Dual Channel RESOLFT Nanoscopy by Using Fluorescent State Kinetics

Abstract

We show that RESOLFT fluorescence nanoscopy, a low light level scanning superresolution technique employing reversibly switchable fluorescent proteins (rsFPs), is capable of dual-channel live-cell imaging that is virtually free of chromatic errors and temporal offsets. This is accomplished using rsEGFP and Dronpa, two rsFPs having similar spectra but different kinetics of switching and fluorescence emission. Our approach is demonstrated by imaging protein distributions and dynamics in living neurons and neuronal tissues.

Novel uses of fluorescent proteins

“…An elegant combination of RESOLFT and time-domain microscopy, dubbed t-RESOLFT, allowed for dual-channel superresolution imaging in living cells of spectrally similar rsEGFP and Dronpa with distinct fluorescent lifetimes and switching kinetics [59]. PAFPs for RESOLFT should also exhibit high switching rate and very low photofatigue over multiple cycles of photoconversion.…”

Linewidth and Writing Resolution

“…This approach is borrowed from the field of super-resolution fluorescence microscopy and is based on the reversible saturable optical fluorescence transition (RESOLFT) concept [21,22]. This approach is borrowed from the field of super-resolution fluorescence microscopy and is based on the reversible saturable optical fluorescence transition (RESOLFT) concept [21,22].…”

“…By optically switching molecules into the "off" state only in the periphery of the light spot, a volume of subdiffraction size with molecules remaining in the "on" state can be obtained [22]. By optically switching molecules into the "off" state only in the periphery of the light spot, a volume of subdiffraction size with molecules remaining in the "on" state can be obtained [22].…”

STED microscopy for nanoscale imaging in living brain slices

“…RESOLFT (reversible saturable optical fluorescence transitions) refers to the general principle behind STED microscopy, where the diffraction barrier is broken by spatially controlling the 'on' and 'off' states of photoswitchable fluorescent proteins [26] or organic dyes [27] in the scanning doughnut configuration or in wide-field imaging [28,29]. This technique uses much lower light intensities than STED microscopy, but requires specifically engineered photoswitchable proteins, which can be difficult to express inside cells.…”