MINimal photon FLUXes (MINFLUX) offers nanometer localization precision, with lower fluorophore requirements than for other super-resolution microscopy (SRM) techniques. Nonetheless, low localization probabilities hamper its application, and use of less bright and photostable fluorophores, including near-infrared (NIR) fluorophores has been difficult to realize. Here, we devised strategies overcoming these limitations. We systematically studied the blinking properties of far-red and NIR cyanine fluorophores, followed by simulations of MINFLUX localizations, over typical time scales (microsecond to 10 milliseconds), sample and excitation conditions for MINFLUX imaging. We identified fluorophore blinking via photoisomerization and photoreduction as the main cause of localization errors, and that use of balanced redox buffers and repetitive excitation beam scans can suppress such errors. Implementing these strategies, we could demonstrate NIR-MINFLUX imaging with nanometer localization precision, thereby also presenting an overall strategy to design optimal sample and excitation conditions, for MINFLUX imaging and for SRM in general.

Reversible dark state transitions in fluorophores represent a limiting factor in fluorescence-based ultrasensitive spectroscopy, are a necessary basis for fluorescence-based super-resolution imaging, but may also offer additional, largely orthogonal fluorescence-based readout parameters. In this work, we analyzed the blinking kinetics of Cyanine5 (Cy5) as a bar-coding feature distinguishing Cy5 from rhodamine fluorophores having largely overlapping emission spectra. First, fluorescence correlation spectroscopy (FCS) solution measurements on mixtures of free fluorophores and fluorophore-labeled small unilamellar vesicles (SUVs) showed that Cy5 could be readily distinguished from the rhodamines by its reversible, largely excitation-driven trans-cis isomerization. This was next confirmed by transient state (TRAST) spectroscopy measurements, determining the fluorophore dark state kinetics in a more robust manner, from how the time-averaged fluorescence intensity varies upon modulation of the applied excitation light. TRAST was then combined with wide-field imaging of live cells, whereby Cy5 and rhodamine fluorophores could be distinguished on a whole cell level as well as in spatially resolved, multiplexed images of the cells. Finally, we established a microfluidic TRAST concept and showed how different mixtures of free Cy5 and rhodamine fluorophores and corresponding fluorophore-labeled SUVs could be distinguished on-the-fly when passing through a microfluidic channel. In contrast to FCS, TRAST does not rely on single-molecule detection conditions or a high time resolution and is thus broadly applicable to different biological samples. Therefore, we expect that the bar-coding concept presented in this work can offer an additional useful strategy for fluorescence-based multiplexing that can be implemented on a broad range of both stationary and moving samples.

Cyanine fluorophores are extensively used in fluorescence spectroscopy and imaging. Upon continuous excitation, especially at excitation conditions used in single-molecule and super-resolution experiments, photo-isomerized states of cyanines easily reach population probabilities of around 50%. Still, effects of photo-isomerization are largely ignored in such experiments. Here, we studied the photo-isomerization of the pentamethine cyanine 5 (Cy5) by two similar, yet complementary means to follow fluorophore blinking dynamics: fluorescence correlation spectroscopy (FCS) and transient-state (TRAST) excitation-modulation spectroscopy. Additionally, we combined TRAST and spectrofluorimetry (spectral-TRAST), whereby the emission spectra of Cy5 were recorded upon different rectangular pulse-train excitations. We also developed a framework for analyzing transitions between multiple emissive states in FCS and TRAST experiments, how the brightness of the different states is weighted, and what initial conditions that apply. Our FCS, TRAST, and spectral-TRAST experiments showed significant differences in dark-state relaxation amplitudes for different spectral detection ranges, which we attribute to an additional red-shifted, emissive photo-isomerized state of Cy5, not previously considered in FCS and single-molecule experiments. The photo-isomerization kinetics of this state indicate that it is formed under moderate excitation conditions, and its population and emission may thus deserve also more general consideration in fluorescence imaging and spectroscopy experiments.

Photodynamic therapy (PDT) fundamentally relies on local generation of PDT precursor states in added photosensitizers (PS), particularly triplet and photo-radical states. Monitoring these states in situ can provide important feedback but is difficult in practice. The states are strongly influenced by local oxygenation, pH and redox conditions, often varying significantly at PDT treatment sites. To overcome this problem, we followed local PDT precursor state populations of PS compounds, via their fluorescence intensity response to systematically varied excitation light modulation. Thereby, we could demonstrate local monitoring of PDT precursor states of methylene blue (MB) and IRdye700DX (IR700), and determined their transitions rates under different oxygenation, pH and redox conditions. By fiber-optics, using one fiber for both excitation and fluorescence detection, the triplet and photo-radical state kinetics of locally applied MB and IR700 could then be monitored in a tissue sample. Finally, potassium iodide and ascorbate were added as possible PDT adjuvants, enhancing intersystem crossing and photoreduction, respectively, and their effects on the PDT precursor states of MB and IR700 could be locally monitored. Taken together, the presented procedure overcomes current methodological limitations and can offer feedback, guiding both excitation and PDT adjuvant application, and thereby more efficient and targeted PDT treatments.

Photoisomerization kinetics of the near-infrared (NIR) fluorophore Sulfo-Cyanine7 (SCy7) was studied by a combination of fluorescence correlation spectroscopy (FCS) and transient state (TRAST) excitation modulation spectroscopy. A photoisomerized state with redshifted emission was identified, with kinetics consistent with a three-state photoisomerization model. Combining TRAST excitation modulation with spectrofluorimetry (spectral-TRAST) further confirmed an excitation-induced redshift in the emission spectrum of SCy7. We show how this red-emissive photoisomerized state contributes to the blinking kinetics in different emission bands of NIR cyanine dyes, and how it can influence single-molecule, super-resolution, as well as Förster resonance energy transfer (FRET) and multicolor readouts. Since this state can also be populated at moderate excitation intensities, it can also more broadly influence fluorescence readouts, also readouts not relying on high excitation conditions. However, this additional red-emissive state and its photodynamics, as identified and characterized in this work, can also be used as a strategy to push the emission of NIR cyanine dyes further into the NIR and to enhance photosensitization of nanoparticles with absorption spectra further into the NIR. Finally, we show that the photoisomerization kinetics of SCy7 and the formation of its redshifted photoisomer depend strongly on local environmental conditions, such as viscosity, polarity, and steric constraints, which suggests the use of SCy7 and other NIR cyanine dyes as environmental sensors. Such environmental information can be monitored by TRAST, in the NIR, with low autofluorescence and scattering conditions and on a broad range of samples and experimental conditions.

Photo-induced dark transient states of fluorophores can pose a problem in fluorescence spectroscopy. However, their typically long lifetimes also make them highly environment sensitive, suggesting fluorophores with prominent dark-state formation yields to be used as microenvironmental sensors in bio-molecular spectroscopy and imaging. In this work, we analyzed the singlet-triplet transitions of fluorescein and three synthesized carboxy-fluorescein derivatives, with one, two or four bromines linked to the anthracence backbone. Using transient state (TRAST) spectroscopy, we found a prominent internal heavy atom (IHA) enhancement of the intersystem crossing (ISC) rates upon bromination, inferred by density functional theory calculations to take place via a higher triplet state, followed by relaxation to the lowest triplet state. A corresponding external heavy atom (EHA) enhancement was found upon adding potassium iodide (KI). Notably, increased KI concentrations still resulted in lowered triplet state buildup in the brominated fluorophores, due to relatively lower enhancements in ISC, than in the triplet decay. Together with an antioxidative effect on the fluorophores, adding KI thus generated a fluorescence enhancement of the brominated fluorophores. By TRAST measurements, analyzing the average fluorescence intensity of fluorescent molecules subject to a systematically varied excitation modulation, dark state transitions within very high triplet yield (>90%) fluorophores can be directly analyzed under biologically relevant conditions. These measurements, not possible by other techniques such as fluorescence correlation spectroscopy, opens for bio-sensing applications based on high triplet yield fluorophores, and for characterization of high triplet yield photodynamic therapy agents, and how they are influenced by IHA and EHA effects.

Guanine (G)-rich nucleic acids are prone to assemble into four-stranded structures, so-called G-quadruplexes. Abnormal GGGGCC repeat elongations, and in particular their folding states, are associated with amyotrophic lateral sclerosis and frontotemporal dementia. Due to methodological constraints however, most studies of G quadruplex structures are restricted to in vitro conditions. Evidence of how GGGGCC repeats form into G-quadruplexes in vivo is sparse. We devised a readout strategy, exploiting the sensitivity of trans-cis isomerization of cyanine dyes to local viscosity and sterical constraints. Thereby, folding states of cyanine-labeled RNA, and in particular G-quadruplexes, can be identified in a sensitive manner. The isomerization kinetics, monitored via fluorescence blinking generated upon transitions between a fluorescent trans isomer and a non-fluorescent cis isomer, was first characterized for RNA with GGGGCC repeats in aqueous solution using fluorescence correlation spectroscopy and transient state (TRAST) monitoring. With TRAST, monitoring the isomerization kinetics from how the average fluorescence intensity varies with laser excitation modulation characteristics, we could then detect folding states of fluorescently tagged RNA introduced into live cells. 

Mitochondrial membranes and their microenviron-ments directly influence and reflect cellular metabolic states but aredifficult to probe on site in live cells. Here, we demonstrate astrategy, showing how the widely used mitochondrial membranelocalizationfluorophore 10-nonyl acridine orange (NAO) can betransformed into a multifunctional probe of membrane micro-environments by monitoring its blinking kinetics. By transient state(TRAST) studies of NAO in small unilamellar vesicles (SUVs),together with computational simulations, we found that NAOexhibits prominent reversible singlet-triplet state transitions andcan act as a light-induced Lewis acid forming a red-emissivedoublet radical. The resulting blinking kinetics are highlyenvironment-sensitive, specifically reflecting local membrane oxy-gen concentrations, redox conditions, membrane charge,fluidity, and lipid compositions. Here, not only cardiolipin concentrationbut also the cardiolipin acyl chain composition was found to strongly influence the NAO blinking kinetics. The blinking kinetics alsoreflect hydroxyl ion-dependent transitions to and from thefluorophore doublet radical, closely coupled to the proton-transfer eventsin the membranes, local pH, and two- and three-dimensional buffering properties on and above the membranes. Following the SUVstudies, we show by TRAST imaging that thefluorescence blinking properties of NAO can be imaged in live cells in a spatiallyresolved manner. Generally, the demonstrated blinking imaging strategy can transform existingfluorophore markers intomultiparametric sensors reflecting conditions of large biological relevance, which are difficult to retrieve by other means. This opensadditional possibilities for fundamental membrane studies in lipid vesicles and live cells