Correlative Optical Spectroscopy and Mass Spectrometry Imaging Methodology to Visualise Drug Distribution in a Soft Tissue Section

We are developing correlative spectroscopic imaging methods to better understand topical drug delivery. The aim is to obtain new mechanistic insights into drug permeation and its bioavailability within the skin layers. This information will be valuable to improve the formulation, design and dosing regimen of pharmaceutical products.

This research addresses the current lack of methods in forming simultaneously on the skin structure and the distribution of drugs in it. Following topical application with high spatial resolution and high sensitivity. Access to such information is essential for the development of superior topical products. This protocol offers label free a rapid high resolution visualization of drug distribution in skin with high chemical sensitivity compared to existing traditional techniques. All this information can be extracted from the same tissue section.

These findings advance correlative imaging by presenting a reliable method that merges optical spectroscopy with mass spectrometry. Developed image registration techniques ensure precise data set alignment, enabling detailed analysis of drug distribution in tissues. This progress supports improved topical drug delivery systems and deeper insights into biological interactions across tissues.

The research raises scientific question about how formulation excipient impact drug transport, factor affecting drug penetration across skin types, long-term drug release and clearance kinetics, and the broad application of raman and MSI correlative imaging to examine other topical drugs and other substances in soft tissues.

[Narrator] For stimulated raman scattering or SRS microscopy, use a water immersion 40 times magnification lens in conjunction with a short working distance air condenser lens. Place the mounted skin tissue section onto the SRS microscope sample stage, ensuring the tissue is on the same face of the microscope slide as the condenser lens. Focus the objective lens on the tissue section, then optimize the condenser height. Set the laser power to appropriate values to obtain a good signal-to-noise ratio without damaging the tissue. Next, acquire a mosaic image of the whole tissue section corresponding to the CH2 stretching band. Then select a region of interest and set the imaging parameter to 512 x 512 pixel resolution, covering an area of 290 x 290 micrometers. Use an imaging speed of 400 hertz X1 zoom, and a line average of one. Now apply consistent gain settings for SRS and SHG fluorescence detectors. Acquire images in the CH stretching region at 2,850 per centimeter corresponding to CH2. Then capture images in the fingerprint region at 1,666 per centimeter corresponding to amide 1. After SRS imaging, immediately transfer the skin sample to the ToF-SIMS setup. Set the negative ion polarity with a duty cycle time of 100 milliseconds, a mass range of 0 to 900 mass to charge and a beam diameter of five micrometers. Use a 20 electron volt electron flood gun at 25 microamperes for charge compensation. Acquire an overall image using the stage macro raster mode, mapping the entire tissue section with a field of view depending on the tissue dimensions. Next, using a field of view of one by 0.5 millimeters with four shots per pixel per frame, and 10 frames per patch corresponding to an ion dose of 1.31 times 10 to the power of 11 ions per square centimeter. Capture images of the region of interest by selecting the appropriate field of view. Set a field of view of 0.5 x 0.5 millimeters with a resolution of 256 x 256 pixels. Acquire one shot per pixel with one frame per scan for a total of 15 scans corresponding to an ion dose density of 3.27 times 10 to the power of 10 ions per square centimeter for quality control check of tissue homogenate samples. After the acquisition, calibrate the resulting mass spectra using H, C, C2 and C3 ions. Using Surface Lab 7.1 software, perform data acquisition and extraction. For image registration, reduce both SIMS and SRS images to three dimensions using non-negative matrix factorization and visualize them as red, green, and blue color channels. Using the MATLAB control point selection tool CP Select, choose matching features between the two non matrix factorization images. Perform registration using the CP2 T form and IM transform functions. Apply in a fine transformation using the SIMS data as the fixed image and the optical spectroscopy data as the moving image. For the drug of interest where multiple peaks are detected in the ToF-SIMS data, sum the intensities of these peaks to create a combined drug ion intensity. Extract the average total ion count from the two homogenate datasets for each skin tissue sample. Using this corresponding value, normalize the ToF-SIMS drug ion intensities in each skin image. After registration, overlay the optical spectroscopy and SIMS data by assigning the respective images to the RGB channels. In the present study, an increased drug signal was detected within the upper approximately 50 micrometer region of the skin after four hours of Voltaren gel application compared to the placebo treated sample. After 16 hours of Voltaren gel application, the drug signal extended into deeper layers of the skin indicating enhanced tissue penetration.