ISM-FLUX: MINFLUX with an array detector*

Slenders, Eli; Vicidomini, Giuseppe

doi:10.1101/2022.04.19.488747

Cited by 4 publications

(9 citation statements)

References 42 publications

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“…In conclusion, iSMLM represents a significant expansion of ISM into the realm of SMLM, akin to ISM extensions of other super-resolution modalities such as STED microscopy [49], MINFLUX [50], or Super-resolution Optical Fluctuation Imaging (SOFI) [51][52][53]. However, iSMLM distinguishes itself with its simplicity, versatility, and seamless incorporation of fluorescence lifetime imaging capabilities.…”

Section: Discussionmentioning

confidence: 99%

Doubling the resolution of fluorescence-lifetime single-molecule localization microscopy with image scanning microscopy

Radmacher,

Nevskyi,

Gallea

et al. 2023

Preprint

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Single-molecule localization microscopy (SMLM) is a widely used super-resolution microscopy technique, renowned for its simplicity and impressive achievable resolution. It is typically based on a wide-field fluorescence microscope and relies on emitter photoswitching to capture individual snapshots of a sample with sparse distributions of fluorescent labels. These labels can then be precisely identified and localized. Recently, we demonstrated that SMLM can also be realized with a fast confocal laser-scanning microscope (CLSM), opening the door to fluorescence lifetime SMLM. This technique has found applications in lifetime-based image multiplexing and metal-induced energy transfer SMLM. In this work, we present an extension of CLSM-based SMLM by incorporating a single-photon detector array into the CLSM. This enables the combination of CLSM-based SMLM with Image Scanning Microscopy (ISM), a powerful technique for doubling the lateral resolution of a laser-scanning confocal microscope. By doing so, we achieve a two-fold improvement in lateral localization accuracy for SMLM in an easy and straightforward manner. Furthermore, we take advantage of the lifetime information provided by our CLSM for colocalizing different targets without any chromatic aberration. This feature becomes particularly crucial when dealing with the few-nanometer resolution achievable with our system. To demonstrate the capabilities of fluorescence lifetime ISM-SMLM (FL-iSMLM), we perform dSTORM and DNA-PAINT experiments on fluorescently labeled cells, showcasing the resolution-doubling and fluorescence lifetime multiplexing of our system. Finally, we have developed an open-source software that allows any interested user to easily implement (FL)-iSMLM into an existing CLSM equipped with a fast array detector. This user-friendly approach enables widespread adoption and application of (FL)-iSMLM in various research settings.

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Section: Discussionmentioning

confidence: 99%

Doubling the resolution of fluorescence-lifetime single-molecule localization microscopy with image scanning microscopy

Radmacher,

Nevskyi,

Gallea

et al. 2023

Preprint

View full text Add to dashboard Cite

show abstract

“…To find the emitter position, we extended the maximum likelihood approach from MINFLUX to the use of an array detector (10). The likelihood function for the emitter at position r E can be written as…”

Section: Resultsmentioning

confidence: 99%

“…However, a constraint of MINFLUX is that the molecule to be localized must reside within the TCP. If the molecule is outside the TCP, its position cannot always be uniquely determined from the data, and the localization uncertainty rapidly grows for a given number of photons (10). This limitation translates into a lack of robustness for the pure MINFLUX concept, which needs to be compensated by implementing iterative localization approaches.…”

Section: Introductionmentioning

confidence: 99%

“…Even though MINFLUX has shown superior results in single-molecule localization and tracking (11)(12)(13), the throughput and robustness might be further improved. We have recently theoretically shown that substituting the singleelement detector with an array detector, such as an asynchronous read-out SPAD array detector, can maximize the localization range of MINFLUX (10). Asynchronous readout SPAD array detectors are transforming confocal laserscanning microscopy (14)(15)(16).…”

Section: Introductionmentioning

confidence: 99%

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Array Detection Enables Large Localization Range for Simple and Robust MINFLUX

Slenders,

Patil,

Held

et al. 2024

Preprint

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The MINFLUX concept significantly enhances the spatial resolution of single-molecule localization microscopy (SMLM) by overcoming the limit imposed by the fluorophore’s photon counts. Typical MINFLUX microscopes localize the target molecule by scanning a zero-intensity focus around the molecule in a circular trajectory, with smaller trajectory diameters yielding lower localization uncertainties for a given number of photons. Since this approach requires the molecule to be within the scanned trajectory, MINFLUX typically relies on a photon-demanding iterative scheme with decreasing trajectory diameters. Although the iterative procedure does not substantially reduce the photon efficiency of MINFLUX, this approach is prone to misplacements of the trajectory and increases the system’s complexity. In this work, we introduce ISM-FLUX, a novel implementation of MINFLUX using image-scanning microscopy (ISM) with a single-photon avalanche diode (SPAD) array detector. ISM-FLUX provides precise MINFLUX localization within the trajectory while maintaining conventional photon-limited uncertainty outside it. The robustness of ISM-FLUX localization results in a larger localization range and greatly simplifies the architecture, which may facilitate broader adoption of MIN-FLUX.

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“…Depending on the spatial arrangement of the excitation beams, molecules in distances of up to 100 nm can be tracked simultaneously. Similar to all MINFLUX experiments, this field of view could be further extended by the use of single-photon avalanche detector arrays, 39 yielding the simultaneous nanometer precise localizations of multiple emitters over a large range of distances. Overall, we envision that further developing these approaches will pave the way for nanometer precise multi-color tracking experiments in living cells.…”

Section: Discussionmentioning

confidence: 99%

Super-resolved FRET and co-tracking in pMINFLUX

Cole,

Zähringer,

Bohlen

et al. 2024

Nat. Photon.

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Single-molecule FRET (smFRET) is widely used to investigate dynamic (bio) molecular interactions taking place over distances of up to 10 nm. With the advent of recent super-resolution methods such as MINFLUX, MINSTED or RASTMIN, the spatiotemporal resolution of these techniques advanced towards the smFRET regime. While these methods do not suffer from the spatial restriction of FRET, they only visualize one emitter at a time, thus rendering fast dynamics of interactions out of reach. Here, we describe two approaches to overcome this limitation in pMINFLUX using its intrinsic fluorescence lifetime information. First, we combined pMINFLUX with smFRET. This enabled us to track a FRET donor fluorophore and simultaneously colocalize its FRET acceptor with nanometer precision. To extend co-localized tracking beyond the FRET range, we developed pMINFLUX lifetime multiplexing, a method to simultaneously track two fluorophores with similar spectral properties but distinct fluorescence lifetimes. We demonstrated its application on both static and dynamic DNA origami systems with a precision better than 2 nm. Within the FRET range, pMINFLUX lifetime multiplexing additionally uses a novel combined phasor-microtimegating approach. This paves the way for nanometer precise co-localized tracking for inter-dye distances between 4 nm and 100 nm, closing the resolution gap between smFRET and co-tracking.

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ISM-FLUX: MINFLUX with an array detector*

Cited by 4 publications

References 42 publications

Doubling the resolution of fluorescence-lifetime single-molecule localization microscopy with image scanning microscopy

Doubling the resolution of fluorescence-lifetime single-molecule localization microscopy with image scanning microscopy

Array Detection Enables Large Localization Range for Simple and Robust MINFLUX

Super-resolved FRET and co-tracking in pMINFLUX

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