5.1.1 Bight-field Imaging
From bright-field images, we observed that after glycerol application the skin became transparent and the hair inside the deep area was more visible in both skin tissues (Fig. 4.4). Glycerol is with a higher refractive index (1.47) than extracellular and intracellular fluids (1.34-1.36). After immersion the skin tissues into anhydrous glycerol, glycerol diffused inside and filled all of the skin tissues to create a higher refractive index matching environment for the scattering particles (such as organelles, protein fibrils, membranes, protein globules, with refractive index 1.39-1.47 [49, 66] ). Therefore, the light scattering was reduced and the image depth increased. This result is similar to and supported by the previous research of G. Vargas et al. in 1999, by observing the rat skin after the glycerol application [9]. By using spectrophotometer and OCT they proved that glycerol will diffuse into the skin to decrease the refractive index mismatch. This result
also correlates with many previous studies [5, 26, 67].
5.1.2 HGM Imaging
With much-reduced scattering and higher transparency in the studied samples, we
thus expect an increase of the SHG intensity at deeper dermis layers. Scattering in skin tissues will not only results in a decrease of excitation intensity, but also defocus the point spread function. The combined effect will not only cause SHG and THG signals to decrease, but will also degrade the system resolution and contrast with increased imaging depth [2]. As a result, in both samples of Case E100-I90, we witnessed that the SHG image intensity in the dermis was greatly enhanced after the optical clearing (Fig. 4.9 and Fig. 4.10). The excitation light was able to travel down to deep regions of the skin with less energy decrement and less wavefront distortion. Moreover, the reduction of scattering also makes it easier for SHG signals to travel back to the detector. Our SHG results correlate well with the bright-field images and with previous studies.
Besides excitation intensity and point spread function [2], THG intensities also strongly depend on the refractive index mismatch inside the excitation focus [68, 69].
After the glycerol application, the transparency effect will not only restore the excitation light and allow easier collection of the generated nonlinear signals, as been indicated by the SHG intensity recovery, but will also decrease the refractive index mismatch. These two different mechanisms will compete for the THG intensity. In Case E100-I90, THG intensity decreased at the epidermis while increased or remain the same at the dermis, indicating that refractive index matching is the dominant effect at the epidermis. Although glycerol will greatly enhance the homogeneity of the tissue and reduce the THG radiation,
in the dermis, a strong reduction of scattering of the whole epidermis will result in a strong increment of excitation intensity and thus eventually the detected THG photons. Thus, the THG image intensity increased or remained the same in the dermis after optical
clearing.
5.1.3 Skin Structure Analysis
The dehydration and shrinkage were proved two of the most important mechanisms of the optical clearing and well reported in previous illustrations [49, 70, 71]. In Case E100-I90-2, we found that the dehydration and shrinkage of tissue enhanced the SHG intensity and resulted in less reduction of THG image intensities at epidermis when compared to Case E100-I90-1 (Fig. 4.10). It is known that the thickness of skin tissues can be affected by glycerol application. First, the strong affinity of glycerol would make the water flux out tissues and lead to dehydration. Second, the permeability coefficients of water and glycerol are different, which are on the order of 10−2cm/min and 10−5 cm/min. This difference made more water flux out than glycerol enter the skin, causing the tissue volume decrease [52]. By moving the skin structure toward the surface due to shrinkage, one thus expects to see further-improved image intensities.
5.2 100% Glycerol Immersion V.S. 50% Glycerol Immersion
In the case of 50% glycerol application with immersion, the SHG intensities at
dermis were also greatly enhanced after the optical clearing (Fig. 4.14). Although with its lower refractive index (1.398)[72] and lower dehydration power compared to 100%
glycerol, the 50% glycerol was also found able to decrease the scattering in the tissue by observing the significantly increased SHG intensity. In the bright light image, the transparency of the skin tissues was enhanced, showing a decrement of the scattering.
However, the 50% glycerol could not introduce such a highly homogeneous environment inside the tissue as 100%. Under the bright light microscopy, the variation of the transparency by using 50% glycerol was less than 100% glycerol. The SHG images and bright images of Case E50-I90 were shown in section 4.3.2.
The most significant difference between these two different concentrations is the variation of the THG intensity. Different from the case of 100% glycerol immersion, of which the THG intensity greatly decreased in the epidermis after OC, the THG intensity of the 50% immersion cases increased or remained the same (
Table 4.13). Although we observed significant reduction of light scattering in both concentrations through SHG and white light microscopy, the OC effect in THG intensity by using 50% glycerol showed the opposite consequences (Fig. 5.1). As discussed above, the THG intensity is affected by the refractive index mismatch inside the excitation focus [68, 69]. The 50% glycerol has a lower osmolarity than 100% glycerol [5, 72] and cannot
introduce such a high homogeneous environment as the 100% glycerol. The optical clearing power of 50% glycerol is known to be less than the 100% glycerol. As a result, for 50% glycerol immersion, there was a compromise between the decrement of the light scattering and the decrement of the refractive index mismatch. The less decrement of refractive index mismatch and the recovery of the light intensity and quality made the THG intensity enhance or remain the same after the OC. Besides, in both two tissues of the 50% immersion cases, the thickness of the SC all greatly decreased by at least 37.5%.
In the skin tissue with thicker SC, Case E50-I90-2, the THG intensity greatly increased at the dermis. To sum up, by immersed the skin tissue inside the 50% glycerol, we could enhance the THG intensity and the image at the epidermis can be better resolved.
Fig. 5.1 The variation of the THG intensity at the middle epidermis layer. We presented and compared the THG intensity variation at the middle epidermis layer of Case E100-I90-1 and Case E50-I90-2. C: control; G: glycerol. (a) In the Case of E100-I90-100% glycerol immersion (Case E100-I90-1), the THG intensity greatly reduced. (b) However, in the case of 50%
glycerol immersion (Case E50-I90-2), the THG intensity greatly increased, opposite from (a). Scale bar = 100 μm.