Review

Authors: Eleanor H Simpson, Thomas Akam, Tommaso Patriarchi...

Fiber photometry is a key technique for characterizing brain-behavior relationships in vivo. Initially, it was primarily used to report calcium dynamics as a proxy for neural activity via genetically encoded indicators. This generated new insights into brain functions including movement, memory, and motivation at the level of defined circuits and cell types. Recently, the opportunity for discovery with fiber photometry has exploded with the development of an extensive range of fluorescent sensors for biomolecules including neuromodulators and peptides that were previously inaccessible in vivo. This critical advance, combined with the new availability of affordable "plug-and-play" recording systems, has made monitoring molecules with high spatiotemporal precision during behavior highly accessible. However, while opening exciting new avenues for research, the rapid expansion in fiber photometry applications has occurred without coordination or consensus on best practices. Here, we provide a comprehensive guide to help end-users execute, analyze, and suitably interpret fiber photometry studies.

Topics: Neurons; Brain; Photometry; Calcium

PubMed: 38103545
DOI: 10.1016/j.neuron.2023.11.016

Authors: Alex A Legaria, Bridget A Matikainen-Ankney, Ben Yang...

Fiber photometry enables recording of population neuronal calcium dynamics in awake mice. While the popularity of fiber photometry has grown in recent years, it remains unclear whether photometry reflects changes in action potential firing (that is, 'spiking') or other changes in neuronal calcium. In microscope-based calcium imaging, optical and analytical approaches can help differentiate somatic from neuropil calcium. However, these approaches cannot be readily applied to fiber photometry. As such, it remains unclear whether the fiber photometry signal reflects changes in somatic calcium, changes in nonsomatic calcium or a combination of the two. Here, using simultaneous in vivo extracellular electrophysiology and fiber photometry, along with in vivo endoscopic one-photon and two-photon calcium imaging, we determined that the striatal fiber photometry does not reflect spiking-related changes in calcium and instead primarily reflects nonsomatic changes in calcium.

Topics: Action Potentials; Animals; Calcium; Corpus Striatum; Mice; Neurons; Photometry

PubMed: 36042311
DOI: 10.1038/s41593-022-01152-z

Review

Authors: Roi Asor, Dan Loewenthal, Raman van Wee...

Mass photometry (MP) is a technology for the mass measurement of biological macromolecules in solution. Its mass accuracy and resolution have transformed label-free optical detection into a quantitative measurement, enabling the identification of distinct species in a mixture and the characterization of their relative abundances. Its applicability to a variety of biomolecules, including polypeptides, nucleic acids, lipids, and sugars, coupled with the ability to quantify heterogeneity, interaction energies, and kinetics, has driven the rapid and widespread adoption of MP across the life sciences community. These applications have been largely orthogonal to those traditionally associated with microscopy, such as detection, imaging, and tracking, instead focusing on the constituents of biomolecular complexes and their change with time. Here, we present an overview of the origins of MP, its current applications, and future improvements that will further expand its scope.

Topics: Photometry

PubMed: 40327438
DOI: 10.1146/annurev-biophys-061824-111652

Authors: Eric D B Foley, Manish S Kushwah, Gavin Young...

The quantification of membrane-associated biomolecular interactions is crucial to our understanding of various cellular processes. State-of-the-art single-molecule approaches rely largely on the addition of fluorescent labels, which complicates the quantification of the involved stoichiometries and dynamics because of low temporal resolution and the inherent limitations associated with labeling efficiency, photoblinking and photobleaching. Here, we demonstrate dynamic mass photometry, a method for label-free imaging, tracking and mass measurement of individual membrane-associated proteins diffusing on supported lipid bilayers. Application of this method to the membrane remodeling GTPase, dynamin-1, reveals heterogeneous mixtures of dimer-based oligomers, oligomer-dependent mobilities, membrane affinities and (dis)association of individual complexes. These capabilities, together with assay-based advances for studying integral membrane proteins, will enable the elucidation of biomolecular mechanisms in and on lipid bilayers.

Topics: Dynamins; Lipid Bilayers; Photometry; Proteins

PubMed: 34608319
DOI: 10.1038/s41592-021-01261-w

Review

Authors: Roi Asor, Philipp Kukura

We review recent advances in our ability to characterise biomolecular structure, interactions and associated dynamics by mass photometry (MP), the label-free detection and mass measurement of individual biomolecules in solution. Molecular counting and identification provides direct access to relative abundance, and thereby affinities, while associated dynamics yield on- and off-rates. The molecular resolution afforded by MP enables these measurements as a function of stoichiometry and assembly at equilibrium, as opposed to the majority of existing solution-based methods. Together with future improvements in terms of assays and technological performance, MP is likely to provide mechanistic details of complex biomolecular processes.

Topics: Molecular Dynamics Simulation; Photometry

PubMed: 35405425
DOI: 10.1016/j.cbpa.2022.102132

Authors: Andrey Formozov, Alexander Dieter, J Simon Wiegert...

Here, we present simultaneous fiber photometry recordings and optogenetic stimulation based on a multimode fused fiber coupler for both light delivery and collection without the need for dichroic beam splitters. In combination with a multi-color light source and appropriate optical filters, our approach offers remarkable flexibility in experimental design and facilitates the exploration of new molecular tools at minimal cost. We demonstrate straightforward re-configuration of the setup to operate with green, red, and near-infrared calcium indicators with or without simultaneous optogenetic stimulation and further explore the multi-color photometry capabilities of the system. The ease of assembly, operation, characterization, and customization of this platform holds the potential to foster the development of experimental strategies for multi-color fused fiber photometry combined with optogenetics far beyond its current state.

Topics: Brain; Optogenetics; Photometry

PubMed: 37056369
DOI: 10.1016/j.crmeth.2023.100418

Review

Authors: Hao Ding, Bin Su, Dechen Jiang...

Understanding biological mechanisms operating in cells is one of the major goals of biology. Since heterogeneity is the fundamental property of cellular systems, single cell measurements can provide more accurate information about the composition, dynamics, and regulatory circuits of cells than population-averaged assays. Electrochemiluminescence (ECL), the light emission triggered by electrochemical reactions, is an emerging approach for single cell analysis. Numerous analytes, ranging from small biomolecules such as glucose and cholesterol, proteins and nucleic acids to subcellular structures, have been determined in single cells by ECL, which yields new insights into cellular functions. This review aims to provide an overview of research progress on ECL principles and systems for single cell analysis in recent years. The ECL reaction mechanisms are briefly introduced, and then the advances and representative works in ECL single cell analysis are summarized. Finally, outlooks and challenges in this field are addressed.

Topics: Single-Cell Analysis; Electrochemical Techniques; Luminescent Measurements; Photometry

PubMed: 35880657
DOI: 10.1002/open.202200113

Authors: Di Wu, Grzegorz Piszczek

Mass photometry (MP) is a relatively new experimental technique with a quickly expanding list of applications. Using optical detection, MP measures the mass of individual molecules to obtain molecular mass distributions of proteins and other biomolecules in solution. The combination of speed, sensitivity, and very low sample consumption with label- and immobilization-free detection sets MP apart from other analytical methods. An increasing number of laboratories incorporates mass photometry as a routine sample analysis technique. However, MP measurements can sometimes be challenging, especially for users without previous experience with single-molecule techniques. Here, we present a protocol for the determination of protein molecular mass distributions by MP. It describes the sample and materials preparation as well as data collection and analysis. The advantages and limitations of this technique and the potential sources of artifacts are also given. This protocol can be used by new MP users and serve as a checklist for laboratories routinely performing MP experiments to guide consistent data collection and documentation.

Topics: Nanotechnology; Photometry; Proteins

PubMed: 33651123
DOI: 10.1007/s00249-021-01513-9

Authors: Suhel Tamboli, Dimitry Topolnik, Risna Radhakrishnan...

Fiber photometry technique allows investigation of in vivo neural activity during behavior allowing understanding of brain-behavior relationship. Here, we provide a protocol for synchronized wireless fiber photometry and video recordings in rodents during behavior. We explain the detailed steps for stereotaxic virus injection, optic fiber cannula implantation, setup for synchronized fiber photometry and behavioral recording, and analysis of photometry data. These protocol steps can be adapted for various animal models, photometry, and behavioral recording systems. For complete details on the use and execution of this protocol, please refer to Tamboli et al. and Amalyan et al..

Topics: Animals; Photometry; Video Recording; Wireless Technology; Mice; Behavior, Animal; Rats; Fiber Optic Technology; Brain

PubMed: 39425933
DOI: 10.1016/j.xpro.2024.103407

Authors: Alex A Legaria, Alexxai V Kravitz

In this issue of , Formozov et al. present an innovative fiber photometry system that uses a fused fiber coupler (FFC) instead of a dichroic mirror to split the excitation and emission light. The FFC-based photometry system is highly flexible and can be easily reconfigured to record from different biosensors.

Topics: Optogenetics; Optical Fibers; Photometry; Biosensing Techniques

PubMed: 37056372
DOI: 10.1016/j.crmeth.2023.100439