Ebook: Multimodal and Nanoscale Optical Microscopy

Optical microscopy is rapidly moving to the nanoscale to investigate biological specimens at molecular level using fluorescence as a mechanism of contrast (Diaspro A., Bianchini P., “Optical nanoscopy”, Riv. Nuovo Cimento, 43 (2020) 385). The latest results demonstrate the potential to provide information at the Angstrom level under standard conditions (Weber M., von der Emde H., Leutenegger M. et al., “MINSTED nanoscopy enters the Ångström localization range”, Nat. Biotechnol., 41 (2023) 569; Reinhardt S. C. M., Masullo L. A., Baudrexel I. et al., “Ångström-resolution fluorescence microscopy”, Nature, 617 (2023) 711). Other optical methods are growing to offer a kind of “tunability” in terms of spatial and temporal resolution to study the delicate and complex relationship between structure and function in cells (Diaspro A., Expedition into the Nanoworld. An Exciting Voyage from Optical Microscopy to Nanoscopy (Springer Nature) 2022). The modern optical microscope operates multimodally at the nanoscale using the decisive advantage provided by artificial intelligence algorithms (Pineda J., Midtvedt B., Bachimanchi H. et al., “Geometric deep learning reveals the spatiotemporal features of microscopic motion”, Nat. Mach. Intell., 5 (2023) 71).

New instruments, challenging theoretical approaches, and unique applications from the molecular level to humans are the link to discuss developments of optical microscopy in the realms of fluorescence, and label-free mechanisms of contrast, which are discussed in this volume.

The first contribution is by Colin J. R. Sheppard regarding image scanning microscopy, a technique of confocal microscopy in which the confocal pinhole is replaced by a detector array that allows a better signal-to-noise ratio. Here are discussed the developments and imaging properties of the image scanning microscope. Barbara Storti and Ranieri Bizzarri explain the relevance in the super-resolution domain of the main photophysical features of photochromic Förster Energy Transfer (pcFRET) and describe a powerful way to analyze data. The evolution of laser scanning microscopy approaches is documented by Giuseppe Vicidomini that introduces the fundamental impact provided by the possibility of performing single-photon detection, for example, by employing the asynchronous read-out available through the use of single-photon avalanche diodes. Luca Lanzanò and colleagues discuss how the analysis of fluorescence lifetimes can be boosted by the phasor analysis to perform super-resolution imaging. The separation of photons by lifetime tuning and the use of an image correlation spectroscopy approach are discussed as evolution of the stimulated emission depletion (STED) approach. The exploration of the eukaryotic nucleus by a multimodal single-molecule microscopy approach is a topic debated by Matteo Mazzocca and Davide Mazza. They critically discuss how to quantify nuclear proteins’ dynamics and interactions in living cells.

The tethered particle motion technique and magnetic tweezers are the topics of Laura Finzi and David Dunlap in the context of single-particle tracking and considering recent methodological advances and the application to the study of DNA supercoiling and transcription.

Brillouin scattering is related to a light-matter interaction that induces a frequency shift in the scattered photons. Antonio Fiore and Giuliano Scarcelli report utilizing this phenomenon as contrast mechanism in optical microscopy, enabling the label-free measurement of mechanical properties of cells, tissues, and biomaterials. Refractive index, speed of sound, and kinematic viscosity, as well as mass density and local absorption, are some of the biophysical applications of Brillouin microscopy. Laura Sironi and colleagues with Giuseppe Chirico contribute to laser microfabrication for optical sensing and imaging in vivo by providing an overview of the basics of photo-polymerization induced by two-photon excitation including two case studies focused on direct laser writing of biocompatible hydrogels and scaffolding for tissue and bone regeneration. Optical elements with rapidly adjustable diffractive properties are building blocks for advanced optical instruments, as discussed by Peter Saggau, who reports about applications in multimodal microscopy and nanoscopy.

The route to the Angstrom level performances of the fluorescence optical microscopes has its crucial role in the development of an original single-molecule localization scheme named MINFLUX, due to the minimal photon flux needed, that utilizes inhomogeneous and dynamic illumination to improve the position estimation precision, reaching isotropic, single-nanometer precisions. Francisco Balzarotti discusses this approach in terms of information theory in the scenario of single-molecule localization and imaging.

This volume is completed by the selected contribution of young researchers. Margherita Angelini and coworkers discuss using gold nanohole array plasmonic metasurfaces for sensing applications exploiting their optical properties and implementing a multi-signal platform. Payvand Arjmand and Marc Guillon debate some drawbacks in super-resolved microscopy related to the limit of penetration into the tissue, the acquisition speed, and the possible photo-bleaching. They address the use of compressed detection using the statistical properties of speckles, focusing on STED microscopy. Lisa Cuneo with Simone Civita and colleagues propose an artificial intelligence approach to separate the signal and background based on a scattering network approach coupled with a convolutional neural network (CNN), trained with images of background to which synthetic images of single molecules are added. The reduction of background is also the topic reported by Alessandro Passera and coworkers with Francisco Balzarotti. They show how to improve imaging speed using DNA-PAINT imaging and MINFLUX nanoscopy. Sabrina Zappone demonstrates fluorescence correlation spectroscopy to study how non-coding RNAs can play a role in alpha-synuclein phase transitions, taking advantage of a novel single-photon-avalanche-diode (SPAD) array detector.

Our bet is that, after reading this volume, you can return to your lab with at least a new idea.

P. Bianchini, A. Diaspro, C.J.R. Sheppard and M. Bouzin

Image scanning microscopy is a technique of confocal microscopy in which the confocal pinhole is replaced by a detector array, giving a much stronger signal. The development and imaging properties of image scanning microscopy are reviewed.

Photochromic FRET (pcFRET) combines the super-resolution capability of FRET technique with the photomodulation of the optical characteristics of the acceptor chromophore, in view of obtaining a fast and reliable assessment of molecular binding in solution spectroscopy and fluorescence microscopy. This paper summarizes the main photophysical features of pcFRET and describes a model to analyze obtainable data.

Quantum technologies are revolutionising photonic research, including optical microscopy. The single-photon laser-scanning microscopy (SP-LSM) paradigm is a prominent example. Here, a novel asynchronous read-out single-photon avalanche diode (SPAD) array detector is integrated into a conventional fluorescence laser-scanning microscope, which enables the recording of fluorescence light photon by photon and links to each photon a series of spatial and temporal signatures. The photon-resolved spatiotemporal information —precluded in conventional LSM, which is usually equipped with a single-pixel detector only— drastically expands the information about the specimen obtained from any LSM-based techniques. For example, SP-LSM can transform a confocal LSM in an effective super-resolved functional imaging system with enhanced optical sectioning ability or, a conventional fluorescence fluctuation spectroscopy experiment, into an information-enriched and more robust experiment. However, these two examples represent the tip of the iceberg of a new series of LSM-based techniques able to leverage the photon-resolved spatiotemporal information provided by the new SP-LSM paradigm.

Fluorescence microscopy is an important tool in many scientific areas. The development of fluorescence super-resolution microscopy has overcome the traditional limitation of spatial resolution to half the wavelength of light, enabling the imaging of biological macromolecules at the nanometer scale. Here, we discuss how the analysis of fluorescence lifetimes and the phasor analysis can be used as an original strategy to perform super-resolution imaging. Specifically we describe a method developed in the context of stimulated emission depletion (STED) microscopy called separation of photons by lifetime tuning (SPLIT) and its different implementations. To quantify the improvement of resolution provided by SPLIT we use a statistical method that evaluates the image quality by image correlation spectroscopy (QuICS). As an example of application, we show SPLIT images of transcription foci in U937-PR9 cells and evaluate the improvement of resolution provided by SPLIT compared to conventional STED.

In the cell nucleus, soluble factors need to search for specific DNA sequences to carry out fundamental processes to perpetuate and sustain life, namely transcription, DNA replication, and DNA repair. Different models have been proposed to describe how nuclear proteins search for their genomic target sequences, but testing these models experimentally requires the capability of measuring with high resolution and single cell sensitivity the diffusion of the searcher as well as the structure/topology of the nuclear environment where the search mechanism takes place. In this paper, we will describe the microscopy techniques, such as single molecule tracking, that can be used to quantify the dynamics and interactions of nuclear proteins in living cells, together with the super-resolution/correlative techniques that allow for providing information about the chromatin structure in situ. We will then discuss the possibility of combining these different approaches to provide multimodal maps of the eukaryotic cell nucleus.

This lecture includes a presentation of the basics of two single-particle tracking techniques: the tethered particle motion (TPM) technique and magnetic tweezers (MT) with some recent methodological advances, as well as a review of three exemplary studies of DNA supercoiling and transcription.

Brillouin scattering spectroscopy, which for decades has been used in applied physics and material research, has recently gained prominence as a microscopy technique in biological research and clinical medicine for the measurement of mechanical properties of cells, tissues and biomaterials. Here, we analyze the complex interaction between light and biological media to endow this novel microscopy technology with several more contrast mechanisms of biophysical nature such as refractive index, speed of sound, and kinematic viscosity as well as mass density and local absorption.

The possibility to fabricate microstructures to be used in the medical field is a reality. Further steps in this direction consist in the fabrication of active microstructures for implants and/or for cellular treatments. Two major areas of interest will be briefly treated here. The first is the one of stimulus-responsive optical polymers, especially hydrogels, that can be shaped by means of laser 3D printing and ablation. These can be used for thermal stimulation, energy transduction and sensing. The composition of these polymeric blends is an essential parameter to tune their properties as actuators and/or sensing platforms and to determine the elasto-mechanical characteristics of the printed hydrogel. A second field of interest is the microfabrication of rigid microstructures that can stand the tissue-induced stresses in implants. The increasing demand of microdevices for nanomedicine and personalized medicine has fostered the quest for an efficient combination of composite and hybrid photo-responsive materials and digital micro/nano-manufacturing. Existing works have exploited multiphoton laser photo-polymerization to obtain fine 3D microstructures in hydrogels in an additive manufacturing approach or exploited laser ablation of preformed hydrogels to carve 3D cavities. The aim of this report is to provide a short overview of the basics of photo-polymerization induced by two-photon excitation and to discuss two case studies. In the first one, we discuss the most recent and prominent results in the field of multiphoton laser direct writing of biocompatible hydrogels that embed active nanomaterials not interfering with the writing process and endowing the biocompatible microstructures with physically or chemically activable features such as photo-thermal activity, chemical swelling and chemical sensing. In the second case, we outline the fabrication steps and the first tests of a novel chip which aims at enabling longitudinal studies of the reaction to the biomaterial implant. The chip is composed of a regular reference microstructure fabricated via two-photon polymerization in SZ2080 resist. The geometrical design and the planar raster spacing largely determine the mechanical and spectroscopic features of the microstructures. The development, in vitro characterization and in vivo validation of the Microatlas is performed in living chicken embryos by fluorescence microscopy 3 and 4 days after the implant; the quantification of cell infiltration inside the Microatlas demonstrates its potential as novel scaffold for tissue regeneration. Altogether, the report aims at giving an introduction to the field of nonlinear excitation fabrication and of its impact in the biomedical field.

Optical elements with rapidly adjustable diffractive properties have become technically mature and are commercially available. This has made them interesting building blocks for advanced optical instruments. These lecture notes summarize the acousto-optic concept of dynamic diffractive optical elements (DDOEs) and describe selected applications for Random-Access 2D/3D Scanning Microscopy, Encoded Multi-beam Microscopy, Standing-Wave Nanoscopy, and Random-Access STED Nanoscopy.

The development of novel measurement schemes usually seeks performance improvements in terms of precision, robustness or speed. Information theory provides tools that allow assessing the maximum precision an estimation procedure can have, thus being a invaluable resource for such developments. Here, I review elements of estimation and information theory —in pragmatic terms— and apply them to MINFLUX single-molecule localization. MINFLUX is a novel localization scheme that utilizes inhomogeneous and dynamic illumination to improve the position estimation precision, reaching isotropic, single nanometer precisions.

Gold nanohole array plasmonic metasurfaces have been tested for sensing applications resorting to surface plasmon resonance and plasmon-enhanced fluorescence techniques. Exploiting the optical properties of such metasurface, we realized a multisignal platform implemented in an operating device.

Super-resolution microscopy is considered as an essential tool for imaging biological tissue because it allows resolving tiny structures beyond the diffraction limit of light. However, this technique has certain drawbacks such as the limit of penetration into the tissue, the acquisition speed and the photo-bleaching. Several techniques have been developed to address and solve these problems. We present a super-resolution microscopy technique based on stimulated emission depletion (STED) using compressed detection thanks to the statistical properties of Speckles.

The presence of background is a well-known problem in Single Molecule Localization Microscopy (SMLM). Indeed, the molecules localization precision depends quadratically on the background intensity. Here we try to isolate the single molecules removing the background through a scattering network representation and a Convolution Neural Network (CNN).

In the present paper we employ fluorogenic DNA-PAINT strands to perform DNA-PAINT MINFLUX imaging of DNA origami. By reducing background, this strategy achieves a higher imaging speed compared to conventional DNA-PAINT strands.

A crucial aspect of eukaryotic cells is the spatiotemporal organization of biochemical reactions. One of the strategies cells adopt to regulate biochemical processes is the phase separation of soluble molecules in liquid-like structures. Interactions between intrinsically disordered proteins, such as alpha-synuclein, and RNA molecules are the driving force of the phase separation processes. Changes in the interaction network in alpha-synuclein could promote its transition from a liquid to a solid state, associated with neurotoxicity. Since commonly used methods cannot detect nanoscale liquid or solid-state structures, the early-stage phase transition of alpha-synuclein is still unknown. To overcome these limitations, fluorescence correlation spectroscopy (FCS) techniques are here proposed as a high-throughput tool to investigate the kinetics behind alpha-synuclein phase transitions, focusing mainly on the consequences of the interaction with RNAs.