This upload contains raw data and simulation software underlying the results presented in a manuscript submitted for peer-review, with the title: Near-infrared MINFLUX imaging enabled by suppression of fluorophore blinking Authored by:C Venugopal Srambickal1,, H Esmaeeli1,, J Piguet1, L Reinkensmeier2, R Siegmund2, A Agostinho3 ,M Bates2, A Egner2, J Widengren1,1 Experimental Biomolecular Physics, Bio-Opto-Nano Unit, Department of Applied Physics, Royal Institute of Technology, SE-10691 Stockholm, Sweden2 Department of Optical Nanoscopy, Institute for Nanophotonics, D-37077 Göttingen, Germany3 Science for Life Laboratory, Department of Applied Physics, Royal Institute of Technology, SE-17165 Solna, Sweden Contributed equally* Corresponding author: [email protected] ABSTRACTMINimal photon FLUXes (MINFLUX) offers super-resolution microscopy (SRM) with nanometer localization precision, with lower fluorophore brightness and photostability requirements than for other SRM techniques. Nonetheless, low localization probabilities have been reported in several MINFLUX studies, and a broader use of less bright and photostable fluorophores, including near-infrared (NIR) fluorophores has been difficult to realize. In this work, we identified fluorophore blinking as a main cause of erroneous (and dismissed) fluorophore localizations in MINFLUX imaging and devised strategies to overcome these effects. We systematically studied the blinking/switching properties of cyanine fluorophores emitting in the far-red or NIR range, over typical time scales (µs-10ms), sample and excitation conditions used in MINFLUX imaging. Subsequent simulations of representative MINFLUX localization procedures showed that trans-cis isomerization, and in particular photo-reduction of the fluorophores, can generate significant localization errors. These localization errors, however, could be suppressed by balanced redox buffers and repetitive excitation beam scans. Implementing these strategies, and replacing the slower, intrinsic switching of the fluorophores needed for the localization by transient binding of fluorophore-labelled DNA strands to complementary DNA strands attached to the targets (DNA-PAINT), we could for the first time demonstrate NIR-MINFLUX imaging with nanometer localization precision. This work presents an overall strategy, where fluorophore blinking characterization and subsequent simulations make it possible to design optimal sample and excitation conditions, opening for NIR-MINFLUX imaging, as well as for a broader use of fluorophores in MINFLUX and related SRM studies.Acknowledgements:This study was supported by the European Union's Horizon 2020 research and innovation program under grant agreement 101017180 (NanoVIB). Files with raw data on which the manuscript is based are grouped into folders according to the figures/tables in the manuscript where the extracted results are presented. Additionally, software developed and used for the simulations are arranged into a separate folder.

This upload contains raw data and simulation software underlying the results presented in a manuscript submitted for peer-review, with the title: Near-infrared MINFLUX imaging enabled by suppression of fluorophore blinking Authored by:C Venugopal Srambickal1,, H Esmaeeli1,, J Piguet1, L Reinkensmeier2, R Siegmund2, A Agostinho3 ,M Bates2, A Egner2, J Widengren1,1 Experimental Biomolecular Physics, Bio-Opto-Nano Unit, Department of Applied Physics, Royal Institute of Technology, SE-10691 Stockholm, Sweden2 Department of Optical Nanoscopy, Institute for Nanophotonics, D-37077 Göttingen, Germany3 Science for Life Laboratory, Department of Applied Physics, Royal Institute of Technology, SE-17165 Solna, Sweden Contributed equally* Corresponding author: [email protected] ABSTRACTMINimal photon FLUXes (MINFLUX) offers super-resolution microscopy (SRM) with nanometer localization precision, with lower fluorophore brightness and photostability requirements than for other SRM techniques. Nonetheless, low localization probabilities have been reported in several MINFLUX studies, and a broader use of less bright and photostable fluorophores, including near-infrared (NIR) fluorophores has been difficult to realize. In this work, we identified fluorophore blinking as a main cause of erroneous (and dismissed) fluorophore localizations in MINFLUX imaging and devised strategies to overcome these effects. We systematically studied the blinking/switching properties of cyanine fluorophores emitting in the far-red or NIR range, over typical time scales (µs-10ms), sample and excitation conditions used in MINFLUX imaging. Subsequent simulations of representative MINFLUX localization procedures showed that trans-cis isomerization, and in particular photo-reduction of the fluorophores, can generate significant localization errors. These localization errors, however, could be suppressed by balanced redox buffers and repetitive excitation beam scans. Implementing these strategies, and replacing the slower, intrinsic switching of the fluorophores needed for the localization by transient binding of fluorophore-labelled DNA strands to complementary DNA strands attached to the targets (DNA-PAINT), we could for the first time demonstrate NIR-MINFLUX imaging with nanometer localization precision. This work presents an overall strategy, where fluorophore blinking characterization and subsequent simulations make it possible to design optimal sample and excitation conditions, opening for NIR-MINFLUX imaging, as well as for a broader use of fluorophores in MINFLUX and related SRM studies.Acknowledgements:This study was supported by the European Union's Horizon 2020 research and innovation program under grant agreement 101017180 (NanoVIB). Files with raw data on which the manuscript is based are grouped into folders according to the figures/tables in the manuscript where the extracted results are presented. Additionally, software developed and used for the simulations are arranged into a separate folder.

This folder contains all raw data underlying the results presented in a manuscript, submitted to Nano Letters, and entitled:Photophysical lock-in detection enables background-free upconversion emission imagingAuthored by:Niusha Bagheria, Chenyi Wangb, Du Guoa, Anbharasi Lakshmanana, Qi Zhua, Xu Chena, Nahid Ghazyanic, Qiuqiang Zhanb, Georgios A. Sotirioud, Haichun Liua, Jerker Widengrena a Experimental Biomolecular Physics, Department of Applied Physics, KTH Royal Institute of Technology, SE-106 91, Stockholm, Swedenb Centre for Optical and Electromagnetic Research, South China Academy of Advanced Optoelectronics, South China Normal University, Guangzhou 510006, P. R. Chinac Faculty of Physics, Kharazmi University, Tehran, Iran.d Department of Microbiology Tumor and Cell Biology Karolinska Institute, SE-171 77, Stockholm, SwedenCorresponding authors:*[email protected], [email protected] data files containing raw data and results of the analysis are grouped according to the different figures in the manuscript where the extracted results are presented.ABSTRACTLanthanide-based upconversion nanoparticles (UCNPs) have attracted considerable attention in biomedical applications, due to their anti-Stokes shifted emission enabling autofluorescence-free signal detection. However, residual excitation light can still interfere with their relatively low brightness. While commonly used lock-in detection can distinguish weak signals from substantial random background, concurrently modulated residual excitation light is not eliminated. This remains a challenge, particularly under demanding experimental conditions.Here, we propose a photophysical lock-in detection (PP-LID) approach based on the discovery that UCNPs can act as frequency mixers in response to intensity-modulated excitation. Particularly, modulated excitation with more than one base modulation frequency can generate additional low-frequency beating-signals. These signals are resolvable by frame-rate-limited cameras, devoid of ambient and residual excitation light, and can be regulated through nanoparticle engineering. Extracting beating-signals by PP-LID thus provides a strategy to significantly enhance signal-to-background conditions in UCNP-based bioimaging and biosensing.Keywords: Upconversion nanoparticles (UCNPs), nonlinearity, modulation, lock-in detection, second harmonic, beating frequency, fast Fourier Transform (FFT)

This folder contains all raw data underlying the results presented in a manuscript, submitted to Nano Letters, and entitled:Photophysical lock-in detection enables background-free upconversion emission imagingAuthored by:Niusha Bagheria, Chenyi Wangb, Du Guoa, Anbharasi Lakshmanana, Qi Zhua, Xu Chena, Nahid Ghazyanic, Qiuqiang Zhanb, Georgios A. Sotirioud, Haichun Liua, Jerker Widengrena a Experimental Biomolecular Physics, Department of Applied Physics, KTH Royal Institute of Technology, SE-106 91, Stockholm, Swedenb Centre for Optical and Electromagnetic Research, South China Academy of Advanced Optoelectronics, South China Normal University, Guangzhou 510006, P. R. Chinac Faculty of Physics, Kharazmi University, Tehran, Iran.d Department of Microbiology Tumor and Cell Biology Karolinska Institute, SE-171 77, Stockholm, SwedenCorresponding authors:*[email protected], [email protected] data files containing raw data and results of the analysis are grouped according to the different figures in the manuscript where the extracted results are presented.ABSTRACTLanthanide-based upconversion nanoparticles (UCNPs) have attracted considerable attention in biomedical applications, due to their anti-Stokes shifted emission enabling autofluorescence-free signal detection. However, residual excitation light can still interfere with their relatively low brightness. While commonly used lock-in detection can distinguish weak signals from substantial random background, concurrently modulated residual excitation light is not eliminated. This remains a challenge, particularly under demanding experimental conditions.Here, we propose a photophysical lock-in detection (PP-LID) approach based on the discovery that UCNPs can act as frequency mixers in response to intensity-modulated excitation. Particularly, modulated excitation with more than one base modulation frequency can generate additional low-frequency beating-signals. These signals are resolvable by frame-rate-limited cameras, devoid of ambient and residual excitation light, and can be regulated through nanoparticle engineering. Extracting beating-signals by PP-LID thus provides a strategy to significantly enhance signal-to-background conditions in UCNP-based bioimaging and biosensing.Keywords: Upconversion nanoparticles (UCNPs), nonlinearity, modulation, lock-in detection, second harmonic, beating frequency, fast Fourier Transform (FFT)

This folder contains all raw data underlying the results presented in a manuscript, entitled:Multiplexed near-IR detection of single-molecule fluorescence fluctuations using a single superconducting nanowire single photon detectorAuthored by:Abhilash Kulkarni 1, Niusha Bagheri 1, Jerker Widengren 1,1 Royal Institute of Technology (KTH), Experimental Biomolecular Physics, Dept. Applied Physics, Albanova University Center 106 91 Stockholm, Sweden Corresponding author ([email protected]) The data files are grouped into the different techniques used to generate them and refer to the figures/tables in the manuscript where the extracted results are presented.  ABSTRACT:Fluorescence-based single-molecule and fluctuation spectroscopy in the near-IR can open for biomolecular dynamic studies in biological media, with suppressed autofluorescence and scattering background. However, further implementation is limited by the lower brightness of NIR fluorophores, and available single-photon detector technologies that are still to be explored and adapted. Superconducting Nanowire Single Photon Detectors (snSPDs) have found increasing use in quantum optics and optical communication applications, much thanks to high sensitivity in the near-IR (NIR), low dark-counts, no after-pulsing, and high time resolution. Here, we present characterization of fluorescence intensity fluctuations from single vesicles and NIR fluorophores, based on fluorescence correlation spectroscopy (FCS), specifically taking advantage of these snSPD properties. We present a concept allowing multiplexed readouts based on only one snSPD, in which the emitted photons are separated by their emission wavelength into different optical paths, thereby translating the emission wavelengths into different arrival times onto the snSPD. This concept allows one-laser-one-detector, dual-color fluorescence cross-correlation spectroscopy (FCCS) measurements, with fluorescence intensity fluctuations of two fluorophore species separately analysed, and cross-correlated. It is shown how two fluorophore species in a sample can be distinguished by their different blinking kinetics, fluorescence lifetimes and/or diffusion properties. Apart from differences in emission spectra, the presented concept for multiplexing using a single detector can also be applied to distinguish emitters by properties such as polarization, coherence lengths, and fluorescence bunching and anti-bunching signatures. It can also be generalized to other modalities than FCS, including single-molecule detection, confocal microscopy and imaging.ACKNOWLEDGMENTSThis work was supported by the Knut and Alice Wallenberg Foundation via Wallenberg Center for Quantum Technology (WACQT), and the Swedish Foundation for Strategic Research (SSF, ITM17-0491, BENVAC RMX18-0041).

This folder contains all raw data underlying the results presented in a manuscript, entitled:Multiplexed near-IR detection of single-molecule fluorescence fluctuations using a single superconducting nanowire single photon detectorAuthored by:Abhilash Kulkarni 1, Niusha Bagheri 1, Jerker Widengren 1,1 Royal Institute of Technology (KTH), Experimental Biomolecular Physics, Dept. Applied Physics, Albanova University Center 106 91 Stockholm, Sweden Corresponding author ([email protected]) The data files are grouped into the different techniques used to generate them and refer to the figures/tables in the manuscript where the extracted results are presented.  ABSTRACT:Fluorescence-based single-molecule and fluctuation spectroscopy in the near-IR can open for biomolecular dynamic studies in biological media, with suppressed autofluorescence and scattering background. However, further implementation is limited by the lower brightness of NIR fluorophores, and available single-photon detector technologies that are still to be explored and adapted. Superconducting Nanowire Single Photon Detectors (snSPDs) have found increasing use in quantum optics and optical communication applications, much thanks to high sensitivity in the near-IR (NIR), low dark-counts, no after-pulsing, and high time resolution. Here, we present characterization of fluorescence intensity fluctuations from single vesicles and NIR fluorophores, based on fluorescence correlation spectroscopy (FCS), specifically taking advantage of these snSPD properties. We present a concept allowing multiplexed readouts based on only one snSPD, in which the emitted photons are separated by their emission wavelength into different optical paths, thereby translating the emission wavelengths into different arrival times onto the snSPD. This concept allows one-laser-one-detector, dual-color fluorescence cross-correlation spectroscopy (FCCS) measurements, with fluorescence intensity fluctuations of two fluorophore species separately analysed, and cross-correlated. It is shown how two fluorophore species in a sample can be distinguished by their different blinking kinetics, fluorescence lifetimes and/or diffusion properties. Apart from differences in emission spectra, the presented concept for multiplexing using a single detector can also be applied to distinguish emitters by properties such as polarization, coherence lengths, and fluorescence bunching and anti-bunching signatures. It can also be generalized to other modalities than FCS, including single-molecule detection, confocal microscopy and imaging.ACKNOWLEDGMENTSThis work was supported by the Knut and Alice Wallenberg Foundation via Wallenberg Center for Quantum Technology (WACQT), and the Swedish Foundation for Strategic Research (SSF, ITM17-0491, BENVAC RMX18-0041).

This folder contains all raw data underlying the results presented in a manuscript, submitted to Nano Letters, and entitled:Lanthanide upconversion nonlinearity: a key probe feature for background-free deep-tissue imagingAuthored by:Niusha Bagheria, Chenyi Wangb, Du Guoa, Anbharasi Lakshmanana, Qi Zhua, Nahid Ghazyanic, Qiuqiang Zhanb, Georgios A.Sotirioud, Haichun Liua, Jerker Widengrena a Experimental Biomolecular Physics, Department of Applied Physics, KTH Royal Institute of Technology, SE-106 91, Stockholm, Swedenb Centre for Optical and Electromagnetic Research, South China Academy of Advanced Optoelectronics, South China Normal University, Guangzhou 510006, P. R. Chinac Faculty of Physics, Kharazmi University, Tehran, Iran.d Department of Microbiology Tumor and Cell Biology Karolinska Institute, SE-171 77, Stockholm, SwedenCorresponding authors:*[email protected], [email protected] data files containing raw data and results of the analysis are grouped according to the different figures in the manuscript where the extracted results are presented.ABSTRACTLanthanide-based upconversion nanoparticles (UCNPs) have attracted considerable attention in biomedical imaging and biosensing applications, largely due to their anti-Stokes shifted emission enabling autofluorescence-free signal detection. However, residual excitation light can still interfere with their relatively low brightness. While commonly used lock-in detection, with excitation light modulation and phase-sensitive detection can distinguish weak signals from substantial random background, concurrently modulated residual excitation light is not eliminated. This remains a challenge, particularly under demanding experimental conditions.In this work, we explore the inherent nonlinear response of UCNP emission and discover that UCNPs can act as frequency mixers in response to intensity-modulated excitation. Thereby, frequency components in the UCNP emission can be distinguished, not present in the excitation and background. Moreover, by modulated excitation with more than one base modulation frequency additional low-frequency beating-signals can be generated. By detecting these signals both random ambient light and residual excitation light can be effectively eliminated, and they also have the important advantage to be resolved, by low-speed detectors, such as cameras. Through nanoparticle engineering we show how these beating-signals can be significantly enhanced and detected in tissue with minimized background levels, providing a strategy to significantly enhance signal-to-background conditions in UCNP-based bioimaging and biosensing.Keywords: Upconversion nanoparticles (UCNPs), nonlinearity, modulation, lock-in detection, second harmonic, beating frequency, Fast Fourier Transform (FFT)

This upload contains raw data and simulation software underlying the results presented in a manuscript submitted for peer-review, with the title: Near-infrared MINFLUX imaging enabled by suppression of fluorophore blinking Authored by:C Venugopal Srambickal1,, H Esmaeeli1,, J Piguet1, L Reinkensmeier2, R Siegmund2, M Bates2, A Egner2, J Widengren1,1 Experimental Biomolecular Physics, Bio-Opto-Nano Unit, Department of Applied Physics, Royal Institute of Technology, SE-10691 Stockholm, Sweden2 Department of Optical Nanoscopy, Institute for Nanophotonics, D-37077 Göttingen, Germany Contributed equally* Corresponding author: [email protected] ABSTRACTMINimal photon FLUXes (MINFLUX) offers super-resolution microscopy (SRM) with nanometer localization precision, with lower fluorophore brightness and photostability requirements than for other SRM techniques. Nonetheless, low localization probabilities have been reported in several MINFLUX studies, and a broader use of less bright and photostable fluorophores, including near-infrared (NIR) fluorophores has been difficult to realize. In this work, we identified fluorophore blinking as a main cause of erroneous (and dismissed) fluorophore localizations in MINFLUX imaging and devised strategies to overcome these effects. We systematically studied the blinking/switching properties of cyanine fluorophores emitting in the far-red or NIR range, over typical time scales (µs-10ms), sample and excitation conditions used in MINFLUX imaging. Subsequent simulations of representative MINFLUX localization procedures showed that trans-cis isomerization, and in particular photo-reduction of the fluorophores, can generate significant localization errors. These localization errors, however, could be suppressed by balanced redox buffers and repetitive excitation beam scans. Implementing these strategies, and replacing the slower, intrinsic switching of the fluorophores needed for the localization by transient binding of fluorophore-labelled DNA strands to complementary DNA strands attached to the targets (DNA-PAINT), we could for the first time demonstrate NIR-MINFLUX imaging with nanometer localization precision. This work presents an overall strategy, where fluorophore blinking characterization and subsequent simulations make it possible to design optimal sample and excitation conditions, opening for NIR-MINFLUX imaging, as well as for a broader use of fluorophores in MINFLUX and related SRM studies.Acknowledgements:This study was supported by the European Union's Horizon 2020 research and innovation program under grant agreement 101017180 (NanoVIB). Files with raw data on which the manuscript is based are grouped into folders according to the figures/tables in the manuscript where the extracted results are presented. Additionally, software developed and used for the simulations are arranged into a separate folder.

This folder contains all raw data underlying the results presented in a manuscript, submitted to Angewandte Chemie, and entitled:Interplay between a heptamethine cyanine dye sensitizer (IR806) and lanthanide upconversion nanoparticlesAuthored by:Haichun Liu1, Abhilash Kulkarni1, Uliana Kostiv1, Elin Sandberg1, Anbharasi Lakshmanan1, Georgios A. Sotiriou2, Jerker Widengren1,*1 Department of Applied Physics, KTH Royal Institute of Technology, Roslagstullsbacken 21, SE-106 91, Stockholm, Sweden2Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, SE-171 77, Stockholm, SwedenCorresponding author:*[email protected] data files containing raw data and results of the analysis are grouped according to the different figures in the manuscript where the extracted results are presented.ABSTRACTLanthanide-doped upconversion nanoparticles (UCNPs) have attractive emission properties but suffer from weak light-absorbing capacities and thereby relatively low brightnesses. This motivates using strongly absorbing dye molecules as antennas and sensitizers. However, despite much effort, understanding of this dye-UCNP interplay is still limited. Major sensitization mechanisms are still under discussion, largely because there is a lack of effective means to observe key factors such as dark state transitions within the dyes. Here, we established a combined spectroscopic procedure to systematically investigate the photophysics behind the dye-UCNP interaction, embracing fluorescence-based transient-state excitation-modulation, lifetime and correlation spectroscopy, and spectrofluorometry/spectrophotometry. With this procedure we studied the heptamethine cyanine dye IR806, a typical UCNP sensitizer, established its photophysical model, deciphered its photophysics in UCL-sensitization-related environments and could identify energy transfer from the IR806 singlet excited state to Yb3+ (UCNP sensitizer ion) as the dominant sensitization mechanism. Our studies suggest that IR806 can form non-emissive H-aggregates at the nanoparticle surfaces, which can be dissociated after certain light excitation duration (typically>100µs). Moreover, buildup of a non-fluorescent, photo-redox state of IR806 after longer irradiation times (10–100ms) can deleteriously affect its UCL sensitization, inferring an optimal excitation duration for dye-sensitized UCNPs, relevant for e.g. optical imaging applications.

This folder contains all raw data underlying the results presented in a manuscript, submitted to Angewandte Chemie, and entitled:Interplay between a heptamethine cyanine dye sensitizer (IR806) and lanthanide upconversion nanoparticlesAuthored by:Haichun Liu1, Abhilash Kulkarni1, Uliana Kostiv1, Elin Sandberg1, Anbharasi Lakshmanan1, Georgios A. Sotiriou2, Jerker Widengren1,*1 Department of Applied Physics, KTH Royal Institute of Technology, Roslagstullsbacken 21, SE-106 91, Stockholm, Sweden2Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, SE-171 77, Stockholm, SwedenCorresponding author:*[email protected] data files containing raw data and results of the analysis are grouped according to the different figures in the manuscript where the extracted results are presented.ABSTRACTLanthanide-doped upconversion nanoparticles (UCNPs) have attractive emission properties but suffer from weak light-absorbing capacities and thereby relatively low brightnesses. This motivates using strongly absorbing dye molecules as antennas and sensitizers. However, despite much effort, understanding of this dye-UCNP interplay is still limited. Major sensitization mechanisms are still under discussion, largely because there is a lack of effective means to observe key factors such as dark state transitions within the dyes. Here, we established a combined spectroscopic procedure to systematically investigate the photophysics behind the dye-UCNP interaction, embracing fluorescence-based transient-state excitation-modulation, lifetime and correlation spectroscopy, and spectrofluorometry/spectrophotometry. With this procedure we studied the heptamethine cyanine dye IR806, a typical UCNP sensitizer, established its photophysical model, deciphered its photophysics in UCL-sensitization-related environments and could identify energy transfer from the IR806 singlet excited state to Yb3+ (UCNP sensitizer ion) as the dominant sensitization mechanism. Our studies suggest that IR806 can form non-emissive H-aggregates at the nanoparticle surfaces, which can be dissociated after certain light excitation duration (typically>100µs). Moreover, buildup of a non-fluorescent, photo-redox state of IR806 after longer irradiation times (10–100ms) can deleteriously affect its UCL sensitization, inferring an optimal excitation duration for dye-sensitized UCNPs, relevant for e.g. optical imaging applications.