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The Structural and Functional

Characterization of
Mitochondria and Lysosomes
Through the Use of Dyes

Matt Granger
Dr. Erwin Huebner
BIOL 4560

The discovery of mitochondria in animals is credited to Richard Altmann, who in 1890
gave them the name bioblasts.1 See figure 6 for Altmann drawing. It was not until 1898 that
they were given the name mitochondria, when Carl Benda used the Greek terms mitos
meaning thread, and chondros meaning granule, in reference to the visual description of
mitochondria.1 Mitochondria in plants would later be discovered by Friedrich Meves, when he
was studying the European waterlily, Nymphaea alba.2
One of the first stains used on mitochondria was by Leonor Michaelis, not yet famous
for his work with Maud Menten on enzyme kinetics, who would use Janus Green B to vitally
stain mitochondria.3,4 In 1910, Claudius Regaud would develop a staining method for
mitochondria which utilized hematoxylin with an iron alum.5 In 1924, Bailey and Davis
developed a method to progressively stain mitochondria utilizing Regauds fluid, washing,
dehydrating, embedding in paraffin, and staining with Mallorys phosphotungstic acidhematoxylin dye.6 This stain results in purple mitochondria against a red background. 6
Construction of a model of how mitochondria function took many years to develop. The
function of one mitochondrial protein, succinate dehydrogenase, was discovered by Torsten
Thurnberg, through the observation of the colour change during methylene blue oxidation.7,8
The function of another mitochondrial enzyme, cytochrome oxidase, was discovered by Arnold
Lazarow and Sherwin Cooperstein through the use of Janus Green B, which is a leuco dye and
would stain mitochondria blue when oxidized.9 The first work to bring together what would
become known as the electron transport chain was accomplished by Keilin in 1925 in his work
showing that cytochrome b was the first electron acceptor.10,11 Keilin and Hartree would have

the order of the respiratory chain enzymes figured out by 1939 when they were able to show
that cytochrome a was actually two
separate proteins.10,12
In 1952, George Palade was the
first to describe the inner foldings of the
mitochondria, known as cristae via the use
of an electron microscope.13
In 1963, Nass and Nass would use
Figure 1. Electron micrograph of mitochondria from
guinea pig pancreas showing the inner and outer
membrane. Image made available by James D. Jamieson
and the Department of Cell Biology, Yale University
School of Medicine.

osmium tetroxide to discover that

mitochondria contain DNA.14 Nearly 20
years later, in 1981, Anderson et. al, would

sequence the Human mitochondrial genome, and discover that it was 16569 base pairs in
length, and contained heavy and light strands.15 37 genes would be discovered on the
mitochondrial genome that code for 22 tRNAs, 12S and 16S ribosomal RNA, cytochrome c
oxidase subunits, and many other proteins.15,16
While many scientists, some described above, have been able to develop staining
procedures for the mitochondria, there have been challenges to overcome to maintain the
morphological and functional properties of mitochondria with respect to fixation and cell
death.17 Pocernich and Butterfield were able to show that the use of acrolein as a fixative
resulted in a loss of pyruvate dehydrogenase and alpha-ketoglutarate dehydrogenase activity.18
While researching post-mortem changes in guinea pigs, Anniko and Bagger-Sjbck found that

the first identifiable signs of autolysis in mitochondria in hair cells began to occur within 10 to
15 minutes after sacrifice.19
Many features and functions of the mitochondria have been able to be characterized
through the use of dyes, such as morphology, which can vary in different tissues from being
ovoid or spherical in hepatocytes of rats, to becoming tubular in fibroblasts in order to protect
themselves from autophagocytosis.20,21 Mitochondrial functions can also be analyzed through
the behaviour of dyes, such as leuco dyes that only exhibit colour when exposed to changes in
light, pH, concentration, temperature.22 Some of these dyes and how they have helped
researchers investigate the mitochondria will be
explored below.
Janus Green B
Janus Green B can be used as a vital stain, and
is made up of a diethylsafranine molecule connected
to a dimethylaniline molecule by way of an Azo

Figure 2. Janus Green B molecule. Courtesy

of Sigma Aldrich.

bond.23 Janus Green B is capable of undergoing multiple reduction reactions when kept in an
anaerobic environment, resulting in the production of violet, yellow, and red dye derivatives.23
Selective staining of mitochondria from pancreatic tissue requires an environment that is
partially anaerobic and which results in some the loss of some of the stain, as Lazarow and
Cooperstein observed in 1953 that only mitochondria near the periphery of the tissue had been
selectively stained blue.23 In 1969, through the use of spectrophotometric methods, Udvardy
and Holland determined that over 90% of Janus Green B was bound to coenzyme Q, a
lipoprotein that has the function of shuttling electrons between complex I and complex II in the

electron transport chain.24, 25 Kensuke Baba developed a method to selectively injure

mitochondria by using a ruby laser to radiate HeLa cells stained with Janus Green B.26 Storb et.
al also used ruby lasers and Janus Green B to damage mitochondria from epithelial and baby
hamster kidney cells and determined that mitochondria were able to recover and reverse the
damage within 4 hours if the damage was not too severe.27
Gomori trichrome stain
The ingredients that the Gomori trichrome stain utilizes are chromotrope 2R,
phosphotungstic acid, and either fast green, light green or aniline blue, mixed in diluted glacial
acetic acid.28 Gomoris trichrome stain will result in nuclei being stained black, collagen being
stained either green or blue, and cytoplasm, keratin and muscle fibers being stained red.28 The
Gomori trichrome stain is a stain that has found use in histopathological analysis of
mitochondrial diseases.29 Gomori trichrome stain has also been useful in the visualizing of
dysfunctional astrocytes that are found in the hypothalamus and are associated with increased
lipid-binding proteins that may be associated with Alzheimers disease.30 When used to stain
muscle fibers, this trichrome stain shows what is known as red ragged fibers, where there is
high subsarcolemmal mitochondrial density due to abnormal cytochrome C oxidase function. 31
Red ragged fibers tend to occur most often in type 1
muscle fiber.32 See Figure 7 for an image of the Gomori
trichrome stain showing this condition.
Methylene Blue
A stain that has actually been used as a form of
treatment for mitochondrial dysfunction due to

Figure 3. Structure of Methylene Blue.

Image courtesy of Sigma-Aldrich Co.

Alzheimers disease is methylene blue (MB), as it is capable of crossing the blood-brain

barrier.33,34 A proposed mechanism that enables methylene blue to alleviate mitochondrial
dysfunction is thought to be from the ability of methylene blue to be reduced to MB by NADPH,
and subsequently be oxidized to MBH2 by cyctochrome
c oxidase.35 The result of this is a decrease in
superoxide radicals, and an increase both cellular
oxygen consumption and the number of cytochrome c
oxidase enzymes.35,36 Like JC-1, methylene blue
Figure 4. Structure of Rhodamine-123.
Courtesy of Thermo Fisher Scientific Inc.

accumulation varies depending on membrane

potential, and is photochemically inactive when the

membrane is hyperpolarized.37
Rhodamine-123 (R123) is another fluorescent dye that can be used to stain
mitochondria.38 Many variations of rhodamine have been developed, such as Rho B, Rho 6G,
Rho 19, Rho 101, Rho 110, Rho 116.39 A derivative of Rho 19, C4R1, has been shown to
uncouple mitochondrial resulting in the stimulation of respiration and a decrease in
mitochondrial membrane potential.40 C4R1 also successfully prevented oxidative stress in the
brain after reperfusion.40 R123 has enabled changes in mitochondrial membrane potential to be
studied as R123 is positively charged and will accumulate in the mitochondria when the interior
is more negatively charged, or hyperpolarized.41 As mitochondria become depolarized,
fluorescence from R123 will be diminished.41 One Rhodamine derivative, dihydrorhodamine 6G

(DHR-6G), is capable of being oxidized to rhodamine 6G (R6G) and fluorescing when oxidized by
reactive oxygen species from cigarette smoke.42
JC-1 is another dye that has enabled the study of mitochondrial membrane potential as
it is capable of changing colour with changes in pH, being orange when there is a high
membrane potential, and being green when there is a low membrane potential, this has made
it a useful stain for use in flow cytometry.43,44 However, there are issues with JC-1 as it is also
capable of changing from green to red as its concentration increases, so depending on how it is
used, concentration needs to be monitored closely.41
Diaminobenzidine is a stain that can be used specifically for cytochrome c oxidase, thus
enabling the determination of the location of the enzyme within mitochondria when analyzed
by an electron microscope.45 Seligman et. al utilized diaminobenzidine to determine that
cytochrome c oxidase was located on the outer leaflet
of the mitochondrial inner membrane facing the
mitochondrial matrix.46
Acridine Orange
10-N-nonyl acridine orange (NAO) is a cationic,
fluorescent dye that can be used to stain mitochondria

Figure 5. Structure of Acridine orange.

Image courtesy of Thermo Fisher
Scientific Inc.

in live cells by binding to the anionic phosholipid, cardiolipin.47,48 Through the use of NAO,
researchers were able to determine the percent of cardiolipin in the of the outer leaflets of the
mitochondrial inner membrane to be approximately 57%.49 NAO has also been able to be

utilized to characterize cellular apoptosis as demonstrated by

Zhang et. al.50 During apoptosis, cytochrome c oxidase is released
from mitochondria, and initiates a signal cascade, resulting in the
activation of caspases.51 Zhang et. al were able to show that rats
given NAO resulted in a decreased cytosolic release of
cytochrome c oxidase and Bid (a proapoptotic protein), and
inhibition of transitional pores that let calcium in.50 By inhibiting
Figure 6. Drawing of
mitochondria by Altmann that
may have inspired Banda in his
naming of mitochondria. From
Altmann, Richard Die
Elementarorganismen und ihre
Beziehungen zu den Zellen.
(1894) Viet p. 160

these events, rats treated with NAO showed reduced myocardial

apoptotic damage during myocardial-ischemia and subsequent
reperfusion as measured by the size of the damage.50 The effect
that chain length of acridine orange 10-alkyl halides has on

targeting mitochondrial cardiolipin has also been investigated. Rodriguez et. al showed that
varying the chain length of the alkyl group from being 1 (methyl-), 6 (hexyl-), or 16 (hexadecyl-)
carbons long, resulted in improved mitochondrial
targeting with 10-hexyl-acridine orange.52 Jacobson et.
al utilized NAO to show that hyperpolarization caused
the dye to be accumulated, and that a depolarization
event occurred, less NAO was retained in the
mitochondria.53 Metachromatic and orthochromatic
staining of mitochondria was also achieved by Erbrich
et. al through the use of acridine orange derivatives.54

Figure 8. Chinese hamster ovary cell

mitochondria stained with Janus Green B.
Photo by Douglas Kline.

Figure 7. Gomori trichrome stain. Normal muscle fibers on the

left. Red ragged muscle fibers on the right. Image courtesy of
Peter Takizawa, Director of Medical Studies, Department of Cell
Biology, Yale School of Medicine.

In 1967, Christian de Duve had a paper published in Subcellular Particles, describing a
new organelle called the lysosome, which were thought to differ from mitochondria in that
they contained acid phosphatases, and were responsible for the acid hydrolysis of
materials.55,56 Through the use of injecting bacterial endotoxins from A. aerogenes into rabbits,
Weissmann and Thomas were able to study the release of acid hydrolases from lysosomes.57
Lysosomes play a role in helping to create antigens that will be presented to the immune
system, thus enabling lymphocytes to target foreign bodies.58 Lysosomes also play a role in
autophagy where they fuse with phagosomes to degrade materials; this function may serve
play a part in neuroprotective mechanisms.59 Weissmann also noted that lysosome morphology
may change depending on whether there was the state of disease or not.60 Cancerous cells
have been observed to have altered levels of lysosomal enzymes such as cathepsins, which
have been visualized with GFP; and acid phosphatases, have been visualized with Nile blue
dye.61,62,63,64 While these cancer-related lysosomal changes can result in dysfunctional
lysosomal enzymatic behavior, because of this there is also an opportunity to use these changes
to target malignant cells.65 Long-lived post-mitotic cells such as cardiomyocytes and neurons
are subject to lysosomal lipofuscin accumulation where processes such as autophagy of
mitochondria are disrupted resulting in the production of excessive reactive oxygen species,
which can result in senescence and cell death.66 According to Staretz-Chacham et. al there are
over 50 different lysosomal storage disorders (LSDs), which have an combined incidence rate
approximately as high as 1 in 1500 live births, to as low as 1 in 7000. 67 Most of these LSDs are a
result of dysfunctional lysosomal acid hydrolysis of molecules, and their subsequent

accumulation that can eventually result in organ failure.68,69 Many of these LSDs do not have
any forms of treatment available, or are difficult to treat because of their location, such as the
nervous system.68,70 Lysosomal autophagy dysfunction and subsequent autophagosome
accumulation has also been implicated in Alzheimers and Parkinsons disease,
respectively.71,72,73 For these reasons, the study of LSDs and lysosomes are of particular
Niemann-Pick Disease
Niemann-Pick type C is a lysosomal disorder due to a mutation in NPC1 or NPC2 genes
that results in cholesterol, gangliosides, and bis-monoacylgylcerol phosphate accumulating in
lysosomes, resulting in dysfunction.74 Techniques have been develop which make use of
recombinant antibodies, and immunofluorescent substrates, often of a proprietary blend,
lysosomal disease morphology can be imaged. The accumulated cholesterol in the lysosome has
been a target for characterizing Niemann-Pick through the use of filipin staining, as well as
utilizing a bacterial toxin that is capable of binding to cholesterol, perfringolysin O.75
Kwiatkowska et. al utilized a recombinant probe
consisting of perfringolysin O (PFO) and glutathione S
transferase (GST) that was successful in indicating the
cholesterol deposits that are common in Niemann-Pick
disease.75 anti-GST IgY-peroxidase antibodies from
chickens were then bound to the recombinant probe and
visualized with SuperSignal West Pico Substrate.75 See
figure 9 for image of GST-PFO stain. A fluorescent

Figure 9. Lysosomal cholesterol labelled

with GST-PFO-antibody complex and
visualized with SuperSignal West Pico


antibiotic, filipin, can also be used to stain lysosomal cholesterol from fibroblasts in NiemannPick disease, to show the accumulation of cholesterol in neuron cell bodies and glia.76 Detection
of Niemann-Pick cholesterol deposits in lysosomes from blood smears have also been
successfully stained with filipin.77 However, this method has proven cumbersome because
fibroblasts must first be cultured, so Wakida et. al developed a technique that is much faster
through the utilization of bone marrow smears as a source for lysosomal cholesterol to perform
the filipin stain.78 See figure 10 below for image of filipin stain.
Rhodamine and Fluorescent Dextrans
A rhodamine based probe was developed by Lu et. al that responded to changes in pH,
and when exposed to an alkaline environment is colorless, but when the pH becomes acidic the
probe begins to fluoresce pink, thus enabling real-time observation of lysosomal pH changes.79
Use of an elastin-rhodamine complex was used to observe the activity of a lysosomal enzyme
known as elastase, from Human granulocytes.80 Another pH sensitive rhodamine probe that
was bound to morpholine was created by Shi et. al, that was able to show lysosomal when
induced by chloroquine.81 Other rhodamine-morpholine probes that become fluorescent after
cleavage by caspase-3 in order to observe apoptosis in cells have also been developed and were
useful both in vitro and in vivo.82 Kang-Kang et. al developed two rhodamine-based probes that
were showed no cytotoxicity, and were sensitive to changes in pH at 4.79, and 5.23,
respectively.83 Fluorescent dextrans have succesfully used as a pH monitor in lysosomes, and
were able to further analyze that the pH of new phagosomes was alkaline, despite an
environmental pH of 6.5 and lysosomal fusion.84 Simultaneous delivery of sulforhodamine and
fluorescent dextrans together to an organelle were used to observe that cargo selection to

lysosomes is a selective process as the two compounds were delivered to lysosomes at

different rates, despite the same initial time of entry.85 Wang and Goren believed that the
temporal difference may have been due to the gelling of the dextrans, which may have
impeded lysosomal movement.85 Mycobactrium avium is capable of surviving inside lysosomes,
in order to take advantage of this for study, Oh and Straubinger labelled the surface of M.
avium with rhodamine and carboxyfluorescein to observe if M. avium changes the pH of
lysosomes in order to survive.86 Through the use of the properties of rhodamine, which is a pH
insensitive dye, while carboxyfluorescein is pH sensitive, and analysis of their fluorescent ratios,
Oh and Straubinger were able to determine that M. avium were exposed to a pH of
approximately 5.7. 86 Ohkuma and Poole used fluorescent dextrans to observe typical
macrophage pH to be in the range of 4.7-4.8.87 Fluorescent dextrans have been successfully
used to observe M. tuberculosis interfere with lysosomal pH has in as well.88 Luzio et. al used
endosomes containing either rhodamine-dextran and Oregon-Green-dextran to observe
homotypic and heterotypic SNARE fusion via confocal microscopy.89 Hasegawa et. al modified
rhodamine by linking it to cyclodextrin, making two hybrids that were sensitive to changes in
pH.90 Koshkaryev modified liposomes with octadecyl-rhodamine B, and used them to deliver a
fluorescent dextrin to lysosomes as a proof-of-concept in treating LSDs, and to demonstrate
that octadecyl-rhodamine B can improve delivery to dysfunctional lysosomes.91 From the same
lab as Koshkaryev, Thekkedeth et. al also successfully have used octadeceyl-rhodamine B to
improve delivery of a therapeutic dose of glucocerebroside to a dysfunctional Gaucher cell in
vitro which resulted in an increased amount of the therapeutic reaching the targeted
lysosomes.92 In 2011, a rhodamine derivative has also been fused to a lactam-ethylenediamine

to behave in a pH-sensitive manner that was also resistant to photo-bleaching by Wu et. al.93
Again in 2011, from the same lab, Li et. al developed a rhodamine-lactam hybrid that was
bound to mesoporous silica nanoparticles in order to observe lysosomal pH changes when
analyzed by flow cytometry.94 Glunde et. al developed a compound, 6'-O-lissamine-rhodamine
B-glucosamine, to fluorescently label lysosomes in order to understand the role they may play
in breast cancer metastasis.95 With this compound they were able to observe the co-localization
of 6'-O-lissamine-rhodamine B-glucosamine with lysosome associated proteins 1 and 2, as well
as CD63.95 Hama et. al developed a method utilizing a avidin-rhodamine X hybrid, where the
probe is activated only after endocytosis and subsequent lysosomal degradation.96 Using this
method, they were able to minimize background noise, and fluorescent pinpoint cancerous
microfoci within mouse peritoneal ovaries.96
Acridine orange
Through the use of acridine orange Urashima et. al were able to stain leukemic cells,
resulting in the cells staining orange or green which correlated to the metabolic activity of the
cells, with the orange cells containing more RNA, and having an improved prognosis of acute
lymphoblastic leukemia.97 Pittis et. al used acridine orange as a fluorescent marker to indicate
phagolysosomal fusion in patients with thalassemia major, and to show the efficacy of the
bacterial siderophore, deferoxamine, in restoring this loss-of-function in phagolysosomal fusion
in patients with thallasemia major.98,99 Okamoto et. al were able to use pH-dependent
quenching of acridine orange to demonstrate that H+-ATPase isolated from rat liver can utilize
dATP as well as dGTP, and that Mn++ can result in 77% of the activity as seen when Mg++ is
used.100 Moorjani et. al measured the change in fluorescence of lysosomes labelled with

acridine orange, in order to better understand the interactions between gp120, an HIV surface
glycoprotein that neutralizes antibodies, and the CD4 receptor.101,102 Mozhenok et. al used
acridine orange to study the effects of bilirubin, chelerythrine, and farmorubicin have on
phagosome-lysosome fusion.103 They came to the conclusion that all three compounds were
biologically active and led to increased cytoskeletal activity which was responsible for the
increased success phagosome-lysosome fusion events.103 Through the pH-sensitive properties
of acridine orange that results in a red color at lower pH, and green at higher pH, Wihlmark et.
al observed that retinal pigment epithelial cells that were more sensitive to irradiation with
blue light as they had considerable loss of lysosomal stability and viability when compared to
controls, possibly due to accumulation of lipofuscin.104 The use of acridine orange to observe
photo-oxidative disruption in a similar fashion as described by Wihlmark et. al, has also been
done in Human fibroblasts, where they determined that photo-oxidation results in the release
of lysosomal enzymes to the cytosol which subsequently leads to necrosis, apoptosis, or a form
of damage that is reparable.105 Etoposide, an inhibitor of DNA topoisomerase II, was used to
induce apoptosis in cells vitally stained with acridine orange and bisbenzimide. 106 The combined
fluorescence of bisbenzimide and acridine orange was superior to either stain used alone.106
Minami utilized acridine orange to study the immunosuppressive effects that different
glycopeptidolipid antigen of various M. avium serotypes have on phagosome-lysosome
fusion.107 The therapeutic potential of acridine orange was investigated by Kusuzaki et. al
through the photoexcitation of acridine orange in lysosomes of multidrug resistant (MDR)
mouse osteosarcoma cells.108 They determined that when photoexcited, acridine orange is
capable of exerting cytotoxic effects, and that further research should be done to determine if

it is a suitable form of therapy.108 Kusuzaki et. al have also investigated the cytocidal induction
via visible light, or X-rays of musculoskeletal sarcomas treated with acridine orange due to nonmalignant cells being able to clear acridine orange due to not possessing an acidic environment
where cancerous cells have a growth advantage.109,110 They found that when acridine orange
accumulated in acidic organelles, like lysosomes, and when it was excited by by visible light or
radiation, that the excited acridine orange generated ozone from cytoplasmic oxygen. 109,111 The
ozone will eventually rupture the lysosomes, resulting in the dumping of lysosomal enzymes,
and cell death.109 Research done by Zdolsek evaluated the effect that a variation in oxygen
levels had on the phototoxicity of acridine orange in lysosomes from mouse macrophages, as
well as the effect that sodium azide, a singlet oxygen quencher, to determine whether the
phototoxicity of acridine orange was due to the production of singlet oxygen or free radicals.112
Zdolsek found that elevated oxygen levels resulted in greater phototoxicity, and that cells
treated with sodium azide showed protection against irradiated acridine orange, indicating that
acridine orange produces singlet oxygen, as corroborated by Amagasa, Kobayashi and Ito, and
Suzuki et. al.112,113,114,115 The accumulation of acridine orange in musculoskeletal tumours was
studied with the intent of determining the mechanism of acridine orange accumulation in
musculoskeletal tumours.116 From observing the changes in extracellular and intracellular pH
via the fluorescence intensity of acridine orange, Matsubara et. al found that the accumulation
of acridine orange in cancerous cells was due to difference in intracellular pH and extracellular
or lysosomal pH.116 The antioxidative and lysosomotrophic properties of an acridine derivative,
9-amino-acridine-propanolol (9-AAP), were investigated in combination with acridine orange as
a fluorescent indicator.117 Dickens et. al found that when fluorescent cells were washed with 915

AAP, that they became more dim, due to alkalization, and that 9-AAP may be useful in
protection against free radicals.117 The use of counting cells with antibodies has proven to be a
useful method, but it is also very time-consuming and expensive.118 Lovelace and Cahill
developed a faster, and less expensive method using acridine orange to as a highly specific
marker for counting microglia cells amongst astrocytes, due to the high lysosome content of
microglia and the selectivity of acridine orange for lysosomes.118 In 1992 Handrock et. al used
acridine derivatives have the ability to increase sulfated glycosaminoglycan storage in
lysosomes possibly due to their acridine ring.119 See figure 11 for image of Human fibroblasts
stained with
acridine orange.f120

Figure 10. Lysosomal cholesterol from fibroblasts stained with filipin. Image
courtesy of Mayo Clinic.

Figure 11. Human fibroblasts stained with acridine orange to

show lysosomes.


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