Correcting chromatic offset in multicolor super-resolution localization microscopy

Abstract

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).

madSTORM: a superresolution technique for large-scale multiplexing at single-molecule accuracy

“…Simultaneous visualization of multiple targets requires fluorescent probes with nonoverlapping spectral profiles, generally restricting fluorescence-based light microscopy to six colors and SMLM to two or three colors (Bates et al ., 2007; Dempsey et al ., 2011; van de Linde et al ., 2011). Moreover, nonlinear chromatic aberration causes misalignment of multicolor images (Pertsinidis et al ., 2010; Erdelyi et al ., 2013). To overcome these limits, previous studies imaged multiple targets using repetitive photobleaching or chemical quenching of sequentially bound fluorophores (Schubert et al ., 2006; Nanguneri et al ., 2012; Gerdes et al ., 2013; Jungmann et al ., 2014; Tam et al ., 2014; Valley et al ., 2015).…”

Correcting field-dependent aberrations with nanoscale accuracy in three-dimensional single-molecule localization microscopy

“…This single, global calibration does not sample field-dependent variations. While in principle it would be possible to scan one or several fluorescent beads throughout the 3D volume, as has been done for 2D registration [50–52], the additional delay from scanning in z (10–50 s for each scan) makes bead scanning more difficult, as nanoscale drift of the microscope stage between scans and bleaching of the beads can introduce systematic differences between calibrations that do not reflect true field-dependent variation of the optical instrument.…”

Origin and compensation of imaging artefacts in localization-based super-resolution microscopy

“…Polychromatic PSF was calculated for each emitter by means of a direct integration method [19,21]. The wavelength ranges were set taking into consideration the transmission windows of the emission filters [22] and were matched to the absorption/emission spectra of the most common dyes that can be excited at 405 nm, 488 nm, 561 nm and 635 nm, respectively. In the image plane the pixel size was typically chosen to be 16 µm, the same as applied during the experiments.…”

“…In case of sequential excitation, the different fluorescent dyes are excited separately in time and the full chip size of a single detector can be used. However, registration of the two images is still necessary because of the lateral chromatic aberration of the imaging system [39]. Such registration can be done with a single bead scanned through the FOV or multiple beads located randomly or in an ordered way.…”