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Detection of mitochondrial dysfunction in vitro by laser-induced autofluorescence

Chandavalli R Raghushaker1, Subhash Chandra1, Sanjiban Chakrabarty2

  • 1Department of Biophysics, Manipal School of Life Sciences, Manipal Academy of Higher Education, Manipal, India.

Journal of Biophotonics
|June 29, 2019
PubMed

Insights

This study introduces laser-induced fluorescence (LIF) to detect mitochondrial dysfunction. LIF measures changes in tryptophan autofluorescence, revealing structural damage in mitochondria exposed to heavy metal stress.

Area of Science:

  • Cellular Biology
  • Biophysics
  • Biochemistry

Background:

  • Mitochondria are vital for cellular functions but susceptible to various stresses.
  • Assessing mitochondrial dysfunction is crucial for understanding cellular health and disease.
  • Current methods for detecting mitochondrial stress can be complex and time-consuming.

Purpose of the Study:

  • To develop and validate a novel method for detecting mitochondrial dysfunction using laser-induced fluorescence (LIF).
  • To investigate the changes in tryptophan autofluorescence of mitochondrial proteins under heavy metal-induced stress.
  • To evaluate the potential of LIF as a sensitive tool for assessing mitochondrial structural integrity.

Main Methods:

  • Isolation and purity assessment of mouse liver mitochondria using lactate dehydrogenase and succinate dehydrogenase (SDH) assays.
  • Induction of mitochondrial stress using heavy metals (sodium arsenite and mercuric chloride).
  • Measurement of tryptophan autofluorescence quenching via laser-induced fluorescence (LIF) at 281 nm excitation.
  • Assessment of mitochondrial functional integrity through SDH and cytochrome c oxidase activity assays.
  • Analysis of protein conformational changes using circular dichroism spectroscopy.

Main Results:

  • Heavy metal treatment caused significant quenching of tryptophan autofluorescence in isolated mitochondria.
  • Impaired activities of SDH and cytochrome c oxidase were observed in stressed mitochondria compared to controls.
  • A notable shift in protein autofluorescence below 1 μg concentration indicated conformational changes.
  • Circular dichroism spectroscopy confirmed heavy metal-induced conformational alterations in mitochondrial proteins.

Conclusions:

  • Laser-induced fluorescence (LIF) serves as a novel and sensitive tool for detecting mitochondrial dysfunction and structural integrity changes.
  • Tryptophan autofluorescence is a reliable indicator of mitochondrial stress induced by heavy metals.
  • LIF technology holds potential for integration with other interdisciplinary modalities for comprehensive cellular analysis.
Keywords:
ANS fluorescenceLDH and SDH assayheavy metalstryptophan autofluorescence

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