This tutorial review presents the most important practical considerations for sample preparation, instrument set-up, image acquisition and data analysis to obtain reproducible SRS measurements.

The mechanistic understanding of skin penetration underpins the design, efficacy and risk assessment of many high-value products including functional personal care products, topical and transdermal drugs. Stimulated Raman scattering (SRS) microscopy, a label free chemical imaging tool, combines molecular spectroscopy with submicron spatial information to map the distribution of chemicals as they penetrate the skin. However, the quantification of penetration is hampered by significant interference from Raman signals of skin constituents. This study reports a method for disentangling exogeneous contributions and measuring their permeation profile through human skin combining SRS measurements with chemometrics. We investigated the spectral decomposition capability of multivariate curve resolution - alternating least squares (MCR-ALS) using hyperspectral SRS images of skin dosed with 4-cyanophenol. By performing MCR-ALS on the fingerprint region spectral data, the distribution of 4-cyanophenol in skin was estimated in an attempt to quantify the amount permeated at different depths. The reconstructed distribution was compared with the experimental mapping of CN, a strong vibrational peak in 4-cyanophenol where the skin is spectroscopically silent. The similarity between MCR-ALS resolved and experimental distribution in skin dosed for 4 h was 0.79 which improved to 0.91 for skin dosed for 1 h. The correlation was observed to be lower for deeper layers of skin where SRS signal intensity is low which is an indication of low sensitivity of SRS. This work is the first demonstration, to the best of our knowledge, of combining SRS imaging technique with spectral unmixing methods for direct observation and mapping of the chemical penetration and distribution in biological tissues.

Digital pathology is revolutionising the analysis of histological features and is becoming more and more widespread in both the clinic and research. Molecular pathology extends the tissue morphology information provided by conventional histopathology by providing spatially resolved molecular information to complement the structural information provided by histopathology. The multidimensional nature of the molecular data poses significant challenge for data processing, mining, and analysis. One of the key challenges faced by new and existing pathology practitioners is how to choose the most suitable molecular pathology technique for a given diagnosis. By providing a comparison of different methods, this narrative review aims to introduce the field of molecular pathology, providing a high-level overview of many different methods. Since each pixel of an image contains a wealth of molecular information, data processing in molecular pathology is more complex. The key data processing steps and variables, and their effect on the data, are also discussed.

The formulation of paediatric medicines faces significant challenges to meet the requirements for safe and accurate administration, while maintaining a suitable taste. Multiparticulate formulations have a strong potential to address these challenges because they combine dose flexibility with ease of administration. Understanding the stability of multiparticulate formulations over storage as a function of time and environmental parameters, such as humidity and temperature, is important to manage their commercialisation and use. In this work, we have expanded the toolkit of available techniques for studying multiparticulates beyond those such as scanning electron microscopy (SEM) and confocal laser scanning microscopy. We include advanced methods of environmentally-controlled SEM to monitor temperature- and humidity-induced changes in-situ, and a variety of Raman spectroscopies including stimulated Raman scattering microscopy to identify and localise the different ingredients at the surface and inside the multiparticulates. These techniques allowed unprecedented monitoring of specific changes to the particulate structure and distribution of individual ingredients due to product aging. These methods should be considered as valuable novel tools for in-depth characterisation of multiparticulate formulations to further understand chemical changes occurring during their development, manufacturing and long-term storage. We envisage these techniques to be useful in furthering the development of future medicine formulations.

High-resolution microscopy underpins the analysis of biological systems across spatial scales, from individual biomolecules to complex tissues. As multimodal imaging workflows and quantitative analysis of bioimage data become increasingly widespread, there is a growing need for materials and methods to calibrate imaging systems and evaluate the fidelity of image data. This article describes the development and characterization of dimensionally traceable, three-dimensional microstructures as microscopy phantoms. Fabricated using two-photon photolithography, the phantoms exhibit broadband visible autofluorescence and Raman scattering across the fingerprint and C–H stretching regions, making them suitable for a wide range of optical imaging techniques, including one- and two-photon excited fluorescence and coherent Raman scattering microscopy. Compatible with both air and liquid imaging media, the phantoms enable calibration under realistic biological imaging conditions. For demonstration, a forked wedge design is used to validate image deconvolution results, while a stacked lattice microstructure enables correction of chromatic offsets and axial scaling errors. The impact of phantom-derived image corrections is illustrated using a representative biological imaging application: analysis of a cell-seeded scaffold using confocal fluorescence microscopy. Overall, these phantoms provide a versatile, accessible platform for calibration, quality control, and validation of multimodal imaging pipelines, supporting improved quantitative optical microscopy.

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.