title: How Confocal Is Confocal Raman Microspectroscopy on the Skin? Impact of Microscope Configuration and Sample Preparation on Penetration Depth Profiles.
abstract: OBJECTIVE
  The aim of the study was to elucidate the effect of sample preparation and microscope configuration on the results of confocal Raman microspectroscopic evaluation of the penetration of a pharmaceutical active into the skin (depth profiling).
  
  
  METHODS
  Pig ear skin and a hydrophilic formulation containing procaine HCl were used as a model system. The formulation was either left on the skin during the measurement, or was wiped off or washed off prior to the analysis. The microscope configuration was varied with respect to objectives and pinholes used.
  
  
  RESULTS
  Sample preparation and microscope configuration had a tremendous effect on the results of depth profiling. Regarding sample preparation, the best results could be observed when the formulation was washed off the skin prior to the analysis. Concerning microscope configuration, the use of a 40 × 0.6 numerical aperture (NA) objective in combination with a 25-µm pinhole or a 100 × 1.25 NA objective in combination with a 50-µm pinhole was found to be advantageous.
  
  
  CONCLUSIONS
  Complete removal of the sample from the skin before the analysis was found to be crucial. A thorough analysis of the suitability of the chosen microscope configuration should be performed before acquiring concentration depth profiles.
authors.affiliation: Department of Pharmaceutical Technology, Faculty of Science, University of Tübingen, Tübingen, Germany.
authors.forename: Dominique Jasmin
authors.initials: DJ
authors.lastname: Lunter
mesh_terms: D000818:Animals; D018613:Microscopy, Confocal / Q000379:methods; D015591:Microspectrophotometry / Q000379:methods; D009924:Organ Culture Techniques; D012867:Skin / Q000166:cytology* / Q000378:metabolism*; D013059:Spectrum Analysis, Raman / Q000379:methods*; D013552:Swine