Live-cell monochromatic dual-label sub-diffraction microscopy by mt-pcSOFI

Abstract

We expand photochromic super-resolution optical fluctuation imaging (pcSOFI) to monochromatic dual-channel sub-diffraction microscopy. Multi-tau (mt-)pcSOFI unmixes spectrally identical reversibly switchable fluorescent proteins (RSFPs) based on their blinking kinetics. We show that mt-pcSOFI can be used to simultaneously image two structures in living cells with existing RSFPs and the newly developed ffDronpa-F.

Quantitative comparison of camera technologies for cost-effective super-resolution optical fluctuation imaging (SOFI)

“…The emission spectrum of ffDronpa peaks at about 515 nm, which coincides well with the region of highest detection efficiency of all three cameras (see figure 1, panels (A) and (B)). In all cases we adjusted the excitation intensity to obtain an emitter τ-value [18] (decorrelation time) of around one exposure time (see figure 1, panel (F)). This value offers the highest signal for reconstruction using conventional second-order SOFI imaging without lag time. )…”

“…Probes that display these dynamics, such as photochromic fluorescent proteins, have been shown to result in a two-to three-fold spatial resolution improvement over the classical diffraction-limited resolution [13,14]. The SOFI framework even allows for multiplexing of several emitters, with highly similar steady-state fluorescence characteristics, using differences in their blinking behavior [18]. In addition to the conventional visualization of fluorophore distributions, SOFI has also been used to visualize biosensor activities with similar spatial resolution enhancements [19,20].…”

Quantitative comparison of camera technologies for cost-effective Super-resolution Optical Fluctuation Imaging (SOFI)

“…The cell suspension was then seeded on 35 mm glass bottom culture dishes (#1.5 thickness, MatTek) to ensure a confluency of 50% to 80% for transfection. Cells were then transfected with pcDNA::MAP4-ffDronpa 18 using FuGENE6 (Promega) according to manufacturer's instructions, and cells were incubated for a maximum of 24 hours before imaging. For the imaging process the media was replaced with PBS.…”

“…Probes that display these dynamics, such as photochromic fluorescent proteins, have been shown to result in a two-to three-fold spatial resolution improvement over the classical diffraction-limited resolution 13,14 . The SOFI framework even allows for multiplexing of several emitters, with highly similar steady-state fluorescence characteristics, using differences in their blinking behavior 18 . In addition to the conventional visualization of fluorophore distributions, SOFI has also been used to visualize biosensor activities with similar spatial resolution enhancements 19,20 .…”

Joint tagging assisted fluctuation nanoscopy enables fast high‐density super‐resolution imaging

“…The spatial resolution improvement comes at the cost of sacrificing the temporal resolution. In recent years, the developments of fluctuation‐based super‐resolution microscopy, such as super‐resolution optical fluctuation imaging (SOFI), super‐resolution radial fluctuation (SRRF), etc., make it easier to obtain a high‐spatial resolution with fewer frame numbers by exploring the inherent blinking nature of fluorophores . SOFI achieves super‐resolution reconstruction through calculating the temporal auto‐cumulants or spatiotemporal cross‐cumulants of the blinking image sequences.…”