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Current Protocols Jan 2023In this series of papers on light microscopy imaging, we have covered the fundamentals of microscopy, super-resolution microscopy, and lightsheet microscopy. This last... (Review)
Review
In this series of papers on light microscopy imaging, we have covered the fundamentals of microscopy, super-resolution microscopy, and lightsheet microscopy. This last review covers multi-photon microscopy with a brief reference to intravital imaging and Brainbow labeling. Multi-photon microscopy is often referred to as two-photon microscopy. Indeed, using two-photon microscopy is by far the most common way of imaging thick tissues; however, it is theoretically possible to use a higher number of photons, and three-photon microscopy is possible. Therefore, this review is titled "multi-photon microscopy." Another term for describing multi-photon microscopy is "non-linear" microscopy because fluorescence intensity at the focal spot depends upon the average squared intensity rather than the squared average intensity; hence, non-linear optics (NLO) is an alternative name for multi-photon microscopy. It is this non-linear relationship (or third exponential power in the case of three-photon excitation) that determines the axial optical sectioning capability of multi-photon imaging. In this paper, the necessity for two-photon or multi-photon imaging is explained, and the method of optical sectioning by multi-photon microscopy is described. Advice is also given on what fluorescent markers to use and other practical aspects of imaging thick tissues. The technique of Brainbow imaging is discussed. The review concludes with a description of intravital imaging of the mouse. © 2023 Wiley Periodicals LLC.
Topics: Animals; Mice; Microscopy, Fluorescence; Intravital Microscopy; Photons; Microscopy, Confocal; Optics and Photonics
PubMed: 36706245
DOI: 10.1002/cpz1.634 -
Cell Sep 2022Genetically encoded voltage indicators are emerging tools for monitoring voltage dynamics with cell-type specificity. However, current indicators enable a narrow range...
Genetically encoded voltage indicators are emerging tools for monitoring voltage dynamics with cell-type specificity. However, current indicators enable a narrow range of applications due to poor performance under two-photon microscopy, a method of choice for deep-tissue recording. To improve indicators, we developed a multiparameter high-throughput platform to optimize voltage indicators for two-photon microscopy. Using this system, we identified JEDI-2P, an indicator that is faster, brighter, and more sensitive and photostable than its predecessors. We demonstrate that JEDI-2P can report light-evoked responses in axonal termini of Drosophila interneurons and the dendrites and somata of amacrine cells of isolated mouse retina. JEDI-2P can also optically record the voltage dynamics of individual cortical neurons in awake behaving mice for more than 30 min using both resonant-scanning and ULoVE random-access microscopy. Finally, ULoVE recording of JEDI-2P can robustly detect spikes at depths exceeding 400 μm and report voltage correlations in pairs of neurons.
Topics: Animals; Interneurons; Mice; Microscopy; Neurons; Photons; Wakefulness
PubMed: 35985322
DOI: 10.1016/j.cell.2022.07.013 -
Advances in Experimental Medicine and... 2021Two-photon fluorescence imaging is a powerful tool for observing the dynamics of cells in vivo in intact tissue and is well suitable for imaging neuronal activity for...
Two-photon fluorescence imaging is a powerful tool for observing the dynamics of cells in vivo in intact tissue and is well suitable for imaging neuronal activity for neuroscience research. Due to the nonlinear two-photon absorption, the optical sectioning ability is inherent, resulting in two-photon images with high signal contrast and signal-to-noise ratio with efficient illumination. In addition, the longer wavelength excitation light in two-photon imaging compared to one-photon imaging suffers less scattering and absorption by tissue, which allows deeper penetration. Today, two-photon microscopy is being rapidly developed to adapt to various biological applications for high-speed, high-resolution, large-volume, long-term imaging in freely behaving animals.
Topics: Animals; Microscopy; Neurons; Optical Imaging; Photons
PubMed: 34053022
DOI: 10.1007/978-981-15-7627-0_3 -
Sensors (Basel, Switzerland) Jan 2022In recent years, the biosensor research community has made rapid progress in the development of nanostructured materials capable of amplifying the interaction between... (Review)
Review
In recent years, the biosensor research community has made rapid progress in the development of nanostructured materials capable of amplifying the interaction between light and biological matter. A common objective is to concentrate the electromagnetic energy associated with light into nanometer-scale volumes that, in many cases, can extend below the conventional Abbé diffraction limit. Dating back to the first application of surface plasmon resonance (SPR) for label-free detection of biomolecular interactions, resonant optical structures, including waveguides, ring resonators, and photonic crystals, have proven to be effective conduits for a wide range of optical enhancement effects that include enhanced excitation of photon emitters (such as quantum dots, organic dyes, and fluorescent proteins), enhanced extraction from photon emitters, enhanced optical absorption, and enhanced optical scattering (such as from Raman-scatterers and nanoparticles). The application of photonic metamaterials as a means for enhancing contrast in microscopy is a recent technological development. Through their ability to generate surface-localized and resonantly enhanced electromagnetic fields, photonic metamaterials are an effective surface for magnifying absorption, photon emission, and scattering associated with biological materials while an imaging system records spatial and temporal patterns. By replacing the conventional glass microscope slide with a photonic metamaterial, new forms of contrast and enhanced signal-to-noise are obtained for applications that include cancer diagnostics, infectious disease diagnostics, cell membrane imaging, biomolecular interaction analysis, and drug discovery. This paper will review the current state of the art in which photonic metamaterial surfaces are utilized in the context of microscopy.
Topics: Biosensing Techniques; Microscopy; Optics and Photonics; Photons; Surface Plasmon Resonance
PubMed: 35161831
DOI: 10.3390/s22031086 -
Nature Methods Jul 2023Genetically encoded voltage indicators (GEVIs) enable optical recording of electrical signals in the brain, providing subthreshold sensitivity and temporal resolution...
Genetically encoded voltage indicators (GEVIs) enable optical recording of electrical signals in the brain, providing subthreshold sensitivity and temporal resolution not possible with calcium indicators. However, one- and two-photon voltage imaging over prolonged periods with the same GEVI has not yet been demonstrated. Here, we report engineering of ASAP family GEVIs to enhance photostability by inversion of the fluorescence-voltage relationship. Two of the resulting GEVIs, ASAP4b and ASAP4e, respond to 100-mV depolarizations with ≥180% fluorescence increases, compared with the 50% fluorescence decrease of the parental ASAP3. With standard microscopy equipment, ASAP4e enables single-trial detection of spikes in mice over the course of minutes. Unlike GEVIs previously used for one-photon voltage recordings, ASAP4b and ASAP4e also perform well under two-photon illumination. By imaging voltage and calcium simultaneously, we show that ASAP4b and ASAP4e can identify place cells and detect voltage spikes with better temporal resolution than commonly used calcium indicators. Thus, ASAP4b and ASAP4e extend the capabilities of voltage imaging to standard one- and two-photon microscopes while improving the duration of voltage recordings.
Topics: Animals; Mice; Calcium; Brain; Lighting; Microscopy; Photons
PubMed: 37429962
DOI: 10.1038/s41592-023-01913-z -
Small (Weinheim An Der Bergstrasse,... Jun 2021Crystalline porous materials have been extensively explored for wide applications in many fields including nonlinear optics (NLO) for frequency doubling, two-photon... (Review)
Review
Crystalline porous materials have been extensively explored for wide applications in many fields including nonlinear optics (NLO) for frequency doubling, two-photon absorption/emission, optical limiting effect, photoelectric conversion, and biological imaging. The structural diversity and flexibility of the crystalline porous materials such as the metal-organic frameworks, covalent organic frameworks, and polyoxometalates provide numerous opportunities to orderly organize the dipolar chromophores and to systemically modify the type and concentration of these dipolar chromophores in the confined spaces, which are highly desirable for NLO. Here, the recent advances in the crystalline porous NLO materials are discussed. The second-order NLO of crystalline porous materials have been mainly devoted to the chiral and achiral structures, while the third-order NLO crystalline porous materials have been categorized into pure organic and hybrid organic/inorganic materials. Some representative properties and applications of these crystalline porous materials in the NLO regime are highlighted. The future perspective of challenges as well as the potential research directions of crystalline porous materials have been also proposed.
Topics: Metal-Organic Frameworks; Optics and Photonics; Photons; Porosity
PubMed: 33734577
DOI: 10.1002/smll.202006416 -
Nature Communications Jun 2022When pursuing sustainable approaches to fabricate photonic structures, nature can be used as a source of inspiration for both the nanoarchitecture and the constituent...
When pursuing sustainable approaches to fabricate photonic structures, nature can be used as a source of inspiration for both the nanoarchitecture and the constituent materials. Although several biomaterials have been promised as suitable candidates for photonic materials and pigments, their fabrication processes have been limited to the small to medium-scale production of films. Here, by employing a substrate-free process, structurally coloured microparticles are produced via the confined self-assembly of a cholesteric cellulose nanocrystal (CNC) suspension within emulsified microdroplets. Upon drying, the droplets undergo multiple buckling events, which allow for greater contraction of the nanostructure than predicted for a spherical geometry. This buckling, combined with a solvent or thermal post-treatment, enables the production of dispersions of vibrant red, green, and blue cellulose photonic pigments. The hierarchical structure of these pigments enables the deposition of coatings with angular independent colour, offering a consistent visual appearance across a wide range of viewing angles.
Topics: Cellulose; Nanoparticles; Nanostructures; Optics and Photonics; Photons
PubMed: 35697688
DOI: 10.1038/s41467-022-31079-9 -
ACS Nano Aug 2022Metasurfaces are 2D artificial materials consisting of arrays of metamolecules, which are exquisitely designed to manipulate light in terms of amplitude, phase, and... (Review)
Review
Metasurfaces are 2D artificial materials consisting of arrays of metamolecules, which are exquisitely designed to manipulate light in terms of amplitude, phase, and polarization state with spatial resolutions at the subwavelength scale. Traditional micro/nano-optical sensors (MNOSs) pursue high sensitivity through strongly localized optical fields based on diffractive and refractive optics, microcavities, and interferometers. Although detections of ultra-low concentrations of analytes have already been demonstrated, the label-free sensing and recognition of complex and unknown samples remain challenging, requiring multiple readouts from sensors, ., refractive index, absorption/emission spectrum, chirality, . Additionally, the reliability of detecting large, inhomogeneous biosamples may be compromised by the limited near-field sensing area from the localization of light. Here, we review recent advances in metasurface-based MNOSs and compare them with counterparts using micro-optics from aspects of physics, working principles, and applications. By virtue of underlying the physics and design flexibilities of metasurfaces, MNOSs have now been endowed with superb performances and advanced functionalities, leading toward highly integrated smart sensing platforms.
Topics: Reproducibility of Results; Optics and Photonics; Refractometry
PubMed: 35960685
DOI: 10.1021/acsnano.2c03310 -
Biosensors Dec 2022Silicon photonic (SiP) sensors offer a promising platform for robust and low-cost decentralized diagnostics due to their high scalability, low limit of detection, and... (Review)
Review
Silicon photonic (SiP) sensors offer a promising platform for robust and low-cost decentralized diagnostics due to their high scalability, low limit of detection, and ability to integrate multiple sensors for multiplexed analyte detection. Their CMOS-compatible fabrication enables chip-scale miniaturization, high scalability, and low-cost mass production. Sensitive, specific detection with silicon photonic sensors is afforded through biofunctionalization of the sensor surface; consequently, this functionalization chemistry is inextricably linked to sensor performance. In this review, we first highlight the biofunctionalization needs for SiP biosensors, including sensitivity, specificity, cost, shelf-stability, and replicability and establish a set of performance criteria. We then benchmark biofunctionalization strategies for SiP biosensors against these criteria, organizing the review around three key aspects: bioreceptor selection, immobilization strategies, and patterning techniques. First, we evaluate bioreceptors, including antibodies, aptamers, nucleic acid probes, molecularly imprinted polymers, peptides, glycans, and lectins. We then compare adsorption, bioaffinity, and covalent chemistries for immobilizing bioreceptors on SiP surfaces. Finally, we compare biopatterning techniques for spatially controlling and multiplexing the biofunctionalization of SiP sensors, including microcontact printing, pin- and pipette-based spotting, microfluidic patterning in channels, inkjet printing, and microfluidic probes.
Topics: Silicon; Optics and Photonics; Antibodies; Lectins; Biosensing Techniques
PubMed: 36671887
DOI: 10.3390/bios13010053 -
Molecular Systems Biology Mar 2021Histological analysis of biological tissues by mechanical sectioning is significantly time-consuming and error-prone due to loss of important information during sample... (Review)
Review
Histological analysis of biological tissues by mechanical sectioning is significantly time-consuming and error-prone due to loss of important information during sample slicing. In the recent years, the development of tissue clearing methods overcame several of these limitations and allowed exploring intact biological specimens by rendering tissues transparent and subsequently imaging them by laser scanning fluorescence microscopy. In this review, we provide a guide for scientists who would like to perform a clearing protocol from scratch without any prior knowledge, with an emphasis on DISCO clearing protocols, which have been widely used not only due to their robustness, but also owing to their relatively straightforward application. We discuss diverse tissue-clearing options and propose solutions for several possible pitfalls. Moreover, after surveying more than 30 researchers that employ tissue clearing techniques in their laboratories, we compiled the most frequently encountered issues and propose solutions. Overall, this review offers an informative and detailed guide through the growing literature of tissue clearing and can help with finding the easiest way for hands-on implementation.
Topics: Animals; Decision Trees; Humans; Optics and Photonics; Organ Specificity; Solvents; Staining and Labeling
PubMed: 33769689
DOI: 10.15252/msb.20209807