Non-Invasive. In Vivo Skin Analysis. Quantification of Product Uptake into Different Skin Depths.

Confocal Raman Spectroscopy

The spectral information in confocal microscopy can be obtained through different techniques such as Absorption, Reflection, Transmission, Emission, Photoluminescence, Fluorescence or Raman spectroscopy. Among those techniques confocal Raman holds a special place. Raman spectroscopy as such is based on the inelastic scattering of monochromatic light when the frequency of photons changes upon interaction with a sample. The photons of the laser light are absorbed by the sample and subsequently reemitted. Frequency of the reemitted photons is shifted up or down in comparison with the original monochromatic frequency, which is known as the Raman effect. The Raman shift provides information about vibrational and rotational energies of molecular bonds.

It was realized that Raman spectroscopy was a convenient probe of the vibrational energy levels within a molecule which easily provides molecular fingerprints. Another unique advantage of Raman spectroscopy is it can be used to selectively excite a needed portion of the molecule by changing the excitation wavelength. On top of that Raman spectroscopy doesn’t required any sample preparation, samples are not destroyed, water bands are usually small and easily subtracted and Raman spectra usually contain sharp bands that are characteristic of the specific molecular bonds in the sample. The intensity of the bands in a Raman spectrum is proportional to the concentration of the corresponding molecules and thus can be used for quantitative analysis.

In biological samples, such as cells and tissues, infrared spectra often show broad spectral features which can give information regarding cellular components. However, Raman spectra give this information as well as more detailed information regarding the constituents of these specific components allowing good specificity for qualitative analysis and for discrimination among similar materials.

The latest trend in high-end confocal Raman microscopy instrumentation is modular systems designed for easy customization of their components and therefore providing the best possible results in terms of chemical differentiation and sensitivity.

In Vivo Skin Analysis & Quantification of Product Penetration


  • Penetration of Actives.
    • Did active penetrate into the skin?
    • How far?
    • How fast?
    • How long does active remain?
  • Quantification of Actives
    • How much of the actives penetrated the skin?
  • Natural Moisturizing Factor (NMF)
  • Water Content in Stratum Corneum (SC)
  • SC Thickness

Case Study

Case Study on Raman

Left panel. In vivo Raman spectra of skin treated with product (blue) and of untreated skin (red).

Right panel. Fit analysis (green) of the treated skin spectrum (blue), using only spectra of intrinsic skin components. The fit residual is shown in red.

Client Objectives:

  • Compare the skin penetration of test product with marketed products.
  • Quantify actives at different depth (0, 5, 10, 15 and 20 microns) over time (2, 4 and 8 h post application)


  • Raman Spectroscopy to substantiate their claim.
  • Technology not readily available to substantiate the claim.


  • Biometrix used their global resources to acquire the technology to conduct the study in a timely and cost effective manner so that the claims can be substantiated.
  • Raman Confocal Spectroscopy IS NOW AVAILABLE IN BIOMETRIX for several applications (single and repeated dose).

Average NMF (of 33 subjects) vs Depth for All Time Pts

Average Methyl-Salicylate (of 33 Subjects) vs Depth for All Time Pts

Stratum Corneum Thickness Over Time


Methyl-salicylate penetrates significantly (p<0.05) into the skin, and is found in the stratum corneum at all 3 time-points

The NMF-concentration was found to continually and significant decrease from 2h (p<0.05)

The water concentration profiles, show a steady increase in water concentration close to the skin surface over 8 hours


Claims Made from Raman Spectroscopy



Natural Moisturizing Factor

SC Thickness

Evaluation of NMF and Water Content of Skin After Application of Serum, Using Raman Confocal Microscopy

Raman Spectroscopy Studies

  • Analysis 1: Water Concentration Profiles
  • Analysis 2: Natural Moisturizing Factors (NMF)
  • Analysis 3:Content parameters measuredPyrrolidone Carboxylic Acid (PCA)
    • Ceramide
    • Glycerol

Spectra were obtained by focusing a low power laser on skin after treatment and measuring the Raman scattered light. A small part of the scattered light is found at wavelengths higher than the incident light. Collected spectra provide information about penetration of the test product and molecular composition of the skin.

Products Tested:

  • Product A (untreated control)
  • Product B
  • Product C
  • Product D

The penetration profiles were defined at skin depths of 0, 5, and 10 microns +/1 micron (Raman spectrum region: 400 to 1800 cm-1).

Bianchini, R. AAD 2018


Raman Measurements of Glycerol Penetration 0, 5 and 10 microns

Measurements at 4 hours

Measurements at 24 hours

San Francisco Institute of Aesthetic and Regenerative Medicine

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