Fig. 3: Measurement of distances between two simultaneously emitting fluorescent molecules by (x,y) scanning with an illumination intensity minimum.
From: Diffraction minima resolve point scatterers at few hundredths of the wavelength
a, Scanning fluorescence microscope with photon-counting detection (APD) of fluorescence passing the dichroic mirror (DM) and a confocal pinhole (PH). The interference of two beams with adjustable phase difference \(\phi\) entering the pupil of the objective lens creates an illumination intensity pattern in the focal plane featuring x orientated or y oriented line-shaped diffraction minima and maxima (MINFLUX set-up). Two fluorophores are sketched as stars. b, Changing \(\phi\) scans the line-shaped minima in the x direction and y direction. c, Top: line-scan principle: linear ramp of \(\phi\) over \(2\uppi\) shifts the minimum across the scatterers, producing a sinusoidal line profile of fluorescence (or scattered signal). Bottom: the continuous line scan is adequately replaced by probing three points near the scatterers with the minimum (MINFLUX recording). d, Normalized counts measured during repeated line scans. The absolute number of counts decreases over time in a stepwise manner as individual fluorophores bleach. Repeated ramping of \(\phi\) over \(2\uppi\) in the x direction and the y direction across the scatterers yields a line-scan stack. e, Averaged counts per line, normalized over the whole stack. Two bleaching steps are clearly visible, marking transitions from two emitting molecules to one and to zero (background). f, Exemplar lines from two molecules and a single emitting molecule show the sinusoidal profile of the fluorescence counts and the spatial COM shift after the first bleaching step. Each line allowed us to extract information based on the photons just near the minimum, just near the maximum or from the entire line. g, Heat map of localizations of the fluorescence COM for two fluorophores at 20 nm distance. Inset: two clusters of localizations are visible showing the COM shift after one fluorophore was bleached. h, Averaged counts per three points MINFLUX measurement for x axis and y axis normalized over the whole measurement. The bleaching steps and fluctuations in fluorophore brightness are clearly visible. i, Normalized counts for each segment. A second order polynomial fit shows a change of the shape of the parabola after the bleaching step as well as a shift of the position of the minimum. Measurement of d relies only on photons from the first (two molecule) segment; the bleaching steps are considered just for an independent control of measured d.
