Quantifying Polymer Deposition
energy ranges: UV, violet, blue, and green. Te instrument is equipped with a motorized stage that is capable of trans- lating the sample in the x-direction, making it possible to obtain distance scans along the hair fiber axis or across a cross section. Te 3 mm distance scans along a hair fiber axis reported here were generated using light microscope images magnified approximately 500× and a diaphragm setting of 10 μm × 20 μm. Since a higher resolution was required to image the cuticle in cross sections of hair fibers, a magnification of 1,000× was used for these scans with a diaphragm setting of approximately 3 μm2
. Table 1 shows the parameter settings
for these measurements. Polymer labeling. In order to detect the polymer, it must
be labeled with an appropriate fluorophore that is capable of re-emitting light when it is excited by incident light. We chose sulforhodamine-b, which is an anionic fluorophore, since it has a large absorption, has a large luminescence, and is highly soluble in water. Te sulfite groups (SO3
2–) are responsible
Figure 2: Diagrammatic view of a microfluometer. The detector (not shown) would normally be a visible-light spectrometer.
source through a wavelength selective (excitation) filter. Short wavelengths passed by the excitation filter reflect from the sur- face of a dichroic mirror (beam splitter) through the microscope objective to bathe the specimen with intense light. Te emitted light of longer wavelength re-radiates spherically in all directions, regardless of the excitation light source direction. If the specimen fluoresces, the emitted light gathered by the objective lens passes back through the dichromatic mirror and is subsequently filtered by a barrier (emission) filter, which blocks the unwanted excitation wavelength [9,10]. Aſter leaving the microscope, the light enters a detector, which in the present case is a spectrometer. Microfluorometry can be used in the quantitative study of
fluorescent molecules (fluorophores) by monitoring the FI from a sample. Te fluorophore absorbs light energy of a specific wavelength and re-emits it at a longer wavelength. Wavelengths of maximum absorption and emission (for example, absorp- tion/emission=485 nm/517 nm) are typical terms used to refer to a given fluorophore. Te absorbed wavelengths depend on the energy transfer efficiency, the time before emission from the fluorophore, and the chemical environment of the fluorophore molecule [10]. Te molecule in the excited state interacts with the surrounding molecules, which allows the emitted fluorescence to be clearly distinguished from the excitation light source. Since light-detecting devices are highly sensitive, even a single photon can be detected. One fluorophore can emit millions of photons per second [11]. Experimental setup. Microfluorometry was performed
using a Leitz MPV 1.1 microscope with a Vertical Ploem Illuminator. A photograph of the instrument used in this work is shown in Figure 3. Te instrument operates in the incident mode in the visible range at a specific wavelength determined by the band-pass filter selected for the following
26
Figure 3: Photograph showing the components of TRI Princeton’s microfluo- rometer.
www.microscopy-today.com • 2019 July
for the molecule’s anionic character. Tis fluorophore does not exhibit pH-dependent absorption or fluorescence over the range of pH from 3 to 10. Figure 4A shows the molecu- lar structure of the sulforodamine-b used for our study. A fluorescence emission spectrum of a hair fiber labeled with sulforhodamine-b is shown in Figure 4B. Cationic polymers in general are highly substantive to hair
because of its 3.67 pH isoelectric point, when the molecule car- ries no net electrical charge, which is also the pH of cosmeti- cally unaltered hair. At any pH above the isoelectric point, the surface of hair has a net negative charge and cationic polymers are attracted to it [1]. Te control hair fibers were treated only with the anionic
sulforhodamine-b that is known to be repelled from the nega- tively charged surface of the hair fibers; therefore, only a small amount of fluorophore electrostatically binds to the untreated hair surface, resulting in a low FI. Applying the polymer. Strands of hair were randomly
selected from a tress (a bunch of hair fibers). Numerous stud- ies have shown that the middle part of a hair fiber experiences
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