On the surface, it seems obvious what the differences are
between discrete and continuous variables. It is as their names imply; a
discrete variable is defined as a “variable that arises upon random chance and
possessing only countable variables” (Biostat Lecture 3), while a continuous
variable is defined as “variable that can take on any value between two
specified numbers” (Biostat lecture 3). These two types of variables can easily
be applied to many of the experiments I conduct around the lab. The number of Drosophila that have developed brain
tumors can easily be considered a discrete variable, while the CT values
obtained in my qPCR can be easily thought of as continuous variables. However,
on closer inspection, these variables are not so obviously defined. Eloquently
expressed in Velleman and Wilkinson’s article “Nominal, Ordinal, Interval, andRatio Typologies are Misleading”, argues that often these categories introduced
by S.S. Stevens, such as nominal, ordinal, interval, and ratio, can be
restrictive, and more importantly, by asserting the scale type “independent of
the questions asked of the data” it in turn, limits what can be asked of the
data generated in an experiment. This close-minded thinking may have
detrimental effects on hypothesis-driven research and limits what subsequent
experiments should be run as a direct result of the data generated in the pilot
experiment. Furthermore, Motulsky introduces an interesting concept in Intuitive Biostatistics that variables
can be much more ambiguous than they originally seem. One such example is that
of color. The perception of color can be thought of as nominal variable with
discrete outputs such as blue, red, or yellow. However, the concept of color as
a nominal variable becomes much more ambiguous once you consider monochromatic
color as a wavelength where it can be considered a ratio variable. Something
that seems so obviously to be a categorical variable can be seen as a
continuous variable as well. This idea of wavelength of color is extremely important
in microscopy work. In many of the fluorphores used in microscopy, they express
their excitation as a single wavelength say 647 nm. On the surface, it is
possible to consider this value as a discrete variable since it is advertised
to be excited at this single wavelength. However, in actuality, photo bleed
through may occur where you observe detection of fluorescence in other
fluorescent channels as well, indicating a possible range of excitation. This
is a real world application of the idea of the pitfalls of thinking so
concretely of the idea of what type of variable it should be and instead
allowing for an open-mind. By understanding the idea that excitation can be
thought of as a range of wavelengths, I can play out microscopy experiments that
includes multiple fluorophores without worry of photo-bleed through.
Excitation of a fluorophore occurs over a range of wavelengths, within which is a peak wavelength where maximum excitation is achieved. Bleed-through is when you excite a system of multiple fluorophores at a certain wavelength appropriate for the target fluorophore, but a non-target fluorophore emission is also detected. This occurs when two conditions are met: (1) the fluorophores are improperly matched such that the excitatory wavelength efficiently excites one fluorophore – as intended – but also inefficiently excites another because the excitatory wavelength falls somewhere in that fluorophore’s excitatory range; and (2) the emission filter on your detector allows a range of wavelengths into the detector that includes the possible emission from both fluorophores. A solution is to use properly-matched fluorophores, which means that the ranges of excitation wavelengths for each overlap minimally. Another solution is to just replace the filter with one that only allows for detection of the emission of the fluorophore of interest. It is not that excitation “can” be thought of as a range of wavelengths; rather, it must be. Knowing this would keep one from worrying about bleed-through only because then they would understand this phenomenon and prepare for it in advance. Please correct me if I have misunderstood something.
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