Long axial-range double-helix point spread functions for 3D volumetric super-resolution imaging.

Single-molecule localization microscopy (SMLM) is a powerful tool for observing structures beyond the diffraction limit of light. Combining SMLM with engineered point spread functions (PSFs) enables 3D imaging over an extended axial range, as has been demonstrated for super-resolution imaging of various cellular structures. However, super-resolving structures in 3D in thick samples, such as whole mammalian cells, remains challenging as it typically requires acquisition and post-processing stitching of multiple slices to cover the entire sample volume or more complex analysis of the data.

Multimodal illumination platform for 3D single-molecule super-resolution imaging throughout mammalian cells.

Single-molecule super-resolution imaging is instrumental in investigating cellular architecture and organization at the nanoscale. Achieving precise 3D nanometric localization when imaging structures throughout mammalian cells, which can be multiple microns thick, requires careful selection of the illumination scheme in order to optimize the fluorescence signal to background ratio (SBR). Thus, an optical platform that combines different wide-field illumination schemes for target-specific SBR optimization would facilitate more precise 3D nanoscale studies of a wide range of cellular structures.

Lysine Demethylase 4A is a Centrosome Associated Protein Required for Centrosome Integrity and Genomic Stability.

Centrosomes play a fundamental role in nucleating and organizing microtubules in the cell and are vital for faithful chromosome segregation and maintenance of genomic stability. Loss of structural or functional integrity of centrosomes causes genomic instability and is a driver of oncogenesis. The lysine demethylase 4A (KDM4A) is an epigenetic 'eraser' of chromatin methyl marks, which we show also localizes to the centrosome with single molecule resolution.

Multimodal illumination platform for 3D single-molecule super-resolution imaging throughout mammalian cells.

Single-molecule super-resolution imaging is instrumental for investigating cellular architecture and organization at the nanoscale. Achieving precise 3D nanometric localization when imaging structures throughout mammalian cells, which can be multiple microns thick, requires careful selection of the illumination scheme in order to optimize the fluorescence signal to background ratio (SBR). Thus, an optical platform that combines different wide-field illumination schemes for target-specific SBR optimization would facilitate more precise, 3D nanoscale studies of a wide range of cellular structures.

Single-molecule imaging in the primary cilium.

The primary cilium is an important signaling organelle critical for normal development and tissue homeostasis. Its small dimensions and complexity necessitate advanced imaging approaches to uncover the molecular mechanisms behind its function. Here, we outline how single-molecule fluorescence microscopy can be used for tracking molecular dynamics and interactions and for super-resolution imaging of nanoscale structures in the primary cilium.

A hierarchical pathway for assembly of the distal appendages that organize primary cilia.

Distal appendages are nine-fold symmetric blade-like structures attached to the distal end of the mother centriole. These structures are critical for formation of the primary cilium, by regulating at least four critical steps: ciliary vesicle recruitment, recruitment and initiation of intraflagellar transport (IFT), and removal of CP110. While specific proteins that localize to the distal appendages have been identified, how exactly each protein functions to achieve the multiple roles of the distal appendages is poorly understood.

PS F: Polarized Spiral Point Spread Function for Single-Shot 3D Sensing.

We propose a compact snapshot monocular depth estimation technique that relies on an engineered point spread function (PSF). Traditional approaches used in microscopic super-resolution imaging such as the Double-Helix PSF (DHPSF) are ill-suited for scenes that are more complex than a sparse set of point light sources. We show, using the Cramér-Rao lower bound, that separating the two lobes of the DHPSF and thereby capturing two separate images leads to a dramatic increase in depth accuracy.

Light Sheet Illumination for 3D Single-Molecule Super-Resolution Imaging of Neuronal Synapses.

The function of the neuronal synapse depends on the dynamics and interactions of individual molecules at the nanoscale. With the development of single-molecule super-resolution microscopy over the last decades, researchers now have a powerful and versatile imaging tool for mapping the molecular mechanisms behind the biological function. However, imaging of thicker samples, such as mammalian cells and tissue, in all three dimensions is still challenging due to increased fluorescence background and imaging volumes.

Increased local tumor control through nanoparticle-mediated, radiation-triggered release of nitrite, an important precursor for reactive nitrogen species.

The efficacy of dose-enhancing gold nanoparticles (AuNPs) is negatively impacted by low tumor uptake, low cell membrane penetration, limited diffusion distance, and short lifetime of radiation-induced secondary particles. To overcome these limitations, we have developed a novel AuNP system capable of radiation-triggered release of nitrite, a precursor of reactive nitrogen species, and report here on the in vivo characterization of this system. AuNPs were functionalized through PEGylation, cell-penetrating peptides (CPP; AuNP@CPP), and nitroimidazole (nIm; AuNP@nIm-CPP).

Novel fibrillar structure in the inversin compartment of primary cilia revealed by 3D single-molecule superresolution microscopy.

Primary cilia in many cell types contain a periaxonemal subcompartment called the inversin compartment. Four proteins have been found to assemble within the inversin compartment: INVS, ANKS6, NEK8, and NPHP3. The function of the inversin compartment is unknown, but it appears to be critical for normal development, including left-right asymmetry and renal tissue homeostasis.

Quantitative Super-Resolution Microscopy of the Mammalian Glycocalyx.

The mammalian glycocalyx is a heavily glycosylated extramembrane compartment found on nearly every cell. Despite its relevance in both health and disease, studies of the glycocalyx remain hampered by a paucity of methods to spatially classify its components. We combine metabolic labeling, bioorthogonal chemistry, and super-resolution localization microscopy to image two constituents of cell-surface glycans, N-acetylgalactosamine (GalNAc) and sialic acid, with 10-20 nm precision in 2D and 3D.

Phosphofructokinase controls the acetaldehyde-induced phase shift in isolated yeast glycolytic oscillators.

The response of oscillatory systems to external perturbations is crucial for emergent properties such as synchronisation and phase locking and can be quantified in a phase response curve (PRC). In individual, oscillating yeast cells, we characterised experimentally the phase response of glycolytic oscillations for external acetaldehyde pulses and followed the transduction of the perturbation through the system. Subsequently, we analysed the control of the relevant system components in a detailed mechanistic model.

Studying Glycolytic Oscillations in Individual Yeast Cells by Combining Fluorescence Microscopy with Microfluidics and Optical Tweezers.

In this unit, we provide a clear exposition of the methodology employed to study dynamic responses in individual cells, using microfluidics for controlling and adjusting the cell environment, optical tweezers for precise cell positioning, and fluorescence microscopy for detecting intracellular responses. This unit focuses on the induction and study of glycolytic oscillations in single yeast cells, but the methodology can easily be adjusted to examine other biological questions and cell types. We present a step-by-step guide for fabrication of the microfluidic device, for alignment of the optical tweezers, for cell preparation, and for time-lapse imaging of glycolytic oscillations in single cells, including a discussion of common pitfalls.

Light sheet approaches for improved precision in 3D localization-based super-resolution imaging in mammalian cells [Invited].

The development of imaging techniques beyond the diffraction limit has paved the way for detailed studies of nanostructures and molecular mechanisms in biological systems. Imaging thicker samples, such as mammalian cells and tissue, in all three dimensions, is challenging due to increased background and volumes to image. Light sheet illumination is a method that allows for selective irradiation of the image plane, and its inherent optical sectioning capability allows for imaging of biological samples with reduced background, photobleaching, and photodamage.

Tilted Light Sheet Microscopy with 3D Point Spread Functions for Single-Molecule Super-Resolution Imaging in Mammalian Cells.

To obtain a complete picture of subcellular nanostructures, cells must be imaged with high resolution in all three dimensions (3D). Here, we present tilted light sheet microscopy with 3D point spread functions (TILT3D), an imaging platform that combines a novel, tilted light sheet illumination strategy with engineered long axial range point spread functions (PSFs) for low-background, 3D super localization of single molecules as well as 3D super-resolution imaging in thick cells. TILT3D is built upon a standard inverted microscope and has minimal custom parts.

3D single-molecule super-resolution microscopy with a tilted light sheet.

Tilted light sheet microscopy with 3D point spread functions (TILT3D) combines a novel, tilted light sheet illumination strategy with long axial range point spread functions (PSFs) for low-background, 3D super-localization of single molecules as well as 3D super-resolution imaging in thick cells. Because the axial positions of the single emitters are encoded in the shape of each single-molecule image rather than in the position or thickness of the light sheet, the light sheet need not be extremely thin. TILT3D is built upon a standard inverted microscope and has minimal custom parts.

Observation of live chromatin dynamics in cells via 3D localization microscopy using Tetrapod point spread functions.

We report the observation of chromatin dynamics in living budding yeast () cells, in three-dimensions (3D). Using dual color localization microscopy and employing a Tetrapod point spread function, we analyze the spatio-temporal dynamics of two fluorescently labeled DNA loci surrounding the locus. From the measured trajectories, we obtain different dynamical characteristics in terms of inter-loci distance and temporal variance; when the locus is activated, the 3D inter-loci distance and temporal variance increase compared to the inactive state.

Entrainment of heterogeneous glycolytic oscillations in single cells.

Cell signaling, gene expression, and metabolism are affected by cell-cell heterogeneity and random changes in the environment. The effects of such fluctuations on cell signaling and gene expression have recently been studied intensively using single-cell experiments. In metabolism heterogeneity may be particularly important because it may affect synchronisation of metabolic oscillations, an important example of cell-cell communication.

Allosteric regulation of phosphofructokinase controls the emergence of glycolytic oscillations in isolated yeast cells.

Unlabelled: Oscillations are widely distributed in nature and synchronization of oscillators has been described at the cellular level (e.g. heart cells) and at the population level (e.

Heterogeneity of glycolytic oscillatory behaviour in individual yeast cells.

There are many examples of oscillations in biological systems and one of the most investigated is glycolytic oscillations in yeast. These oscillations have been studied since the 1950s in dense, synchronized populations and in cell-free extracts, but it has for long been unknown whether a high cell density is a requirement for oscillations to be induced, or if individual cells can oscillate also in isolation without synchronization. Here we present an experimental method and a detailed kinetic model for studying glycolytic oscillations in individual, isolated yeast cells and compare them to previously reported studies of single-cell oscillations.

Sustained glycolytic oscillations in individual isolated yeast cells.

Unlabelled: Yeast glycolytic oscillations have been studied since the 1950s in cell-free extracts and intact cells. For intact cells, sustained oscillations have so far only been observed at the population level, i.e.