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Methods to Study Mitochondrial Structure and

Mitochondria are intricate highly organized cellular organelles, which play crucial
roles not only in energy homeostasis but also in many signalling, biosynthetic
and cell death pathways. Furthermore, mitochondria are extremely dynamic
organelles that constantly divide and combine and travel inside the cell. For
these reasons, it is not surprising that a broad range of procedural approaches
have been developed to gauge mitochondrial activities.
This review aims to discuss the major cytochemical, biochemical, and molecular
biological techniques used to inspect mitochondrial dynamics with a focus on the
more recently developed technologies.

High Resolution Imaging of Mitochondria in Live Cells

It is difficult to study the mitochondria using phase contrast or differential
contrast optics. Therefore, over the last few decades many vital stains
have been identified to label the mitochondria in living cells. This method
is based on development of particular fluorescent probes to stain
mitochondria or to label the different proteins present inside the
A number of fluorescent probes listed in Table 1 have been increasingly used to
quantitatively assess the overall cardiomyocyte membrane potential, oxidative
stress, mitochondrial number, apoptosis, and Ca 2+ concentrations.
Table 1 Fluorescent dyes to study mitochondria.
Fluorescent Dyes

maximum (nm)

3,3 Dihexyloxacarbocyanin
iodide (DiOC 6 )
5,5 ,6,6 -Tetrachloro-1,1 ,3,3
iodide (JC-1)




527 and 590

Methods to Study Mitochondrial Structure and

Nonyl acridine orange (NAO)
Rhodamine 123 (Rhod 123)



Tetramethylrhodamine ethyl
ester (TMRE)
Tetramethylrhodamine methyl
ester (TMRM)





With recent advancements in the field of nanoscopy or super-resolution

fluorescence technologies have been instrumental in reducing the limiting role of
diffraction in a lens-based optical microscopy. With the help of these
technologies, it has recently been demonstrated the distribution of different
proteins in mitochondria and the flow of the mitochondrial inner membrane in
live cells with a high precision.

High Resolution Electron Microscopy and Electron

For the last six decades Electron Microscopy has become an indispensable tool to
study mitochondrial ultrastructure and activities. The first results were given by
Palade and Sjostrand (1953) where they showed mitochondria as doublemembrane enclosed organelles.
The conventional transmission Electron Microscopy produces two dimensional
images of objects, and for this reason 3 dimensional imaging techniques like
Electron Tomography have been successful. This new advancement has been
able to give 3 dimensional reconstructions of mitochondria at the molecular
The most recent technology in this field has been cryo-ET. Cryo-ET is
accomplished using frozen samples and is devoid of damages induced by
chemical fixation, staining and dehydration.

Methods to Study Mitochondrial Structure and


Fig 1 Transmission electron microscopy of cardiac mitochondria.

Methods to Study Mitochondrial Structure and

Biochemical and Molecular Biological Methods

In Vitro Assessment of Mitochondrial Function

The In Vitro method of measuring activities of various mitochondrial
enzymes is used to estimate the functionality of steps involved in the
metabolism of mitochondria. There are two major in vitro approaches that
are employed: polarographic measurement of measurement of oxygen
consumption and a bioluminescent measurement of ATP production.
When the substrate luciferin is oxidized by firefly luciferase in an ATPdependent manner, light signals are generated which are proportional of
ATP concentrations. These emissions can be measured by a luminometer
to compute the rates of production of ATP.
An alternate approach is the polarographic measurement of oxygen
utilization in mitochondria in the presence of particular substrates. The
polarographic technique for quantifying mitochondrial oxidative changes
requires the following four basic machineries: an oxygen electrode, a
constant voltage source, a closed reaction vessel, and a recorder.

In Vivo Assessment of Mitochondrial Functions

Many non-invasive methods based on magnetic resonance spectroscopy
(MRS) have come up as useful techniques to study mitochondrial function

Methods to Study Mitochondrial Structure and

Fig: Scheme of human cardiac MRS analysis.
in vivo in many human tissues. In this method we measure magnetic
resonance signals from MR visible nuclei, such as phosphorus, hydrogen,
and sodium.
This technique allows examination of mitochondrial energy metabolism. 31
P-MRS allows in vivo identification of PCr and ATP dynamics, whereas 13 CMRS assesses the TCA cycle, glycolysis, or b -oxidation.

Conclusion and Future Progress

The various techniques discussed above have greatly enhanced our
understanding of mitochondrial functional role in physiology and
pathophysiology. Light microscopy has been used for a long time to
examine mitochondria in numerous human tissues, including the heart.
The improvement of microscopic instruments in the last decades and the
generation of a range of fluorescence stains to mark mitochondria on the
whole and their proteins have made feasible the study of these organelles
in live cells.

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3. Polarographic Assays of Mitochondrial Functions, Ye Xiong, Patti L.

Peterson, and Chuan-pu Lee
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