Time-resolved molecule counting by photon statistics across the visible spectrum

Grußmayer, Kristin S.; Herten, Dirk‐Peter

doi:10.1039/c7cp00363c

Cited by 21 publications

(35 citation statements)

References 24 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Since the photon correlation SNR increases monotonically with fluorescence quantum yield (QY) and the excitation repetition rate, labels should exhibit a high QY under close-to-saturation conditions. Photostability for a duration in the scale of a second, necessary for high SNR and to avoid scanning artifacts, has been demonstrated for colloidal QDs, solid-state defects and a variety of dye molecules routinely used in bio-imaging 31,36,38 . Finally, in order to achieve high photon rates under saturation conditions, the emitter's excited state lifetime should preferably be below a microsecond.…”

Section: Discussionmentioning

confidence: 99%

Super-resolution enhancement by quantum image scanning microscopy

et al. 2018

View full text Add to dashboard Cite

The principles of quantum optics have yielded a plethora of ideas to surpass the classical limitations of sensitivity and resolution in optical microscopy. While some ideas have been applied in proof-of-principle experiments, imaging a biological sample has remained challenging mainly due to the inherently weak signal measured and the fragility of quantum states of light. In principle, however, these quantum protocols can add new information without sacrificing the classical information and can therefore enhance the capabilities of existing super-resolution techniques. Image scanning microscopy (ISM), a recent addition to the family of super-resolution methods, generates a robust resolution enhancement without sacrificing the signal level. Here we introduce quantum image scanning microscopy (Q-ISM): combining ISM with the measurement of quantum photon correlation allows increasing the resolution of ISM up to two-fold, four times beyond the diffraction limit. We introduce the Q-ISM principle and obtain super-resolved optical images of a biological sample stained with fluorescent quantum dots using photon antibunching, a quantum effect, as a resolution enhancing contrast mechanism. Main TextThe diffraction limit, as formulated by Abbe, sets the attainable resolution in far-field optical microscopy to about half of the visible wavelength 1 , hindering its applicability in life science studies at very small scales. Over the past two decades, several super-resolution methods have successfully overcome the diffraction limit, including emission depletion microscopy, localization microscopy and structured illumination microscopy 2-6 . The continuous and rapid improvement in detector technology has enabled two more recent developments in the field of super-resolution microscopy, which are the center of this work: quantum super-resolution microscopy and image scanning microscopy (ISM). As for the first, a surge of interest in super-resolution imaging based on quantum optics concepts 7-13 , inspired and facilitated by the progress in high temporal resolution imagers, resulted in a few successful proof-of-principle demonstrations 7,8,14 . The second, ISM, relies on a small array of fast detector and offers a two-fold enhancement of resolution 15,16 . Since ISM is compatible with a standard confocal microscope architecture it has already been integrated into commercial products.While all super-resolution modalities violate at least one of the basic assumptions of the Abbe theory, many rely on breaking more than one. For instance, stimulated emission depletion (STED) and saturated structured illumination microscopy (SSIM) breach both the assumption of a linear response of a fluorophore to the excitation light and that of a uniform illumination field 17,18 . In contrast, the few demonstrations of quantum super-resolution microscopy 7,8,14 relied solely on violating the implicit assumption, underlying Abbe's derivation, that light behaves as waves rather than particles. ISM, as well, depends on violating a single assumption, a u...

show abstract

Section: Discussionmentioning

confidence: 99%

Super-resolution enhancement by quantum image scanning microscopy

et al. 2018

View full text Add to dashboard Cite

show abstract

“…(Fig. 4e) [25][26][27]56 . As already mentioned, some higher DOL antibodies showed stepwise photobleaching ( Supplementary Fig.…”

Section: Photon Antibunching Experiments Of Multiple-al647-labeled Anmentioning

confidence: 99%

“…For sufficiently short laser pulses the number of photon-pairs detected per laser pulse can be used to determine whether the emission is from one or more independently emitting quantum systems. Therefore, we used the classical Hanbury-Twiss and Brown coincidence setup 54 in combination with pulsed excitation to determine the number of independently emitting Al647 molecules per multiple-labeled antibody [25][26][27][28][29]55,56 . To record single-molecule fluorescence transients, single antibodies were selected from an image scan and positioned in the laser focus of a confocal microscope.…”

Section: Photon Antibunching Of Multiple-al647-labeled Antibodies In mentioning

confidence: 99%

“…Since the intensity of the central peak contains information about the number of independently emitting molecules, the Nc/Nl,av ratio can be used for molecular counting. For example, neglecting background, Nc/Nl,av ratios of 0.0, 0.5, 0.67, and 0.75 are expected for 1-4 independently emitting fluorophores, respectively [25][26][27][28][29]55,56 . From the interphoton times histogram recorded from a single double-stranded DNA construct labeled with a single Al647 molecule we determined Nc/Nl,av ratios of 0.03 ± 0.004 and 0.18 ± 0.04 in PBS and photoswitching buffer, respectively ( Supplementary Figs.…”

Section: Photon Antibunching Of Multiple-al647-labeled Antibodies In mentioning

confidence: 99%

See 1 more Smart Citation

Multiple-labeled antibodies behave like single emitters in photoswitching buffer

Helmerich

Beliu

Sauer

2020

Preprint

View full text Add to dashboard Cite

The degree of labeling (DOL) of antibodies has so far been optimized for high brightness and specific and efficient binding. The influence of the DOL on the blinking performance of antibodies used in direct stochastic optical reconstruction microscopy (dSTORM) has so far attained limited attention. Here, we investigated the spectroscopic characteristics of IgG antibodies labeled at DOLs of 1.1- 8.3 with Alexa Fluor 647 (Al647) at the ensemble and single-molecule level. Multiple-Al647-labeled antibodies showed weak and strong quenching interactions in aqueous buffer but could all be used for dSTORM imaging with spatial resolutions of ∼ 20 nm independent of the DOL. Photon antibunching experiments in aqueous buffer demonstrate that the emission of multiple-Al647-labeled antibodies switches from classical to non-classical light in photoswitching buffer. We developed a model that explains the observed blinking of multiple-labeled antibodies and can be used advantageously to develop improved fluorescent probes for dSTORM experiments.

show abstract

“…3 Among them are methods relying on brightness calibration, 4 counting of photobleaching steps, 5,6 localization microscopy, [7][8][9][10] or photon antibunching. 11,12 To date, photobleaching step analysis (PBSA) and brightness estimation are most widely used in biological applications of molecular counting, due to their simplicity in data acquisition and the relatively straight-forward interpretation. 13 PBSA has the advantage that no calibra-tion measurements are necessary and that it is relatively robust to variations in molecular brightness.…”

Section: Introductionmentioning

confidence: 99%

Photobleaching step analysis for robust determination of protein complex stoichiometries

Hummert

Yserentant

Fink

et al. 2020

Preprint

Self Cite

View full text Add to dashboard Cite

The composition of cellular structures on the nanoscale is a key determinant of macroscopic functions in cell biology and beyond. Different fluorescence single-molecule techniques have proven ideally suited for measuring protein copy numbers of cellular structures in intact biological samples. Of these, photobleaching step analysis poses minimal demands on the microscope and its counting range has significantly improved with more sophisticated algorithms for step detection, albeit at an increasing computational cost. Here, we present a comprehensive framework for photobleaching step analysis, optimizing both data acquisition and analysis. To make full use of the potential of photobleaching step analysis, we evaluate various labelling strategies with respect to their molecular brightness and photostability. The developed analysis algorithm focuses on automation and computational efficiency. Moreover, we benchmark the framework with experimental data acquired on DNA origami labeled with defined fluorophore numbers to demonstrate counting of up to 35 fluorophores. Finally, we show the power of the combination of optimized trace acquisition and automated data analysis for robust protein counting by counting labelled nucleoporin 107 in nuclear pore complexes of intact U2OS cells. The successful in situ application promotes this framework as a new resource enabling cell biologists to robustly determine the stoichiometries of molecular assemblies at the single-molecule level in an automated fashion.

show abstract

Time-resolved molecule counting by photon statistics across the visible spectrum

Cited by 21 publications

References 24 publications

Super-resolution enhancement by quantum image scanning microscopy

Super-resolution enhancement by quantum image scanning microscopy

Multiple-labeled antibodies behave like single emitters in photoswitching buffer

Photobleaching step analysis for robust determination of protein complex stoichiometries

Contact Info

Product

Resources

About