Resolving Water, Proteins, and Lipids from In Vivo Confocal Raman Spectra of Stratum Corneum through a Chemometric Approach

Summary

Here, we present a protocol for collection of confocal Raman spectra from human subjects in clinical studies combined with chemometric approaches for spectral outlier removal and the subsequent extraction of key features.

Abstract

Development of this in vivo confocal Raman spectroscopic method enables the direct measurement of water, proteins, and lipids with depth resolution in human subjects. This information is very important for skin-related diseases and characterizing skin care product performance. This protocol illustrates a method for confocal Raman spectra collection and the subsequent analysis of the spectral dataset leveraging chemometrics. The goal of this method is to establish a standard protocol for data collection and provide general guidance for data analysis. Preprocessing (e.g., removal of outlier spectra) is a critical step when processing large datasets from clinical studies. As an example, we provide guidance based on prior knowledge of a dataset to identify the types of outliers and develop specific strategies to remove them. A principal component analysis is performed, and the loading spectra are compared with spectra from reference materials to select the number of components used in the final multivariate curve resolution (MCR) analysis. This approach is successful for extracting meaningful information from a large spectral dataset.

Introduction

In clinical studies, in vivo confocal Raman spectroscopy has shown its unique ability for determining stratum corneum thickness and water content^1,2,3,4, and tracking the penetration of active materials topically applied to the skin^5,6. As a noninvasive approach, confocal Raman spectroscopy detects molecular signals based on vibrational modes. Thus, labeling is not needed⁷. In vivo confocal Raman spectroscopy provides chemical information with depth resolution....

Protocol

This study was approved by the institutional review committee of Beijing Children’s Hospital in compliance with the ethical guidelines of the 1975 Declaration of Helsinki. It was conducted according to ICH guidelines for Good Clinical Practice. The study took place from May to July 2015.

1. Collection of in vivo confocal Raman spectra from human subjects with atopic dermatitis

1. Include subjects in compliance with the following criteria.
   1. Include subjects between the ages of 4–18.
   2. Include subjects with mild to moderate atopic dermatitis (score of 2 or 3 according to the Physician’s global as....

Results

In this clinical study, in vivo confocal Raman spectra were collected from 28 subjects from 4–18 years old. A total of 30,862 Raman spectra were collected with the data collection protocol mentioned above. This large spectral dataset contains 20% spectral outliers as shown in Figure 4A. The low signal-to-noise outlier spectra were removed after determining the skin surface, followed by the PCA to identify the spectra with room light features. The third factor in this PCA model is identif.......

Discussion

During the data collection, as described in section 2 and 3 of the protocol, each depth profile was collected in an area with contact between the instrument window and the skin by finding the darker areas from the microscopic images highlighted in the red circles in Figure 2C. Once these areas were located, it was critical to start the depth profile above the skin surface to accurately determine the location of the skin surface for the data analysis procedure. The location o.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Bovine Serum Albumin	Sigma-Aldrich
Cholesterol	Sigma-Aldrich
Cholesterol 3-sulfate sodium	Sigma-Aldrich
D-Erythro-Dihydrosphingosine	Sigma-Aldrich
DI water			Purified with Milipore(18.2MΩ)
Gen2-SCA skin analyzer	River Diagnostics, Rotterdam, The Netherlands	Gen2
Matlab 2018b	Mathwork	2018b
N-behenoyl-D-erythro-sphingosine	Avanti Polar Lipids, Inc.
N-Lignoceroyl-D-erythro-sphinganine(ceramide)	Avanti Polar Lipids, Inc.
Oleic Acid	Sigma-Aldrich
Palmitic Acid	Sigma-Aldrich
Palmitoleic Acid	Sigma-Aldrich
PLS_Toolbox version 8.2	Eigenvector Research Inc.	8.2
RiverICon	River Diagnostics, Rotterdam, The Netherlands	version 3.2
Squalene	Sigma-Aldrich
Stearic Acid	Sigma-Aldrich