Most of the curves related to the dermis optical properties show high
standard deviation bars that are overlapped for the consecutive values
(unlike the epidermis), thus hardening the interpretation of the
observable kinetic changes (Figure 4 and 5 (d, e, f), Table 2). This is
possibly due to the combined effect of measurement uncertainties
propagated through the Jacques model. Additional reason might be the
unstable LC-OCT probe positioning, which was constantly reapplied to
tested skin sites without the possibility to use any kind of adhesive
position-tracking labels due to mechanical manipulations with the skin
surface in the beginning of the experimental protocol, making it
impossible to analyze exactly the same tissue volume throughout the
experiment. Another reason might be the morphological variation between
the dermal layers of the volunteers and a more significant contribution
of multiple scattering to the dermal layer linear fit (as compared to
epidermis).[16]
The uncertainties mentioned in the previous paragraph are described in
detail in the work of Waszczuk et al .[16]First, there are uncertainties on the optical properties, estimated for
a calibration phantom using double integrating spheres, which are then
used in LC-OCT images processing. Second, there is an effect of both
linear fit accuracy and variation between different images of the same
sample on the µ eff and ρ parameters.
Finally, all these uncertainties affect the resulting LC-OCT estimation
of scattering coefficient and scattering anisotropy factor. However,
despite it was mentioned by Waszczuk et al . that when propagating
through the model of Jacques the errors were amplified for high values
of µ s(λ 800) andg(λ 800) , this method is still well suited
to samples with relatively high scattering anisotropy
(g(λ 800) factor ranged between 0.7 and
0.9) and scattering coefficientsµ s(λ 800) up to 12
mm-1, i.e. biological tissues such as skin.
Epidermis scattering coefficient demonstrated ~ 20%
decrease for the control condition. But with a mean standard deviation
over time of 25% of the observed values (Table 2) and the significant
overlapping, it can be concluded that control conditions in general do
not significantly affect the skin optical properties. However, it can
also be seen that some OCA caused noticeable clearing effect in dermis.
For example, a mixture of both sugars with DMSO caused a decrease in
epidermis scattering \(µ_{s}^{\text{epi}}\) (Figure 4(b)). Sucrose/DMSO
mixture effect resulted into 28±18% decrease after 21 minutes of
experimental protocol. Such decrease overcomes a moderate overall
standard deviation (mean ~12% for all timepoints).
Mixture of Glucose and DMSO demonstrated more pronounced effect –
40±3% of epidermal scattering decrease (from 8.3 ± 1.3
mm-1 to 5 ± 0.9 mm-1 for three
volunteers), which is greater than the mean variation of data over the
time for this OCA – 10%. Both mixtures demonstrated as well an
increase in scattering anisotropy of a dermal layer. However, as it was
mentioned above and can be seen in Table 2, dermis values have a
relatively high standard deviation, making it onerous to conclude that
there is a clearing effect on dermis. So, only the general behavior can
be mentioned. The result for Glucose/DMSO mixture is in a good agreement
with observations made in our previous publication, where this mixture
was considered as one of the most efficient among nine OCA in terms of
in-depth increase of LC-OCT image intensity and
contrast.[26] Such increase was assumed to be
caused by the epidermis scattering decrease, that is quantitatively
assessed in the present study. Other notable results are related to the
mixtures of Glucose and PEG with PG as a permeation enhancer. Glucose/PG
mixture (Figure 4(c)) caused 23% decrease of epidermal scattering with
the mean ~11% data variation over time and PEG/PG mixture
caused 33% decrease with ~10% mean data variation.
PEG/OA/PG mixture demonstrated the most pronounced\(µ_{s}^{\text{epi}}\) decrease (Figure 4(a), blue triangle data
points). Already after 21 minutes of experimental protocol (10 minutes
after ultrasound-assisted clearing was over), this parameter decreased
by 33 ± 17% for 3 volunteers, from 9.9 ± 1.1 mm-1down to 6.6 ± 1.7 mm-1. It can be seen that the
decrease pattern in this case is the most confident among the other OCA.
Moreover, for the first 6 measurement points (up to t = 21 min), the
mean standard deviation is only ~5% of the data (overall
SD is ~9% of the data). Considering all the mentioned
uncertainties and high standard deviation bars in the case of other
mixtures, PEG/OA/PG mixture demonstrated the most significant clearing
effect. That is as well in good agreement with previous
observations,[26] where PEG/OA/PG mixture
demonstrated the best increase (40%) in image in-depth intensity and
contrast after 10 minutes of ultrasound-assisted clearing.
Concerning the scattering anisotropy parameter, none of the OCA
demonstrated significant changes in g epidermiswith relatively low SD values. Together with decrease in\(µ_{s}^{\text{epi}}\) one can conclude that OCA clearing effect on
epidermis expressed and limited to matched RI of scattering particles
and interstitial fluid. This RI matching effect is a well-investigated
cause of reduction in tissue scattering mentioned in the
literature.[17] On the contrary, dermal scattering
does not change significantly due to high SD, but the tendency of dermal
scattering anisotropy g dermis towards increase
indicates a different influence of tested OCA on skin dermis than to the
epidermis in vivo . Mie theory explains that such behavior is
related to an increase in the size of the scattering particles rather
than to RI matching.[19] This applies to the
dermis as there are bundles of collagen, which are probably swollen
under the action of OCA mixtures, causing light to scatter in the
forward direction.
Our results obtained on in vivo human skin demonstrate the
possibility of LC-OCT quantitative estimation of changes in skin
scattering coefficientµ s(λ 800) and
scattering anisotropy factor g(λ 800) ,
caused by biocompatible optical clearing (involving the reduced
concentrations of clearing agents used together with chemical and
physical permeation enhancers). Moreover, the mixture of PEG/OA/PG has
shown the best results. Although our current results were not validated
with integrating spheres measurements (as the study was carried out on
skin in vivo ), this was done on phantoms in the study of Waszczuket al. , demonstrating a good correlation between the values
obtained using integrating spheres and application of Jacques’ model to
LC-OCT images.[16]