MINFLUX, or minimal fluorescence photon fluxes microscopy, is a super-resolution light microscopy method that images and tracks objects in two and three dimensions with single-digit nanometer resolution. MINFLUX uses a structured excitation beam with at least one intensity minimum – typically a doughnut-shaped beam with a central intensity zero – to elicit photon emission from a fluorophore. The position of the excitation beam is controlled with sub-nanometer precision, and when the intensity zero is positioned exactly on the fluorophore, the system records no emission. Thus, the system requires few emitted photons to determine the fluorophore's location with high precision. In practice, overlapping the intensity zero and the fluorophore would require a priori location knowledge to position the beam. As this is not the case, the excitation beam is moved around in a defined pattern to probe the emission from the fluorophore near the intensity minimum. Each localization takes less than 5 microseconds, so MINFLUX can construct images of nanometric structures or track single molecules in fixed and live specimens by pooling the locations of fluorescent labels. Because the goal is to locate the point where a fluorophore stops emitting, MINFLUX significantly reduces the number of fluorescence photons needed for localization compared to other methods. A commercial MINFLUX system is available from abberior instruments GmbH.
Principle MINFLUX overcomes the Abbe diffraction limit in light microscopy and distinguishes individual fluorescing molecules by leveraging the photophysical properties of fluorophores. The system temporarily silences (sets in an OFF-state) all but one molecule within a diffraction-limited area (DLA) and then locates that single active (in an ON-state) molecule. Super-resolution microscopy techniques like stochastic optical reconstruction microscopy (STORM) and photoactivated localization microscopy (PALM) do the same. However, MINFLUX differs in how it determines the molecule's location. The excitation beam used in MINFLUX has a local intensity minimum or intensity zero. The position of this intensity zero in a sample is adjusted via control electronics and actuators with sub-nanometer spatial and sub-microsecond temporal precision. When the active molecule located at r → m {\displaystyle {\vec {r}}_{m}} is in a non-zero intensity area of the excitation beam, it fluoresces. The number of photons n {\displaystyle n} emitted by the active molecule is proportional to the excitation beam intensity at that position.
In the vicinity of the excitation beam intensity zero, the intensity I {\displaystyle I} of the emission from the active molecule when the intensity zero is located at position r → {\displaystyle {\vec {r}}} can be approximated by a quadratic function. Therefore, the recorded number of emission photons is:
n ( r → , r → m ) = c I = c ( r → − r → m ) 2 {\displaystyle n({\vec {r}},{\vec {r}}_{m})=cI=c({\vec {r}}-{\vec {r}}_{m})^{2}}
where c {\displaystyle c} is a measure of the collection efficiency of detection, the absorption cross-section of the emitter, and the quantum yield of fluorescence. In other words, photon fluxes emitted by the active molecule when it is located close to the zero-intensity point of the excitation beam carry information about its distance to the center of the beam. That information can be used to find the position of the active molecule. The position is probed with a set of K {\displaystyle K} excitation intensities { I 0 , . . . , I K − 1 } {\displaystyle \{I_{0},...,I_{K-1}\}} . For example, the active molecule is excited with the same doughnut-shaped beam moved to different positions. The probing results in a corresponding set of photon counts { n 0 , . . . , n K − 1 } {\displaystyle \{n_{0},...,n_{K-1}\}} . These photon counts are probabilistic; each time such a set is measured, the result is a different realization of photon numbers fluctuating around a mean value. Since their distribution follows Poissonian statistics, the expected position of the active molecule can be estimated from the photon numbers, using, for example, a maximum likelihood estimation of the form:
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