized at multiple wavelengths. Finally we present a rigorous and practical calibration protocol to correct for chromatic optical offset, and demonstrate its efficacy for the imaging of transferrin receptor protein colocalization in HeLa cells using two-color direct stochastic optical reconstruction microscopy (dSTORM).

An imaging method and optical system, referred to as a line-scanning tomographic optical microscope (LSTOM) using a combination of line-scanning technique and CT reconstruction principle, is proposed and studied theoretically and experimentally. In our implementation a narrow focus line is scanned over the sample and the reflected light is measured in a confocal arrangement. One such scan is equivalent to a transverse projection in tomography. Repeating the scanning procedure in several directions, a number of transverse projections are recorded from which the image can be obtained using conventional CT reconstruction algorithms. The resolution of the image is independent of the spatial dimensions and structure of the applied detector; furthermore, the transfer function of the system is isotropic. The imaging performance of the implemented confocal LSTOM was compared with a point-scanning confocal microscope, based on recorded images. These images demonstrate that the resolution of the confocal LSTOM exceeds (by 15%) the resolution limit of a point-scanning confocal microscope.

Line-scanning tomographic optical microscopy (LSTOM) requires precise rotation of the scanning line. We demonstrate a method that applies translation-invariant optical elements (polarizer and birefringent plate) to minimize the rotation error. An astigmatic line produced by means of a focused beam through a birefringent plate is used as line illumination. A comparative theoretical and experimental study is presented using an LSTOM system.

A line-scanning tomographic optical microscope system requires precise rotation of the scanning line. Center of rotation error introduced by both the imprecision of optical and mechanical components is studied experimentally and via simulations. It was shown that a practical tolerance limit can be chosen where the influence of the investigated error on the reconstructed image quality remains insignificant. An effective and simply practical solution was presented to keep the center of rotation error below this tolerance limit and the spatial resolution of the reconstructed image close to the diffraction limit.

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