Name: Correlative Optical Spectroscopy and Mass Spectrometry Imaging Methodology to Visualise Drug Distribution in a Soft Tissue Section (Video) | JoVE Journal
Uploaded: 2025-06-20T13:20:00.000+00:00
Duration: 7 min 5 s
Description: Learn correlative optical spectroscopy and mass spectrometry imaging for visualizing drug distribution in soft tissue sections. This method enables high-resolution skin imaging with sensitive drug detection for improved topical pharmaceutical development.

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5 minutes

This study was funded by Haleon CH SARL. Contributions of NAB and RHG were partially supported by the Food and Drug Administration (FDA) of the U.S. Department of Health and Human Services (HHS) through a financial assistance award (1-U01-FD006533). This work was funded by the UK Department of Business, Energy, and Industrial Strategy through the projects NMS/ID74 and NMS/ST18 of the UK National Measurement System. The contents of this article are those of the authors and do not necessarily represent the official views of, nor an endorsement by, FDA/HHS or the U.S. Government. NAB is grateful for the support from the Community for Analytical Measurement Science for the 2020 CAMS Fellowship Award funded by the Analytical Chemistry Trust Fund.

Ethical statement: Human skin samples were obtained from Tissue Solutions Ltd, Glasgow, UK, by Charles River Laboratories (CRL) in Edinburgh, UK. All anonymous samples were acquired in compliance with the legal and ethical standards of the collection country, with ethical approval and informed consent from the donors or their closest relatives.
1. Skin sectioning
A cryostat was used to produce sections of the skin tissues.
<ol>
	<li>Transport the treated skin disks out of the -20 &#176;C freezer and cut a small piece (~1.0 cm x 1.0 cm) from the center of the disk.</li>
	<li>Mount the sample on the chuck in the cryostat over a frozen water droplet. Do not use optimal cutting temperature (OCT) compound or other embedding media.</li>
	<li>Set the blade and sample temperature at - 20 &#176;C and - 16 &#176;C, respectively.</li>
	<li>Section 30 &#181;m-thick section onto a glass coverslip (#1.5, Fisher Scientific).</li>
	<li>Perform SRS and ToF-SIMS immediately or alternatively, the sample may be vacuum-packed for storage at -80 &#176;C.</li>
	<li>Desiccate for 20 min prior to use.</li>
</ol>
2. SRS imaging
SRS, SHG and TPEF microscopy was performed using a coherent Raman microscope. Two picosecond laser pulses were generated by a 1031.2 nm Stokes beam, overlapped spatially and temporally with a tunable pump beam. The Stokes beam, modulated at 20 MHz, facilitated the detection of stimulated Raman loss signals via transmission. A silicon-based detector, coupled with a lock-in amplifier, was utilised for signal detection. Another channel was employed to collect second harmonic and emitted fluorescence signals, employing a photomultiplier tube for detection.
<ol>
	<li>Install suitable objective and condensers lenses for multiphoton tissue imaging, in this case a water immersion 40x&#160;magnification lens (1.1 NA) in conjunction with a short working distance air condenser lens (0.9 NA).</li>
	<li>Place the mounted tissue section onto the SRS microscope sample stage so the tissue is on the same face of the microscope slide as the condenser lens. Focus the objective lens on the tissue section, and then optimize the condenser height.</li>
	<li>Set the laser power to appropriate values to obtain good signal-to-noise ratio without damaging the tissue. In this example, approximately 10 mW for the pump beam and 30 mW for the Stokes beam were applied at the sample. Refer to Tsikritsis et al for practical considerations for SRS measurements4.</li>
	<li>Acquire a mosaic image of the whole section with 512 x&#160;512 pixels resolution corresponding to 290 &#181;m x&#160;290 &#181;m at X1 zoom with an imaging speed of 400 Hz and line average of 1 at 2850 cm-1 corresponding to CH2 stretching.</li>
	<li>Within the mosaic image of the whole section, choose a region of interest (ROI) with the following considerations: (i) it is positioned at the center, avoiding the outermost 20 % of the sample perimeter where there could be un-dosed skin which was clamped in the Franz cell apparatus; (ii) it is sufficiently flat to be examined within the same optical depth level; (iii) it has distinct structural features that would assist its visual identification when the sample is transferred to ToF-SIMS.</li>
	<li>Apply consistent gain settings across the study for SRS and SHG/fluorescence detectors, and acquire images in (i) the C-H stretching region (at 2945 cm-1, 2850 cm-1 and 2650 cm-1 corresponding to CH3, CH2 and off-resonance control, respectively); (ii) the fingerprint region (at 1666 cm-1 and 1723 cm-1 corresponding to Amide I and off-resonance control, respectively); (iii) the wavenumber corresponding to the on- and off-resonance related to the applied drug; in this example, at 1586 cm-1 and 1530 cm-1 corresponding to C=C and off-resonance, respectively.</li>
</ol>
After SRS imaging, immediately transfer the sample to the ToF-SIMS setup for analysis.
3. ToF-SIMS imaging
Time-of-flight secondary ion mass spectrometry imaging was performed using a 30 keV Bi3+ primary ion beam with a current of 0.2 pA.
<ol>
	<li>Choose appropriate acquisition settings; in this example, negative ion polarity was used, with a 100 ms duty cycle time, a mass range of m/z 0-900, and a beam diameter of 5 &#181;m. A 20 eV electron flood gun at 5 &#181;A was used for charge compensation.</li>
	<li>Acquire an overall image by the stage macro raster mode to map the entire tissue section with a field of view depending on the tissue dimensions (in our case we used 2.5 to 3.0 mm x&#160;8.5 to 9.0 mm). This overall image is formed from a mosaic of smaller images. In this example, each had a field of view of 0.5 mm x&#160;0.5 mm (256 x&#160;256 pixels) with 1 ion beam shot per pixel, resulting in an ion dose of 8.18 x&#160;108 ions/cm2.</li>
	<li>Utilize the same method to capture images of the ROI by selecting an appropriate field of view; in this instance, it was 1.0 mm x&#160;0.5 mm, with 4 shots per pixel per frame and 10 frames per patch, corresponding to an ion dose of 1.31 x&#160;1011&#160;ions/cm2.</li>
	<li>To ensure quality control, analyze tissue homogenate samples immediately before and after examining each skin section. Use a field of view of 0.5 mm x&#160;0.5 mm (256 x&#160;256 pixels) with 1 shot per pixel and 1 frame per scan, conducting a total of 15 scans. This setup corresponds to an ion dose density of 3.27 x&#160;1010&#160;ion/cm2.</li>
	<li>After acquisition, the resulting mass spectra can be calibrated using H&#8722;, C&#8722;, C2&#8722;, and C3&#8722; ions.</li>
	<li>Data acquisition and extraction were performed using Surface Lab 7.1 software.</li>
</ol>
4. Image registration and data analysis using MATLAB
<ol>
	<li>Reduce SIMS and SRS images to 3 dimensions each using non-negative matrix factorisation to be visualised as red, green, and blue color channels.</li>
	<li>Choose matching features between these two non-matrix factorization images by selecting the Matlab control point selection tool &#34;cpselect&#34; (Matlab, Mathworks, 2019b and Image Processing Toolbox).</li>
	<li>Use the &#34;cp2tform&#34;&#160;and &#34;imtransform&#34; functions to perform registration, applying an affine transformation with the SIMS data as the fixed images and the optical spectroscopy data as the moving images.</li>
	<li>Where multiple peaks are detected in the ToF-SIMS data for the drug of interest, sum together the intensities of these peaks to create a combined drug ion intensity.</li>
	<li>Extract the average total ion count of the two homogenate datasets (before and after) for each corresponding skin tissue sample. Using this corresponding value, normalise the intensities in the ToF-SIMS data of the drug ion in each skin image.</li>
	<li>After registration, create an overlay of the optical spectroscopy and SIMS data by assigning the respective images to the RGB channels: red (SRS image for CH2), green (SHG image for collagen), and blue (SIMS image for diclofenac, including signals from various ions at m/z 214.04, 216.04, 250.02, 252.02, 294.01, and 296.01).</li>
</ol>

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MBB is an employee of Haleon CH SARL, the funding institution of the study.

This method allows correlative imaging with nonlinear optical spectroscopy and mass spectrometry imaging to detect drug distribution in soft tissue with sub-micron spatial resolution and high sensitivity. Importantly, imaging is achieved without the use of potentially interfering labels that would be required for fluorescence-based microscopies. Further validation of the workflow involved the conventional hematoxylin and eosin (H&#38;E) staining of a tissue section to elucidate the skin structure and permitted registrati...

Understanding the rate, extent, and drivers of the distribution of active pharmaceutical ingredients within soft biological tissues following their topical or systemic application is crucial to elucidate the mechanisms underlying tissue-specific drug uptake, metabolism, and clearance, (i.e., a deeper understanding of drug pharmacokinetics). Here, we present a novel correlative imaging method integrating nonlinear optical spectroscopies and mass spectroscopy imaging for label-free visualisation of the tissue structure and the distribution of topical drugs within the same tissue sample1.
Raman spectroscopy (RS) has emerged as a valuable tool for mapping drug distribution within cells and tissues with high spatial resolution and molecular specificity2. RS relies on the inelastic scattering of light3 by a sample of interest permitting a detailed chemical characterisation via the characteristic vibrational modes of molecules therein, without the need for external labels. However, the long acquisition times to produce high-resolution images can potentially compromise the integrity of sensitive biological samples. Additionally, when probing deeper tissue layers, scattering and absorption of light by the tissue can attenuate the Raman signal, further limiting its sensitivity. Stimulated Raman Scattering (SRS) microscopy represents a significant advancement over conventional RS, leveraging pulsed laser excitation and detection of the stimulated Raman signal4. SRS microscopy allows 2-D and 3-D rapid image acquisition with high spatial resolution and - when performed in tandem with second harmonic generation (SHG) and two photon excited fluorescence (TPEF) microscopies - can provide a comprehensive image of connective tissues, collagen, and elastin1,5,6,7,8,9. SHG microscopy is a nonlinear optical technique that relies on the interaction of light with non-centrosymmetric structures in a sample, generating a signal at half the wavelength of the incident light. It is particularly useful for imaging ordered structures like collagen in biological tissues. TPEF is another nonlinear microscopy technique where two lower-energy photons simultaneously excite a fluorophore, causing it to emit fluorescence. TPEF allows deep tissue imaging with reduced photodamage, as the excitation light is in the near-infrared range, which penetrates deeper into biological samples.
Mass spectrometry imaging (MSI) is a powerful analytical technique that combines the capabilities of mass spectrometry with spatial information, allowing label-free identification of a wide range of molecules10,11,12. Time-of-flight secondary ion mass spectrometry (ToF-SIMS) operates by bombarding the sample surface with high-energy primary ions, causing the ejection of secondary ions from the surface. These secondary ions are then accelerated toward a time-of-flight analyser, where their mass-to-charge ratios are determined. This analysis allows for the identification of molecular fragments and isotopic variations with high sensitivity and resolution. Valuable applications of ToF-SIMS have been demonstrated in biological and pharmaceutical research for the study of chemicals and biomolecules in cells13, tissues14,15, and organs15.
Both SRS and ToF-SIMS have been used independently to study the distribution of drugs in different skin models. However, it is most valuable to use these techniques in combination to benefit from their complementarity. In this study, we describe a workflow to combine the optical microscopies (SRS, TPEF and SHG) with ToF-SIMS to obtain images of the skin morphology with sub-micron spatial resolution (provided by the optical methods) overlayed with the drug signal detected with superior sensitivity (provided by the ToF-SIMS). The approach has been used to address the challenging example of visualising the distribution of diclofenac in excised skin tissues treated with a commercial topical formulation (Voltaren gel). The criteria for success included high chemical sensitivity to detect diclofenac at its therapeutic concentration, and sufficient spatial resolution to resolve the skin structure. Although diclofenac content in different skin layers can be quantified after topical treatment through laborious and technically demanding methods such as tape stripping followed by quantification16, or via open flow microperfusion17, these approaches cannot elucidate excipient-dependent diclofenac partitioning, penetration, retention, and depth distribution in the individual skin layers, which are essential factors aiding the product development phase.
Prior to the measurement methodology described below, Voltaren Forte gel (containing 2.32 % diclofenac diethylammonium) or a corresponding placebo gel (supplied by Haleon CH SARL) was applied on the skin surface for different periods (4, 12, 16, or 24 h). After removal of any residual formulation, SRS and ToF-SIMS measurements on the same skin samples were performed; full details of the methodology are described in the original manuscript1.

<table><tbody><tr><td>Coherent Raman microscope&amp;nbsp;</td><td>Leica Microsystems</td><td>https://www.leica-microsystems.com/products/confocal-microscopes/p/leica-tcs-sp8-cars/</td><td></td></tr><tr><td>Human skin samples&amp;nbsp;</td><td>Tissue Solutions Ltd, Glasgow</td><td></td><td></td></tr><tr><td>Leica CM 1850 Cryostat&amp;nbsp;</td><td>Leica Biosystems</td><td>https://www.leicabiosystems.com/en-fr/histology-equipment/cryostats/leica-cm1850/</td><td>Discontinued</td></tr><tr><td>PicoEmerald-S laser system</td><td>APE Berlin</td><td>https://www.ape-berlin.de/en/cars-srs/</td><td></td></tr><tr><td>ToF SIMS 5</td><td>IONToF GmbH</td><td>https://spectral.se/products/tof-sims-5</td><td></td></tr></tbody></table>

correlative optical spectroscopy and mass spectrometry imaging methodology to visualise drug distribution in a soft tissue section

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

A new correlative imaging methodology combining sub-micron spatial resolution with sensitive drug detection by optical spectroscopy and mass spectrometry imaging has been developed to visualise chemical distribution upon drug application in soft tissue. In this example, the method has been tested on excised skin tissue after in vitro topical application of a commercial nonsteroidal anti-inflammatory drug product for 16 hours. Non-destructive optical spectroscopic methods, including stimulated Raman scattering, second harmonic generation, and two-photon fluorescence microscopies, were first employed to map the skin structure and morphology. Subsequently, the same skin tissue samples were analysed via time-of-flight secondary ion mass spectrometry (ToF-SIMS), which enabled enhanced sensitivity for the detection of diclofenac across the outermost skin layers - epidermis and dermis. Image registration methods were devised to integrate the optical and mass spectrometric data. This approach combines label-free, high-resolution visualisation of tissue structure with sensitive chemical detection, and represents a valuable tool for investigating drug distribution in skin and potentially, in other soft biological tissues.

Figure 1 presents the results of feature-based image registration, displaying the registered overlay images of SRS, SHG, and SIMS for two skin samples-one treated with a placebo (Figure 1a) and the others with Voltaren gel for 4 hours (Figure 1b) and 16 hours (Figure 1c). In this image, the summed drug intensity signal (from ToF-SIMS measurements) is depicted in blue, li...

This study presents a method for correlative imaging of drugs in soft tissues using nonlinear optical spectroscopy and mass spectrometry imaging. Combining high-resolution skin imaging with sensitive drug detection offers a valuable approach to study drug distribution in the skin, critical for the development of superior topical products.

A new correlative imaging methodology combining sub-micron spatial resolution with sensitive drug detection by optical spectroscopy and mass spectrometry ...

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A mass spectrometry imaging (MSI) source operated at atmospheric pressure was developed by coupling mid-infrared laser desorption and electrospray post-ionization. Exogenous ice matrix was used as the energy-absorbing matrix to facilitate resonant desorption of tissue-related material. This manuscript provides a step-by-step protocol for performing IR-MALDESI MSI of whole-body neonatal mouse.

Whole-body Mass Spectrometry Imaging by Infrared Matrix-assisted Laser Desorption Electrospray Ionization (IR-MALDESI)

The assembly and use of a multimodal microendoscope is described which can co-register superficial tissue image data with tissue physiological parameters including hemoglobin concentration, melanin concentration, and oxygen saturation. This technique can be useful for evaluating tissue structure and perfusion, and can be optimized for individual needs of the investigator.

Multimodal Imaging and Spectroscopy Fiber-bundle Microendoscopy Platform for Non-invasive, In Vivo Tissue Analysis

Here, we describe a protocol using laser capture microdissection coupled with LC/MS analysis to spatially-quantify drug distributions within pulmonary tuberculosis granulomas. The approach has broad applicability to quantifying drug concentrations within tissues at high spatial detail.

Spatial Quantification of Drugs in Pulmonary Tuberculosis Lesions by Laser Capture Microdissection Liquid Chromatography Mass Spectrometry (LCM-LC/MS)

A novel method of sample preparation was developed to accommodate cell and tissue coculture to detect small molecule exchange using imaging mass spectrometry.

Capturing Small Molecule Communication Between Tissues and Cells Using Imaging Mass Spectrometry

In the era of cancer immunotherapy, interest in elucidating tumor microenvironment dynamics has increased strikingly. This protocol details a mass spectrometry imaging technique with respect to its staining and imaging steps, which allow for highly multiplexed spatial analysis.

Expanding the Comprehension of the Tumor Microenvironment using Mass Spectrometry Imaging of Formalin-Fixed and Paraffin-Embedded Tissue Samples

A novel sample preparation method is demonstrated for the analysis of agar-based, bacterial macrocolonies via matrix-assisted laser desorption/ionization imaging mass spectrometry.

Investigation of Microbial Cooperation via Imaging Mass Spectrometry Analysis of Bacterial Colonies Grown on Agar and in Tissue During Infection

Utilizing a multimodal platform combining label-free optical imaging modalities, we have developed a protocol for visualizing and quantifying cellular dynamics and metabolism. Through imaging via multiphoton fluorescence, second harmonic generation, and stimulated Raman scattering microscopy, we can generate a holistic overview of the cellular and molecular environment.

Multimodal Optical Imaging Platform for Studying Cellular Metabolism

Dopamine replacement pharmacotherapy using L-DOPA is the most commonly used symptomatic treatment of Parkinson’s disease, but is accompanied by side effects including involuntary abnormal movements, termed dyskinesia 1. Here, a protocol for MALDI imaging mass spectrometry is presented that detects changes in rat brain neuropeptide levels related to dyskinesia.

MALDI Imaging Mass Spectrometry of Neuropeptides in Parkinson's Disease

Learn correlative optical spectroscopy and mass spectrometry imaging for visualizing drug distribution in soft tissue sections. This method enables high-resolution skin imaging with sensitive drug detection for improved topical pharmaceutical development.

Correlative Optical Spectroscopy and Mass Spectrometry Imaging Methodology to Visualise Drug Distribution in a Soft Tissue Section (Video) | JoVE Journal

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

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