Single Molecule Spectroscopy and Superresolution Imaging XVIII

Here we investigate the effect of proximity of a Surface Enhanced Raman Scattering (SERS) substrate on the intensity of the Raman signal from an analyte layer. inspired by Tip-Enhanced Raman Scattering (TERS), we investigated what happens if we deposit the monolayer onto the concave side of a lens and then contact it to the top of a SERS substrate, to create a SERS "sandwich" (with the lens). We call this nano/micro-cavity sandwich device a "Gradient SERS" (G-SERS) sandwich. When contact is made, in the center of the device, the two surfaces produce "Newton's Rings", due to optical interference. We observe strong SERS enhancement of the Raman signal especially at the point where the analyte contacts the SERS substrate and compare this signal to the Raman signals from different "rings".In the center of the rings, where the lens is the closest to the SERS substrate, the intensity is highest. We also observe an additional Raman enhancement due to cavity enhanced Raman scattering (CERS), when the cavity resonance crosses one of the Raman transitions.

Super-resolution microscopy has revolutionized our ability to visualize structures below the diffraction limit, crucial for studying complex targets like chromatin. Chromatin's hierarchical organization, ranging from nanometers to micrometers, is effectively examined using SMLM methods such as STORM. Our sequential immunolabeling protocol enables reliable multi-label investigations in the dense nuclear environment, combining multiplexed localization datasets with robust spatial analysis and joint density analysis algorithms. By coupling this multi-label SMLM with gene-specific labeling using RASER-FISH, we can analyze transcriptional activity and chromatin reorganization at sub 200-nm length scales and understand the role of transcription in chromatin organization.

Understanding the molecular mechanisms of biomolecular condensate formation through liquid-liquid phase separation is crucial for deciphering cellular cues in normal and pathological contexts. Recent studies have highlighted the existence of sub-micron assemblies, known as nanocondensates or mesoscopic clusters, in the organization of a significant portion of the proteome. However, as smaller condensates are invisible to classical microscopy, new tools must be developed to quantify their numbers and properties. Here, we establish a simple analysis framework using single molecule fluorescence spectroscopy to quantify the formation of nanocondensates diffusing in solution. We used the low-complexity domain of TAR DNA-binding protein 43 (TDP-43) as a model system to show that we can recapitulate the phase separation diagram of the protein in various conditions. Single molecule spectroscopy reveals rapid formation of TDP-43 nanoclusters at ten-fold lower concentrations than described previously by microscopy.

Nitrogen-vacancy (NV) centers in nanodiamonds can be applied as single fluorescent quantum sensors. Our goal is to exploit the spin properties of the luminescent NV- center to reveal the dynamics of biological systems. The extraordinary properties such as very high photo-stability and non-blinking behaviour allow for optical detection of magnetic resonance due to the NV triplet spin states and nanoscale distance measurements. Using a confocal anti-Brownian electrokinetic trap (ABEL trap), we determined NV brightness, spectral ratio, diffusion coefficient, surface charge and multiexponential fluorescence lifetimes for each nanodiamond one by one in solution (I. Perez at al., Proc. of SPIE 12849, 1284906, 2024). For 25 years we have studied subunit rotation of the membrane enzyme FoF1-ATP synthase in solution by intramolecular single-molecule FRET (smFRET) and increased observation times to about a second with the ABELtrap (I. Perez et al., Intl. J. Mol. Sci. 24, 88442, 2023). Now, monitoring fluorescence lifetime changes of the NV- center due to local magnetic fields enables us to record conformational changes of a diffusing single protein for hundreds of seconds.

While conventional fluorescence spectroscopy is concerned with the spectral features of fluorescence emission, time-resolved fluorescence spectroscopy (TRFS) also captures the temporal behaviour allowing obtaining spectro-temporal fluorescence waveforms of the excited molecules, unique to each (organic) molecule. Fluorescence lifetime depends on both radiative (i.e. fluorescence) and non-radiative (i.e. quenching, FRET) processes. Therefore, TRFS may also provide information about pH, ion concentrations or oxygen in the local environment of fluorophores, important in tissue assessment. This work introduces a full calibration procedure of a compact system that integrates a miniaturized spectrograph layout with a high-speed time-gated image sensor employing Current-Assisted Photonic Sampler (CAPS) technology. In addition, enhancing the spectral and temporal characteristics of the measured fluorescence, spectrum and decay, using deconvolution techniques to correct for the point spread function (PSF) and the instrument response function (IRF) of the system.

Molecular interactions are key in cellular signalling. They are usually ruled by the organization and mobility of the involved molecules. However, the direct and non-invasive observation of the interactions in the living cell membrane is often impeded by principle limitations of conventional far-field optical microscopes, for example with respect to limited spatio-temporal resolution and information content. Here, we present an advanced optical microscopy study involving tools such super-resolution STED microscopy in combination with spectral imaging and fluorescence correlation spectroscopy (FCS) or single-molecule tracking on a MINFLUX or iSCAT (interferometric SCATtering) microscope. We highlight how these approaches can reveal novel aspects of membrane bioactivity such as of the existence and function of potential lipid rafts and during pathogen invasion, but also reveal limitations.

Fluorescence correlation spectroscopy (FCS) is essential for studying molecular interactions and dynamics at the single-molecule level. Integrating Single-Photon Avalanche Diode (SPAD) arrays with time-resolved instrumentation in confocal microscopy enhances FCS capabilities. We evaluate FCS applications using a cooled 23-pixel SPAD-array, developed with Pi Imaging Technologies, as an add-on to the confocal microscope Luminosa. This SPAD array enables simultaneous detection of multiple fluorescence signals with high temporal resolution. Any pixel within the SPAD array can be chosen for advanced FCS analyses, providing spatially resolved information on molecular diffusion and dynamics, such as spot-variation FCS and spatial pixel cross-correlation. Time-correlated single-photon counting (TCSPC) offers insights into fluorescence lifetimes and photon bunching, improving the understanding of complex biological mechanisms. Moreover, SPAD array detection enhances optical resolution for imaging via image scanning microscopy (ISM). In conclusion, integrating SPAD arrays with time-resolved detection advances confocal microscopy, enabling new FCS modalities and superresolution imaging.

Fluorescence lifetime correlation spectroscopy (FLCS) uses lifetime information to distinguish different species within a mixture and remove background and after-pulsing artifacts in regular FCS. However, there is little documentation regarding the sensitivity of this technique to obtain reliable data at low fluorophore concentrations in the sub-picomolar range, which is crucial for detecting trace amounts of biomarkers. Our goal is to figure out the physical parameters that determine the limit of detection of FLCS and design the best-performing microscope setup by finding the best compromise between fluorescence brightness and detection volume. Our approach could push the detection limit of FLCS to sub picomolar range. We apply this to study biotin-streptavidin kinetics which is of utmost importance for biotechnologies but is difficult to measure due to its ultra-high affinity.

We present the application of adaptive optics (AO) to intentionally introduce controlled aberrations, affecting two advanced microscopy techniques: Stimulated Emission Depletion (STED) imaging and Fluorescence Correlation Spectroscopy (FCS). FCS provides insights into molecular dynamics and interactions, while STED microscopy enables super-resolved sub-diffraction imaging. By combining the strengths of both techniques, STED FCS emerges as a powerful method that merges the high spatial resolution of STED imaging with the temporal resolution of FCS, allowing for the study of dynamic processes with exceptional precision at the nanoscale. However, in biological experiments, probing deeper into samples like tissue often introduces optical aberrations, which degrade the quality of both imaging and spectroscopy data. In recent years, adaptive optics have addressed this issue, utilizing active optical elements such as deformable mirrors (DMs) and spatial light modulators (SLMs). We here present our investigations on the influence of aberrations on STED microscopy imaging and (STED-)FCS measurements, where we have deliberately induced different aberrations and explored the outcome.

By combining advances in microscopy with novel functional materials, new applications of imaging in neuroscience and cancer detection become possible. In this talk, I will discuss some recent findings using multi-functional fluorescent organic small molecules in two distinct application spaces: imaging and stimulation of neural systems and cancer therapeutic screening. In addition, some recent work using computational methods to analyze the viability of spheroids and organoids without labels and in a longitudinal manner may be presented.

The NovaFLIM software boosts FLIM, FLIM-FRET, and anisotropy analysis efficiency for z-stacks, time-lapse series, and tiled images acquired with PicoQuant’s Luminosa confocal microscope. It also supports data from MicroTime 200 and LSM upgrade kits for Nikon, Olympus, and Zeiss systems. With GPU-accelerated batch analysis, processing time is significantly reduced, and advanced export options are available. 1D and 2D histograms of fitted parameters allow for precise ROI definition and comparison of images with different morphologies. Flexible ROI handling based on histograms and phasor plots opens new possibilities. Additionally, NovaISM enables ISM-FLIM analysis using the PDA-23 add-on for Luminosa. Image Scanning Microscopy (ISM) with SPAD arrays enhances resolution by 1.5-1.7x and improves contrast by rejecting out-of-focus light. This leads to a better signal-to-noise ratio, higher lifetime contrast, and faster, gentler imaging of live samples.

We developed a novel, ultra-compact interferometric SIM microscope providing multi-color, high-speed 2D-, TIRF- and 3D-SIM and compatible with short coherence length or picosecond laser sources. This system was combined with a custom electro-optic (EO) wide-field FLIM detector providing a high contrast, significant lower noise and cost-efficiency compared to established FLIM-cameras. This method provides a high imaging speed with a large field of view (FOV). We applied the system on various samples including live-cells with a FOV of 60 µm and a spatial resolution of 130 nm. The lifetime accuracy with this system is 300 ps with a precision of 100 ps.

Historically, Time Correlated Single Photon Counting (TCSPC) has been hindered by a critical tradeoff between speed and distortion. Classical theory dictates that accurate light waveform reconstruction requires the detector count rate to be limited to 1-5% of the laser frequency, making TCSPC inherently slow. Efforts to speed up TCSPC, such as multichannel systems, post-processing algorithms for pile-up correction, and the exploitation of faster Single Photon Avalanche Diodes (SPADs), often face implementation and application-specific challenges. In this work, we present the first experimental validation of a novel TCSPC approach that overcomes detector dead time and illumination intensity limitations, achieving high-speed operation without pile-up distortion. By combining real-time system status information with classic TCSPC data, we reach an unprecedented count rate of 60% of the excitation frequency with near-zero distortion using off-the-shelf modules.

Living cells are complex, dynamic, and heterogeneous environment with compartmentalized ionic strength, which influence protein activities. Here, I will highlight our recent single-molecule studies on the sensitivity of mCerulean3-linker-mCitrine construct (or RD) to changes in the environmental ionic strength using fluorescence correlation spectroscopy (FCS). The donor (mCerulean3) and the acceptor (mCitrine) of RD are linked by two oppositely charged alpha helices and a flexible random coil region. At the single-molecule level, we hypothesis that the molecular brightness intact RD is smaller than that of the enzymatically cleaved counterpart due to Förster resonance energy transfer (FRET), under the excitation and detection of the donor. Our results indicate that the energy transfer efficiency of RD decreases as the in vitro ionic strength increases. These single-molecule studies are compared with previous time-resolved fluorescence as a benchmark approach for FRET studies. These in vitro single-molecule studies are important step towards the development of protein-based FRET sensors for in vivo studies.

Super-resolution localization microscopy requires precise drift correction to maintain accurate focus during the extended acquisition time. Here, we present a marker-free drift correction technique based on displacement analysis of oblique bright-field features from the sample. Our method can track sample drift in real time with sub-nanometer precision in all three dimensions. We compared our method against conventional techniques and demonstrated its high precision in SMLM imaging across various biological samples.

Super-resolution (SR) fluorescence imaging is generally incompatible with live-sample imaging. Single Molecule Localization Microscopy (SMLM) provides high resolution but requires multiple raw images and specialized fluorophores. Image Scanning Microscopy (ISM), though efficient, offers limited spatial resolution. We introduce a new SR technique that shares the benefits of ISM and SMLM, and can achieve high-speed, long-term SR imaging with low photon requirements. We apply this to the imaging of neural activity in jellyfish and the monitoring of mitochondrial changes in cancer cells.

Biological systems operate at the nanoscale, from DNA and proteins to larger assemblies. Nanoscopic imaging is crucial for biomedical research, especially for understanding chromatin's 3D conformation, a key regulator of gene expression. Current methods use fluorescent labeling, disrupting functions and lacking label density consistency. We introduce DNA Spectroscopic Photon-Localization Intrinsic-Contrast Nanoscopy, a label-free, 3D genomic imaging technology with nanometer resolution. Utilizing DNA's stochastic autofluorescence and photon localization, the platform provides 2D and 3D imaging. Spectral regression and classification algorithms enhance the spatial resolution and distinguish chromatin types and genomic domains, advancing towards potentially unveiling the complete 3D structure of a cell.

We introduce a four dual wedge prisms (DWPs) design for high-throughput single-molecule spectroscopy in single-molecule localization microscopy (SMLM). This system enhances photon collection and spectral resolution, enabling detailed analysis of dye spectral heterogeneity. Our findings reveal significant spectral variability among fluorophores, critical for molecular diagnostics, biological imaging, and materials science. By characterizing this heterogeneity, we can select stable fluorophores with minimal overlap and explore environmental factors affecting spectral properties, ensuring reliable results. Leveraging this variability also enhances multiplexed imaging and novel sensing mechanisms. This presentation will cover the optical setup, data acquisition, and implications for single-molecule spectroscopy.

We introduce a set of novel multiplexing approaches using organelle-specific nanotextures called NanTex for monochromatic, super-resolved image data. Initially based on nanoscale Haralick-feature extraction we progressed to an AI-approach using Unet feature generation relying on probabilistic demixing rather than image segmentation and therefore showing more promise in regions of heavy overlap. Furthermore, we applied our method to MINFLUX data without retraining, showing the relevance of the concept.

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. 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 MINFLUX.

We present a silicon nitride photonic integrated circuit (PIC) that combines the generation of both the stimulation and depletion beams for STED microscopy, using optical phased arrays with wavelength-based steering. The stimulation and depletion beams are generated by precisely controlling the phase of light emitters arranged in concentric circular arrangements together with a beam-shaping objective, effectively replacing the bulky and complex free-space optics used in conventional STED microscopy setups. This innovative method requires only an objective lens and a PIC positioned at its focal point to illuminate the samples. We present the theoretical framework and modeling results, predicting submicron (<300 nm) lateral resolution. Furthermore, we extend the design to show compatibility with both 2D and 3D STED microscopy applications. Finally, we detail the PIC layout, including the implementation and design of the necessary integrated components for the proposed PIC.

Generating structured illumination patterns is crucial for various state-of-the-art optical microscopy techniques, including Structured Illumination Microscopy (SIM). However, current patterns generation methods, often based on diffraction gratings or spatial light modulators, are often slow, bulky, or highly sensitive to the alignment, which hinders their widespread adoption. We propose an integrated, monolithic device created on a glass substrate using Femtosecond Laser Micromachining. This device can generate and move a highly stable structured pattern across the detection plane of a microscope, and enables widefield microscopes to perform SIM thanks to its simple use as an add-on.

Super-resolution microscopy has enabled studies that probe protein spatial distribution at the nanoscale. This, in turn, has made it possible to study the distribution of post-translational modifications in the nucleus. Cancer is associated with widespread alterations in gene expression. It is of interest to classify the resulting change in the spatial distribution of post-translational modifications. However, there is a lack of studies that examine the interactions of multiple histone modifications in a single nucleus. We quantified the individual distribution and clustering behaviors of H3K27me3 and H3K27ac and classified the level of contact between these histone modifications using spectroscopic single-molecule localization microscopy (sSMLM). We also associated the detected changes in these parameters with degrees of cancer malignancy and with drug-induced perturbations in methylation machinery.

Combining single-molecule localization microscopy (SMLM) with engineered point spread functions (PSFs) enables 3D nanoscale imaging over an extended axial range. However, localizing single molecules in 3D in whole mammalian cells remains challenging as it often requires acquisition and post-processing stitching of multiple slices to cover the entire cell volume, or more complex analysis of the data. Here, we demonstrate simplified imaging and analysis workflows by 3D single-molecule super-resolution (SR) imaging using long axial-range double-helix (DH)-PSFs. We experimentally benchmark the localization precisions of short- and long axial-range DH-PSFs at different photon levels, as well as the resolution and image acquisition and analysis speeds for whole-cell 3D SR imaging using simple fitting-based analysis. Furthermore, we demonstrate that the achievable image acquisition speed can be drastically improved by implementing the deep learning-based approach DeepSTORM3D for localization of dense emitters.

Super-resolution fluorescence microscopy, combined with ultra-low irradiation intensities and neural network-based image restoration, allows extensive imaging of living cells while minimizing photobleaching and phototoxicity. By targeting the dynamic endoplasmic reticulum (ER), this approach enables continuous observation of ER dynamics for several hours with second-level temporal resolution. This methodology supports quantitative analysis of ER structural changes over varying timescales and facilitates fast 3D live-cell STED microscopy, providing comprehensive insights into organelle dynamics in living cells.

Genomics-based diagnostic methods that are rapid, accurate, and cost-effective are crucial for progress in precision medicine, with applications in diagnosing infectious diseases, cancer, and rare diseases. One promising technology in this domain is optical genome mapping (OGM), which can detect structural variations, perform epigenomic profiling, and identify microbial species. OGM is based on imaging linearized DNA molecules that are stained with fluorescent labels, that are then aligned to a reference genome. In this talk I will outline our recent efforts towards improving computational aspects of OGM. This includes: 1. Localizing dense fluorescent labels using deep learning, which significantly improves alignment of short molecules (<50 kb) 2. Rapidly and accurately mapping DNA fragment images to a reference genome directly (with no molecule localization step), using a novel transformer-encoder architecture. 3. Using information theory to design optimal labels for OGM. Optimal labels show potential of an order of magnitude improvement in mapping accuracy.

A major breakthrough in Parkinson’s disease research was announced with the identification of the first biomarker and the development of an early diagnosis tool. Alpha-synuclein (αSyn) aggregates, detected in the biofluids of patients with Parkinson’s disease, have the ability to catalyze their own aggregation, leading to an increase in the number and size of aggregates. This self-templated amplification is used by newly developed assays to diagnose Parkinson’s disease and turned the presence of αSyn aggregates into a biomarker of the disease. The seed amplification assays can now diagnose Parkinson’s disease with high sensitivity and specificity and detect prodromal individuals before diagnosis. We have developed a single molecule counting method and instrument to perform diagnostics at the single molecule level. The accurate counting of aggregates in biofluids provides the first quantitative measure of the amyloid load in cerebrospinal fluid. Our work seeks to push this exciting development further by using single molecule “fingerprinting” of these aggregates to stratify patients.

We are presenting study towards increasing spectroscopy resolution by improving spectral and temporal laser source performance. Our experimental setup combined advantages of special seed sources laser driving scheme providing with stable repetition rate, high precision, and stability of the ps-pulses (<100ps@1MHz) together with a high-power optical amplification technique based on specially developed tapered double clad active fiber. This first-time proposed approach allowed to maintain temporal predictability of laser pulse arrived at the sample and mitigate any spectral disturbance while amplifying to relatively high pulse energy of 1.5uJ as well as power parameter stability. Thus, we fundamentally meet fast synchronization requirements of time-resolved and correlation spectroscopy principles. The temporal characteristics of the pulse train, together with high pulse energy, make it possible to effectively discriminate secondary emission of material: fluorescence and nonlinear scattering (including stimulated Raman). The obtained laser output characteristics at 1064/532nm (second harmonic) satisfy most of the needs of a widest range of IR/VIS spectroscopy and imaging applications

Chromatin is organized as a disordered polymer and has a hierarchical functional structure at different length-scales. Spectroscopic single molecule localization microscopy (sSMLM), with ~20 nm resolution and the ability to provide molecular specificity by labeling different targets, is widely exploited in studying chromatin organization. However, it suffers from localization uncertainty introduced by external labels and the optical properties of dyes can lead to varying interpretations of genome structure, as there may not be a direct one-to-one correlation between the fluorophore localization and the actual structure. Here we use a physically informed polymer model for chromatin and corresponding electron microscopy results as ground truth, and simulate fluorophore localizations through Monte-Carlo process to identify the effect of label size and optical properties of the dyes used in sSMLM. We then investigated the chromatin conformation within domains on EdU labeled HCT116 cells and developed a generative AI model to reconstruct chromatin domain information.

Super-resolution microscopy has revolutionized molecular imaging by providing detailed insights at the nanoscale. This work introduces three dual wedge prism (DWP) assemblies for enhanced spectroscopic single-molecule localization microscopy (SMLM). These configurations—2D-DWP for high-precision localization, 3D-DWP for volumetric data, and 3D-Symmetric DWP for improved signal-to-noise ratio—optimize photon budget and imaging quality. Designed for plug-and-play integration, these assemblies simplify super-resolution microscopy and enable multiplexed imaging in various biological samples. The presentation covers technical specifications, installation, and imaging results, emphasizing their benefits and applications in biological research.

Super-resolution microscopy is widely used to quantify the spatial distribution of histone modifications. However, many features of nucleosome organization exist close to or below the resolution limit of super-resolution, and measurements made directly from super-resolution images are prone to biases from repeated blinking of individual fluorophores and the length of the probe used. To address these limitations, we have developed a simulated ground truth chromatin model upon which we simulate probe binding, fluorophore blinking dynamics, noise, and image reconstruction. This model can be used to investigate the effects of biases and to validate histone quantification techniques.

We present a versatile FPGA-based Time-Correlated Single Photon Counting (TCSPC) and time-tagging device with high accuracy, ultra high bandwidth FPGA interface, and White Rabbit synchronization. This device achieves a digital time resolution of 1 ps and single-channel timing uncertainty of 2 ps rms, with short dead-time. It supports eight input channels and a sync channel. Triggering can be programmed using edge trigger or vertex-finding Constant Fraction Discriminator (CFD), optimizing performance for detectors like superconducting nanowire single photon detectors (SNSPD) and hybrid photodetectors (HPD). A high-speed data interface for external FPGA boards allows preprocessing of the time-tagging data stream, mitigating data bottlenecks. White Rabbit synchronization provides a deterministic, Ethernet-based timing network with sub-nanosecond accuracy, enabling precise synchronization over large distances. The instrument supports communication, configuration, and data processing via a GUI or enhanced Python API. Its low jitter, high channel count, high bandwidth, and precise synchronization make it ideal for optical quantum technologies and high-speed fluorescence lifetime imaging.

The Universal Laser Engine combines: * a multi-wavelength flat top-hat illumination * a set of high-speed switching lasers * picosecond pulsed lasers for fluorescence lifetime applications. We have proven the applicability of our laser solution for both. wide-field/TIRF and confocal fluorescence microscopy. It allows to combine multiple methods for simultaneous measurements, such as alternating multi-color excitation schemes, super-resolution and lifetime approaches. We were able to implement novel biophysical methods, such as parallelized and camera-controlled single-molecule detection and wide-field fluorescence lifetime imaging recordings.

Confocal microscopy is a vital tool across academic fields due to its sectioning capability, especially when paired with time-resolved single photon detectors and time-correlated single photon counting (TCSPC). It underpins advanced techniques like fluorescence lifetime imaging (FLIM) and fluorescence correlation spectroscopy (FCS). Recently, high-performance SPAD-arrays with a few dozen pixels have emerged. Combined with multi-channel TCSPC devices, these open new possibilities for time-resolved confocal sensing. We present PicoQuant’s latest multi-channel TCSPC device and a cooled 23-pixel SPAD-array developed with Pi Imaging Technologies. This combination enhances superresolution techniques such as image scanning microscopy (ISM), improving signal-to-noise ratio (SNR) and lateral/axial resolution. These gains are fully compatible with lifetime information for species separation. Additionally, advanced data processing applied to FLIM-ISM provides further performance improvements. The use of small arrays in superresolution is just the beginning, with many more applications anticipated as this technology becomes more accessible.

Confocal fluorescence microscopy offers axial sectioning and fluorescence lifetime detection but is diffraction-limited. Single-molecule localization microscopy (SMLM) enables super-resolution but typically lacks photon counting-based lifetime imaging. Our goal was to combine super-resolution imaging with fluorescence lifetime contrast and confocal sectioning for multiplexing, improved axial resolution (MIET-SMLM), or FRET imaging. We implemented DNA-PAINT and dSTORM on the confocal Luminosa microscope. Autofocus and temperature stability were essential for accurate image acquisition. We analyzed confocal FLIM movies to generate super-resolved images, using photon arrival times for lifetime contrast. In MIET-PAINT, lifetime values were converted to axial positions, while in FRET-PAINT, lifetimes revealed FRET events. We achieved super-resolved FLIM, 3D imaging with MIET-PAINT, and FRET-PAINT on a commercially available microscope, enabling easy adoption by other labs.