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Running head: FLOURESCENT CELL DYES

Fluorescent Cell Dyes:


Development, Use, and Applications in Imaging and Spectroscopy
Fernanda Lugo
University of Texas at El Paso

UNIV 1301, Fall 2014


Professor C.V Garcia

FLOURESCENT CELL DYES: DEVELOPMENT, USE, AND APPLICATIONS IN


IMAGING AND SPECTROSCOPY

Thesis Statement:
The many fields of research in Biology being done today depend mainly on the
observation of cells and their reactions, but how can we observe cells? Microscopy is the main
method, but its basic forms only allow one to see the outline of cells, not the minute detailed
organelles inside. Understanding many illnesses and pathologies depends on understanding the
functionality and action of all organelles; so to see clearly inside cells, microbiology seeks the
assistance of the physical and chemical phenomenon denominated fluorescence. Fluorescence
refers to a molecules ability to refract light a certain way (called a chromophore) and to re-emit
a light by means of quantum excitation (fluorophores). In Biology this allows scientists to paint
cells with a certain compound that travels to a specific organelle, so that the organelle is then
visible. In this essay, the means by which cells intake dyes will be explained, and the phenomena
that permit the fluorescence itself will be clarified.
Introduction:
The biomedical focus of the 21-century is aimed at reducing disease tracking and
diagnosing them early on and eliminating them. Among the various diseases in this time and age,
one that has been troublesome and elusive to a cure for so long is cancer. There are billions of
people working on developing and testing drugs, but another issue with cancer is that it is a
complex thing to track and diagnose. The major way of detecting cancer nowadays is by MRIs
tumors, CT scans, and often time it is required to have invasive biopsies to confirm. Most times
it is quite uncertain whether a tumor is cancerous or benign.
A developing field of research is now focusing
on non-invasive ways to detect malignant growth in

FLOURESCENT CELL DYES: DEVELOPMENT, USE, AND APPLICATIONS IN


IMAGING AND SPECTROSCOPY

bodies: Fluorescent cell dyes. Cell dyes in general have the property to attach and be attracted to
specific sites in tissue or cells depending on their affinity; certain dyes paint mitochondria,
certain dyes paint lysosomes, certain dyes like propidium iodide only paint dead cells and so on.
Another application of dyes is that as they migrate through a tissue or a cell, they can work as
signaling devices to the specific biochemical group or property that the moiety of the dye will
bond to, similar to the way watercolors on a waxed up paper will not really paint until it finds a
portion of the sheet that is different.
So far, cell dyes in general have been explained, but fluorescent cell dyes have another
fascinating property: they are generally poly-aromatic hydrocarbons or heterocyclic, which
means their crystalline arrangement facilitates their ability to fluoresce, as the name suggests.
Fluorescence is described as the emission of light by a substance as a result of radiation from the
light spectrum. Physicists and chemists describe it more complexly as the excitation of electrons
on a substance that jumps to another orbital level and emits a photon, but for all intents and
purposes as biologists, in this writing it will be referred to as something that shines when you use
shorter wavelength emissions on it. At the cellular level, this property is very valuable for
microscopy, as one can clearly observe greater details with bright colored emissions than with
regular visible light on a microscope.
Fluorescent Probes for Cancer Tracking Research:
A fluorescent probe is a fluorophore
designed to respond to a specific stimulus
or to localize within a specific region of a
biological specimen. So how do scientists

FLOURESCENT CELL DYES: DEVELOPMENT, USE, AND APPLICATIONS IN


IMAGING AND SPECTROSCOPY

determine what moiety on which molecule will transport the dye to a certain specified place in
the cell? Which organelle will the dye migrate to if say an amino or thiol group is added perhaps
on this end of the hydrocarbon? An active targeting mode can be further engineered by
chemical functionalization of vesicle surfaces with biological moieties (like antibodies, peptides,
DNA/RNA, small molecules). More importantly though is to which tissue will it migrate, which
will be analyzed further on (Papalia, 2014).
But what is known for most existing dyes is the methods of absorption of the dye through
the membrane. Primarily by experimentation, scientists can determine the patterns of absorption
of certain groups of molecules, and then one can see the tendency of such group to go to a certain
type of cell or a certain organelle. Certain dyes are taken in by passive transport, but the majority
due to the size of their molecules, require endocytosis: a method that allows the passage of large
molecules by forming vesicles extended portions of the phospholipid bilayer that act as carriers
for the molecule until it is processed by the Golgi to realize there is no threat, and is then let to
drift in the cytosol.
Some compounds are known to be apoptosis inducing or simply very toxic to certain
types of cells, and these studies are primarily how medicines are developed. An example of this
is NIR-emissive (near infrared) compositions that are too large to be cleared via renal filtration,
which results in high fluorescence background noise and increased potential for in vivo toxicity
(Mikhail, 2007). Extensive research and experimentation is done on how cytotoxicity of a certain
compound affects cancer adhering cells and metastatic cancer cells, and always a normal control
cell, so the desired compound for a cancer treatment would attack cancer and not all other types
of cells.

FLOURESCENT CELL DYES: DEVELOPMENT, USE, AND APPLICATIONS IN


IMAGING AND SPECTROSCOPY

Using natures own compounds such as the


naturally green fluorescent protein (GFP) discovered in
1961, initiated extensive research in the area of the
naturally fluorescent proteins. As a result, Martin Chalfie,
Osamu Shimomura and Roger Y. Tsien share the 2008
Nobel Prize in Chemistry for the work on the discovery of
GFP and its application as a tagging tool in bioscience (Brovko, 2010). This is the goal of cancer
research, and with the aid of fluorescent spectroscopy dye development scientists can bind
compounds and medicines with little risk of cytotoxicity; this in turn will allow us to attack
cancer cells with these complexes of fluorescent dyes/medicines that will paint them, and then
the fluorescent signaling will tell a great deal about the development of the illness. It simplifies
the task of finding the needle in a haystack that is cancer cells. Their genetic errors and typos that
inhibit the apoptotic cycle are often very well hidden from the surrounding tissue, so the use of
dyes has spectacularly aided in the detection of such misshapen cells.
What helps in identifying tumor growth is aided ironically by the physiology of the
tumor itself. Tumors have a tendency to devolvement in epithelial cells, which means they
seldom grow deeper than 2mm before having to resort to feeding directly of the blood source.
Once they have made contact to a blood source this is called the tumor microvasculature which
not only which leaves he tumor at greater risk for being exposed by the sentinels of the body:
lymphocytes and phagocytes , but is also often leaky. Vasculature of tumors is often leaky and
accumulates molecules in the blood stream to a greater extent than normal tissue. The enhanced
permeability and retention effect is ironically a benefit to the objective of cell dyes for cancer

because as nanometer-sized polymersomes that are small enough to traverse compromised

FLOURESCENT CELL DYES: DEVELOPMENT, USE, AND APPLICATIONS IN


IMAGING AND SPECTROSCOPY

endothelial cell barriers, and passively accumulate in tumor tissues are easily detected. Sadly this
condition of leaky aggregation of bloodstream tissues is also a great danger for the surrounding
tissues as it often leads to hypoxic environments, which happen to be a cause for cancer
themselves. Hypoxia leads to genetic instability, which is associated with cancer progression, via
down regulating nucleotide excision repair (NER) and mismatch repair (MMR) pathways
(Mikhail, 2010).
The risk of using fluorescent probes to find tumor growth in vivo has been undergoing
research development, but the use of many types of aromatic carbon ligands with powerful fluorescing
properties (quantum yield) has been limited by difficulties in obtaining nanocrystals that are

biocompatible. To address this problem, a new technique that copies the natural way vesicles are
formed in endocytosis is being engineered: encapsulating these individual chromophores and
nanocrystals in phospholipid block-copolymer micelles to be used in both in vitro and in vivo
imaging. In research done by Dubertret in his 2002 paper of experimentation with quantum dots,
a specific fluorophore he saw results that show: When conjugated to DNA, the nanocrystalmicelles acted as in vitro fluorescent probes to hybridize to specific complementary sequences.
Exciting results show the promise of decreasing toxicity and any potential damage, while not
mitigating at all the absorption of the fluorescent probe.

FLOURESCENT CELL DYES: DEVELOPMENT, USE, AND APPLICATIONS IN


IMAGING AND SPECTROSCOPY

Conclusion:
The importance of developing fluorescent dyes is inherently dependent on the links this
field of spectroscopy has in related fields like physics, chemistry, biochemistry, medicine and
pharmacy. Dyes for cell and tissue imaging could be used for tracking and detecting tumor
growth in a noninvasive way, as fluorescence microscopy allows the deep penetration of nondamaging doses of radiation to make compounds like fluorophore probes emit light back to the
sensory technology, such as an MRI or CT scan.
Learning about the physical and chemical properties of such compounds will help
advances in all fields, not only in spectroscopy, as the applications of fluorophores are nearly as
diverse as its natural occurrence and that of similar phenomena like bioluminescence and
naturally fluorescing proteins. Fluorescent dyes may be the progress we need in our health
industry, because their benefits of simple noninvasive diagnosis and tracking of tumor growth,
and the medicine linking technology could help save lives in the fields of medicine in the near
future.

References
Dubertret B, Skourides P, Norris DJ, Noireaux V, Brivanlou AH, Libchaber A. In vivo imaging
of quantum dots encapsulated in phospholipid micelles. Science. 2002 Nov
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FLOURESCENT CELL DYES: DEVELOPMENT, USE, AND APPLICATIONS IN


IMAGING AND SPECTROSCOPY

Lubov Brovko, Bioluminescence and Fluorescence for In Vivo Imaging, Published: Society of
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9780819482488 | Print ISBN13: 9780819482471


http://ebooks.spiedigitallibrary.org/book.aspx?bookid=77
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Papalia, T., Siracusano, G., Colao, I., Barattucci, A., Aversa, M. C., Serroni, S., et al. (2014).
Cell internalization of BODIPY-based fluorescent dyes bearing carbohydrate residues. Dyes
& Pigments, 110, 67-71. doi:10.1016/j.dyepig.2014.05.022
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