A two-photon fluorescent probe for lysosomal zinc ions
Hyo‐Jun LeeChang‐Woo ChoHyewon SeoSubhankar SinghaYong Woong JunKyung‐Ha LeeYoungseob JungKyong‐Tai KimSeongjun ParkSung Chul BaeKyo Han Ahn
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The selective detection of zinc ions in lysosomes over that in cytosol is achieved with a fluorescent probe, which enabled the fluorescence imaging of endogenous zinc ions in lysosomes of NIH 3T3 cells as well as mouse hippocampal tissues by two-photon microscopy under excitation at 900 nm.Keywords:
Two-photon excitation microscopy
Image formation in three-photon fluorescence microscopy is analysed. The point spread function, three-dimensional optical transfer function and axial resolution are considered. The results are generalized to multiphoton fluorescence. Three-photon fluorescence is particularly amenable to resolution improvement by use of pupil filters.
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One-photon fluorescence microscopy is an important biological and biomedical imaging technique. This chapter provides a comprehensive introduction of one-photon microscopy to help researchers maximize the effectiveness of their imaging experiments. This chapter first introduces fluorescence generation and the diffraction limit as background. It then outlines the basic operating principles of multiple one-photon microscopy configurations. Specific configurations include wide-field microscopy, light-field microscopy, confocal microscopy, light-sheet microscopy, and super-resolution microscopy. This chapter concludes by discussing multiple specific applications of one-photon fluorescence microscopy in neuroscience, matching the capabilities of the various microscope configurations with their role in obtaining novel information from biological samples.
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Cytosolic Ca2+ levels are maintained at low nanomolar concentrations, and disruption of Ca2+ homeostasis is associated with cell/tissue damage. Thus, methods have been developed to accurately assess cellular Ca2+ levels, each with intrinsic advantages and disadvantages. Here, we present in detail a ratiometric fluorometric method for cytosolic Ca2+ measurement in cultured melanoma cells using Fura 2-AM cell loading and fluorescence microscopy imaging. For complete details on the use and execution of this protocol, please refer to Esteves et al. (2020).
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Multiphoton microscopy has been a powerful tool in brain research, three-photon fluorescence microscopy is increasingly becoming an emerging technique for neurological research of the cortex in depth. Nonhuman primates play important roles in the study of brain science because of their neural and vascular similarity to humans. However, there are few research results of three-photon fluorescence microscopy on the brain of nonhuman primates due to the lack of optimized imaging systems and excellent fluorescent probes. Here we introduced a bright aggregation-induced emission (AIE) probe with excellent three-photon fluorescence efficiency as well as facile synthesis process and we validated its biocompatibility in the macaque monkey. We achieved a large-depth vascular imaging of approximately 1 mm in the cerebral cortex of macaque monkey with our lab-modified three-photon fluorescence microscopy system and the AIE probe. Functional measurement of blood velocity in deep cortex capillaries was also performed. Furthermore, the comparison of cortical deep vascular structure parameters across species was presented on the monkey and mouse cortex. This work is the first in vivo three-photon fluorescence microscopic imaging research on the macaque monkey cortex reaching the imaging depth of ∼1 mm with the bright AIE probe. The results demonstrate the potential of three-photon microscopy as primate-compatible method for imaging fine vascular networks and will advance our understanding of vascular function in normal and disease in humans.
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Two-photon excitation microscopy
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Abstract Two‐photon fluorescence microscopy allows three‐dimensional imaging of biological specimens in vivo . Compared with confocal microscopy, it offers the advantages of deeper tissue penetration and less photodamage but has the disadvantage of slightly lower resolution.
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Efficient two-photon (2PA) absorbing dyes and bioconjugates were used in two-photon fluorescence microscopy (2PFM) of cells, tissue sections, and excised tumors. Results show the utility of these dyes in studying biological processes.
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