Spectroscopic single-molecule localization microscopy (sSMLM) combines super-resolution microscopy and spectroscopy. Its single molecule sensitivity and high spectral precision have made it uniquely valuable for several applications, including multicolor imaging, chemical characterization, polarity sensing, and multiplexed single particle tracking. However, widespread adoption is hampered by a lack of standardization in optical implementation, calibration techniques, and image processing. We demonstrate our lab’s efforts to develop tools that simplify adoption and optimize photon efficiency, including protocols for calibration techniques, a user-friendly imageJ plugin for image processing, and a fabricated monolithic beam splitter and prism designed to fit into a microscope body with minimal optical alignment.

The ability to measure fluorophore molecular orientation and mobility can provide valuable information on the local physical and chemical environment. Analysis of polarized images can determine the orientation and mobility of fluorophores with well-defined uncertainties and bias, as well as enabling the use of two to four times greater emitter density per image frame than PSF engineering methods do. This study presents extensive Monte Carlo simulation and experimental data to determine quantitatively the degree of coupling among orientations and orientational mobility, and the dependence of orientational uncertainty on photon count and background noise. These results may be used for the rational design of experimental protocols and conditions to yield the levels of precision and accuracy necessary to effectively explore a wide range of physical phenomena.

The retinal pigment epithelium (RPE) is a monolayer of pigmented cells critical for sight and any intracellular damage in this monolayer will compromise the integrity of the entire eye, leading to blindness and other health problems. Super-resolution microscopy provides an opportunity to image damaged and healthy RPE tissue down to the molecular scale. Previous studies are limited in scope, relying on cultured RPE cells due to the difficulties in imaging tissue slices. Here, we report the first super-resolution imaging of flat-mounted whole albino mouse retinal pigment epithelium using single-molecule localization microscopy (SMLM). After optimizing the labeling protocols, we imaged Phalloidin, β-Tubulin, ZO-1 Tight Junctions, Peroxisome marker (PMP70), Histone-H4, and TOM20 mitochondrial proteins both separately and in simultaneous two-color spectroscopic SMLM (sSMLM) imaging of Phalloidin and ZO-1 as well as TOM20 and β-Tubulin. This work lays the foundation for future investigations of multiple intracellular interactions within damaged RPE monolayers at the molecular level.

We present a novel technique for volumetric super-resolution imaging. Our technique, which is based on the principles of single molecule localization microscopy, utilizes a mirror cavity with a series of pinholes on one of the mirrors allowing for simultaneous optical sectioning of different imaging planes. In addition, we employ a unique machine learned algorithm for 3D localization of events that occur between different imaging planes. Our technique enables high-resolution imaging of thicker volumes than what is currently available using other single molecule localization techniques.

Stochastic optical reconstruction microscopy (STORM) achieves super-resolution imaging by blinking individual dye molecules in thiol-containing media. While STORM is well-established for imaging thin biological specimens, its application to thick tissues has been limited by light scattering. Mounting media with an oil refractive index have shown promise in improving imaging depth and resolution in optical microscopy, but such buffers have not been explored for STORM. Here, we present a 3-pyridinemethanol-based STORM buffer with a refractive index matching standard immersion oil. Our buffer demonstrates comparable performance to conventional STORM buffers and exceptional stability for 5 weeks, enabling imaging of numerous cells on a single slide and larger field-of-view imaging. With perfect index matching, our buffer simplifies imaging and holds potential for lightsheet STORM applications in thick tissues.

Fluorescence laser-scanning microscopy (FLSM) is a widely utilized tool in life-science research. In recent years, this technique has undergone a profound transformation, thanks to the introduction of novel single-photon avalanche diode (SPAD) array detectors. This study reveals the exciting possibilities of combining the SPAD array detector with single-molecule techniques. We propose a real-time single-molecule tracking architecture, where the SPAD array effortlessly localizes the molecule of interest, and the beam scanning architecture effectively maintains the molecule at the center of the microscope's detection volume. This approach enables comprehensive three-dimensional tracking throughout the entire cell, offering valuable insights into molecular nano-environments, interactions, and structural changes through fluorescence lifetime information. Furthermore, utilizing the same FLSM system, we present a novel sequential structure illumination single-molecule localization microscope (similar to MINFLUX). This advanced technique achieves localization precision in the few-nanometer range while simultaneously providing the molecule's fluorescence lifetime.

Oligomers of disease-causing amyloid proteins (such as Alzheimer’s Amyloid beta or ’Aβ’) are generally amphiphilic and their interactions with lipid membranes are possibly the origin of their toxicity. However, how oligomers of different stoichiometries or different mutants differ in their interaction with the membrane, and how these differences correlate with their toxicity, has largely remained beyond the reach of existing experimental techniques. Here we use Q-SLIP, a single-molecule tool that can resolve the surface exposure of different parts of individual oligomers, and different radical-labeled lipids that act as quenchers, to address these questions.

Defect engineering of 2D materials offers enormous opportunities to tune material properties. This presentation will show two types of substitutional defects in 2D materials, self-limited along the out-of-plane and in-plane directions, respectively. The first type is atomic substitution: a nitrogen atom substituting a chalcogen atom in 2D transition metal dichalcogenides (TMDs), which yields new distinct photoluminescence features well separated from the free excitons of 2D TMDs. The second type is layer substitution: an entire layer of chalcogen atoms in 2D TMD substituted by another type of chalcogen atoms, namely, Janus TMDs. Due to the intrinsic vertical dipole, Janus TMDs form unconventional interaction with adjacent materials including other 2D material layers. These unconventional interactions were probed by optical signature changes such as ultra-low frequency Raman modes and photoluminescence yield change. Engineering such substitutional defects in 2D materials promises potential for optoelectronic devices and quantum information platforms.

The development of super-resolution imaging techniques have extended the resolving power to around 10 - 50 nm. However, most current super-resolution imaging techniques need exogenous fluorescent dyes as imaging contrast, whose essential weakness of labeling includes imprecise spatial localization, and perturbation of the sample. In 2017, Dong et al., demonstrated that the intrinsic fluorescence of DNA under visible light excitation has similar photo-switching properties to the organic dyes used in single-molecule localization microscopy. In this paper, we measured the fluorescence spectra of poly-G (guanine) of different lengths (5, 8, 12, 16 base-pair), 20 base-pair single-stranded DNA molecules (poly-A, G, C, T), as well as double-stranded DNA (AT chain, GC chain), under multiple wavelengths. The 20 base-pair AT, GC spectra can be classified with an accuracy of more than 90%, which demonstrates the molecular specificity of the double stranded DNA polymers via its intrinsic fluorescence. Our work paves the way for developing spectroscopic intrinsic-contrast localization optical nanoscopy for chromatin study.

In localization microscopy, the positions of individual nanoscale point emitters (e.g. fluorescent molecules) are determined at high precision from their point-spread functions (PSFs). This enables highly precise single/multiple-particle-tracking, as well as super-resolution microscopy, namely single molecule localization microscopy (SMLM). In this talk I will describe advances to localization microscopy that we have recently achieved using deep learning, both in analysis (image processing) and in optimal imaging-system design. Specific topics to be discussed include: volumetric (3D) SMLM and single particle tracking by deep-learning-based PSF engineering, high-throughput in-flow colocalization in live cells, dynamic SMLM (blinking-to-video), and optical genome mapping. A novel method for additive-manufacturing of phase masks for wavefront shaping will also be discussed.

In recent years, Image Scanning Microscopy (ISM) has emerged as a powerful technique for achieving super-resolution bio-imaging across various applications. Particularly noteworthy is the implementation of a single-photon detector array, enabling the utilization of Lifetime Image Scanning Microscopy, which has proven to be highly effective. In our study, we present a novel approach that combines ISM with direct Stochastic Optical Reconstruction Microscopy (dSTORM), resulting in a doubling of the localization precision in Single Molecule Localization Microscopy (SMLM). Additionally, we capitalize on the available lifetime information provided by ISM, allowing for multilabel fluorescence measurements without the detrimental effects of chromatic aberration, even at resolutions significantly surpassing the diffraction limit. Moreover, we introduce a freely available add-on to previously employed open-source tools for single particle tracking and localization, enhancing the accessibility and utility of our methodology. This add-on serves as a valuable resource for the research community, facilitating the adoption and further advancement of the combined ISM and dSTORM technique.

Simultaneous measurement of the 3D orientation and the 3D position of a single fluorescent molecule can be achieved by Point Spread Function (PSF) engineering. However, this 5D problem is complex to optimize and time consuming when solved with classical approaches. To overcome this problem, we developed a deep learning approach that allows us to obtain an optimized phase mask as well as an Analysis Neural Network that estimates the 5 parameters of immobilized single molecules with a reduced computation time. Our method shows an axial precision of about 30 nm and an orientation precision of about 10 degrees, and it can be applied to complex problems such as molecular orientation in membranes.

This study explores uncertainties in fluorescence labeling, a complication in Single-Molecule Localization Microscopy (SMLM) image interpretation. We examine variability caused by antibody and fluorophore attachment, orientation, and photobleaching, focusing on protein tagging and indirect immunofluorescence, techniques known for their specificity but prone to introducing variable label densities. We use a Monte Carlo (MC) model to simulate SMLM images, providing a 'ground truth' for comparison. This model also investigates the balance between labeling size and density, considering the possibility of single fluorophore attachment in protein tagging and multiple fluorophores in indirect immunofluorescence. We propose methods to quantify the effects of labeling strategies on image quality and accuracy, considering parameters such as labeling linker length and fluorophore photoswitching. Our work enhances the accuracy of SMLM image interpretation and guides the selection of labeling strategies, advancing super-resolution microscopy.

Fluorescence Lifetime Imaging (FLIM) has become more attractive in recent years as it offers increased specificity in many assays as well as the possibility of multiplexing the read out of many markers with a small number of detectors. Here we present how FLIM modalities are implemented in Luminosa, the new single-photon counting confocal microscope by PicoQuant. Thanks to a dynamic binning format and GPU-based algorithms FLIM images of 1024x1024 can be analysed in a few seconds. The FLIM analysis workflow suggests the best fitting model based on statistical arguments and requires minimal user interaction making these modalities become accessible to new users who can then confidently start working with FLIM and incorporate it into their research toolbox combining the strengths of phasor plots with decay fitting.

Time Correlated Single Photon Counting (TCSPC) has been historically subject to the count-rate vs distortion tradeoff. Several attempts to work around this limitation have been reported in the literature, either based on multichannel systems or on post-processing correction algorithms. In this work, we’ll show how distortion can be avoided by exploiting additional information on the system status acquired during the whole experiment. We’ll provide evidence that a new research line can finally combine all the advantages of TCSPC with very high speed. Starting from on-field results, we’ll present the novel technique providing design guidelines for next-generation ultrafast TCSPC acquisition systems.

Time-correlated single-photon counting (TCSPC) allows to achieve picosecond-precision measurements for low-light signals. However, TCSPC suffers from pile-up distortion, constraining the acquisition rate to 1-5% of the laser rate. To overcome the issue, our research focuses on high-rate TCSPC methodologies: in 2017 we reported a hardware acquisition approach, that has been translated into a real system, guaranteeing low distortion at 32 Mcps. This talk provides an overview on the research project, and in particular on the two validation campaigns carried out in fluorescence and lidar measurements, and on our first on-field experiment, i.e. the application of the technique to a single-pixel camera.

Using a novel imaging device—NCam—fluorophore lifetime measurements can be captured simultaneously with wide-field microscopy methods. Because NCam records single-photon events with spatial and temporal information, the localization precision can be improved compared to camera-based imaging or scanning confocal instruments. We demonstrate this new imaging capability by examining the fluorescence behavior of quantum dots.

SPADλ is a linear single-photon detector array with 320×1 single-photon avalanche diode (SPAD) pixels, featuring thermo-electric cooling for reduced noise. These SPADs offer a low dark count rate and wide detection spectrum. Equipped with microlenses, they achieve a peak photon detection efficiency of 45% at 520 nm. This system can count photons at 4 Gcps and provides time-tagging and time-gating for time-resolved detection. With 80 TDC channels, it achieves time-tagging precision averaging better than 130 ps full width half maximum (FWHM). Ideal for flow cytometry, fluorescence lifetime imaging (FLIM), and Raman spectroscopy applications.