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Related Concept Videos

Confocal Fluorescence Microscopy01:16

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Confocal microscopy is an advanced microscopic technique. The prime advantage of the confocal microscope over other microscopy techniques is its ability to block the out-of-focus light from the illuminated samples using pinholes. It is widely used with fluorescence optics to obtain high-resolution, sharp contrast images. Unlike optical microscopes, confocal microscopes use a focused beam of light laser to scan the entire sample surface at different z-planes. These microscopes are, therefore,...
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Super-resolution fluorescence microscopy (SRFM) provides a better resolution than conventional fluorescence microscopy by reducing the point spread function (PSF). PSF is the light intensity distribution from a point that causes it to appear blurred. Due to PSF, each fluorescing point appears bigger than its actual size, and it is the PSF interference of nearby fluorophores that causes the blurred image. Various approaches to achieving higher resolution through SRFM have recently been...
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Phase-Contrast Microscopes
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Total internal reflection fluorescence microscopy or TIRF is an advanced microscopic technique used to visualize fluorophores in samples close to a solid surface with a higher refractive index, such as a glass coverslip. TIRF only allows fluorophores in proximity to the solid surface to be excited. When light from a medium with a lower refractive index (such as air) hits the glass coverslip at a critical angle, the light undergoes total internal reflection stead of passing through the glass.
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Chromis-1, a Ratiometric Fluorescent Probe Optimized for Two-Photon Microscopy Reveals Dynamic Changes in Labile

Daisy Bourassa1, Christopher M Elitt2, Adam M McCallum1

  • 1School of Chemistry and Biochemistry and Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology , Atlanta, Georgia 30332, United States.

ACS Sensors
|February 13, 2018
PubMed
Summary

A new fluorescent probe, chromis-1, enables sensitive detection of zinc ions in live cells using two-photon excitation microscopy (TPEM). This probe reveals changes in cellular zinc homeostasis during oligodendrocyte development.

Keywords:
fluorescent probelive cell imagingneurosciencetwo-photon excitation microscopyzinc homeostasis

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Area of Science:

  • Biomedical Optics
  • Cellular Neuroscience
  • Fluorescent Probe Development

Background:

  • Two-photon excitation microscopy (TPEM) offers advantages for live-cell imaging over confocal microscopy, including reduced phototoxicity and enhanced depth penetration.
  • Limited availability of specialized fluorescent probes hinders TPEM's full potential for metal ion imaging.
  • Zinc ions play critical roles in brain physiology and pathophysiology, necessitating tools for their study.

Purpose of the Study:

  • To develop and characterize a novel fluorescent probe optimized for TPEM-based zinc ion detection in live cells.
  • To investigate the dynamic changes in labile zinc concentrations during oligodendrocyte development.
  • To assess the probe's utility for studying zinc homeostasis under various physiological and pathological conditions.

Main Methods:

  • Design and synthesis of chromis-1, a donor-acceptor fluorophore-based, membrane-permeant Zn(II)-selective probe.
  • Characterization of probe photophysical properties, including two-photon cross-section and spectral response upon Zn(II) binding.
  • Application of chromis-1 for ratiometric imaging of intracellular labile Zn(II) in mouse fibroblast and oligodendrocyte cell cultures using TPEM.

Main Results:

  • Chromis-1 exhibits a high affinity (Kd = 1.5 nM) and wide dynamic range for Zn(II), suitable for live-cell imaging.
  • The probe demonstrates a large spectral shift upon Zn(II) binding, enabling ratiometric measurements.
  • TPEM imaging with chromis-1 revealed a decrease in cellular zinc availability during oligodendrocyte differentiation, indicating altered zinc homeostasis.

Conclusions:

  • Chromis-1 is a robust and sensitive fluorescent probe optimized for TPEM, enabling visualization of labile zinc dynamics in live cells.
  • The probe facilitates the study of zinc's role in cellular processes, particularly in the developing brain.
  • Findings highlight significant changes in zinc homeostasis during oligodendrocyte maturation, underscoring the probe's potential for diverse biological investigations.