The authors would like to thank Dr. Fotis Iliopoulos and Daniel Greenfield of the Evans&#39; Group for their discussion and proofreading of this manuscript. In addition, the authors would like to acknowledge support from LEO Pharma. Figure 2 was created with BioRender.com.

<ol><li>Finnin, B., Walters, K. A., Franz, T. J. In vitro skin permeation methodology. In Transdermal and topical drug delivery: principles and methodology. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aFinnin%2C+B+%22Transdermal+and+topical+drug+delivery%3A+principles+and+practice%22">Transdermal and topical drug delivery: principles and practice</a>. Benson, H. E., Watkinson, A. C. , John Wiley & Sons. Hoboken NJ USA. 85-108 (2012).</li>
<li>Shin, S. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((On+the+road+to+development+of+an+in+vitro+permeation+test+%28IVPT%29+model+to+compare+heat+effects+on+transdermal+delivery+systems%3A+exploratory+studies+with+nicotine+and+fentanyl%5BTitle%5D)+AND+%22Pharmaceutical+Research%22%5BJournal%5D)">On the road to development of an in vitro permeation test (IVPT) model to compare heat effects on transdermal delivery systems: exploratory studies with nicotine and fentanyl.</a> Pharmaceutical Research. 34 (9), 1817-1830 (2017).</li>
<li>Hossain, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Preparation%2C+characterisation%2C+and+topical+delivery+of+terbinafine%5BTitle%5D)+AND+%22Pharmaceutics%22%5BJournal%5D)">Preparation, characterisation, and topical delivery of terbinafine.</a> Pharmaceutics. 11 (10), 548(2019).</li>
<li>Santos, L. L., Swofford, N. J., Santiago, B. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vitro+permeation+test+%28IVPT%29+for+pharmacokinetic+assessment+of+topical+dermatological+formulations%5BTitle%5D)+AND+%22Current+Protocols+in+Pharmacology%22%5BJournal%5D)">In vitro permeation test (IVPT) for pharmacokinetic assessment of topical dermatological formulations.</a> Current Protocols in Pharmacology. 91 (1), 79(2020).</li>
<li>Iliopoulos, F., Caspers, P. J., Puppels, G. J., Lane, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Franz+cell+diffusion+testing+andquantitative+confocal+Raman+spectroscopy%3A+In+vitro-in+vivo+correlation%5BTitle%5D)+AND+%22Pharmaceutics%22%5BJournal%5D)">Franz cell diffusion testing andquantitative confocal Raman spectroscopy: In vitro-in vivo correlation.</a> Pharmaceutics. 12 (9), 887(2020).</li>
<li>Cordery, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Topical+bioavailability+of+diclofenac+from+locally-acting%2C+dermatological+formulations%5BTitle%5D)+AND+%22International+Journal+of+Pharmaceutics%22%5BJournal%5D)">Topical bioavailability of diclofenac from locally-acting, dermatological formulations.</a> International Journal of Pharmaceutics. 529 (1-2), 55-64 (2017).</li>
<li>Pensado, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Stratum+corneum+sampling+to+assess+bioequivalence+between+topicalacyclovir+products%5BTitle%5D)+AND+%22Pharmaceutical+Research%22%5BJournal%5D)">Stratum corneum sampling to assess bioequivalence between topicalacyclovir products.</a> Pharmaceutical Research. 36 (12), 1-16 (2019).</li>
<li>Zhang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dermal+delivery+of+niacinamide-in+vivo+studies%5BTitle%5D)+AND+%22Pharmaceutics%22%5BJournal%5D)">Dermal delivery of niacinamide-in vivo studies.</a> Pharmaceutics. 13 (5), 726(2021).</li>
<li>Bodenlenz, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Open+flow+microperfusion+as+a+dermal+pharmacokinetic+approach+to+evaluate+topical+bioequivalence%5BTitle%5D)+AND+%22Clinical+Pharmacokinetics%22%5BJournal%5D)">Open flow microperfusion as a dermal pharmacokinetic approach to evaluate topical bioequivalence.</a> Clinical Pharmacokinetics. 56 (1), 91-98 (2017).</li>
<li>Eirefelt, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Evaluating+dermal+pharmacokinetics+and+pharmacodymanic+effect+of+soft+topical+PDE4+inhibitors%3AOpen+flow+microperfusion+and+skin+biopsies%5BTitle%5D)+AND+%22Pharmaceutical+Research%22%5BJournal%5D)">Evaluating dermal pharmacokinetics and pharmacodymanic effect of soft topical PDE4 inhibitors:Open flow microperfusion and skin biopsies.</a> Pharmaceutical Research. 37 (12), 1-12 (2020).</li>
<li>Stagni, G., O'Donnell, D., Liu, Y. J., Kellogg, J. D. L., Shepherd, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Iontophoretic+current+and+intradermal+microdialysis+recovery+in+humans%5BTitle%5D)+AND+%22Journal+of+Pharmacological+and+Toxicological+Methods%22%5BJournal%5D)">Iontophoretic current and intradermal microdialysis recovery in humans.</a> Journal of Pharmacological and Toxicological Methods. 41 (1), 49-54 (1999).</li>
<li>Garcia Ortiz, P., Hansen, S. H., Shah, V. P., Menne, T., Benfeldt, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Impact+of+adultatopic+dermatitis+on+topical+drug+penetration%3A+assessment+by+cutaneous+microdialysis+and+tape+stripping%5BTitle%5D)+AND+%22Acta+Dermato-Venereologica%22%5BJournal%5D)">Impact of adultatopic dermatitis on topical drug penetration: assessment by cutaneous microdialysis and tape stripping.</a> Acta Dermato-Venereologica. 89 (1), 33-38 (2009).</li>
<li>Joshi, A., Patel, H., Joshi, A., Stagni, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pharmacokinetic+applications+of+cutaneous+microdialysis%3A+Continuous%2Bintermittent+vs+continuous-only+sampling%5BTitle%5D)+AND+%22Journal+of+Pharmacological+and+Toxicological+Methods%22%5BJournal%5D)">Pharmacokinetic applications of cutaneous microdialysis: Continuous+intermittent vs continuous-only sampling.</a> Journal of Pharmacological and Toxicological Methods. 83, 16-20 (2017).</li>
<li>Kuzma, B. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Evaluation+of+local+bioavailability+of+metronidazole+from+topical+formulations+using+dermal+microdialysis%3A+Preliminary+study+in+a+Yucatan+mini-pig+model%5BTitle%5D)+AND+%22European+Journal+of+Pharmaceutical+Sciences%22%5BJournal%5D)">Evaluation of local bioavailability of metronidazole from topical formulations using dermal microdialysis: Preliminary study in a Yucatan mini-pig model.</a> European Journal of Pharmaceutical Sciences. 159, 105741(2021).</li>
<li>Begley, R., Harvey, A., Byer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((L.Coherent+anti-Stokes+Raman+spectroscopy%5BTitle%5D)+AND+%22Applied+Physics+Letters%22%5BJournal%5D)">L.Coherent anti-Stokes Raman spectroscopy.</a> Applied Physics Letters. 25 (7), 387-390 (1974).</li>
<li>Evans, C. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chemical+imaging+of+tissue+in+vivo+with+video-rate+coherent+anti-Stokes+Raman+scattering+microscopy%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">Chemical imaging of tissue in vivo with video-rate coherent anti-Stokes Raman scattering microscopy.</a> Proceedings of the National Academy of Sciences of the United States of America. 102 (46), 16807-16812 (2005).</li>
<li>Hill, A. H., Manifold, B., Fu, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tissue+imaging+depth+limit+of+stimulated+Raman+scattering+microscopy%5BTitle%5D)+AND+%22Biomedical+Optics+Express%22%5BJournal%5D)">Tissue imaging depth limit of stimulated Raman scattering microscopy.</a> Biomedical Optics Express. 11 (2), 762-774 (2020).</li>
<li>Feizpour, A., Marstrand, T., Bastholm, L., Eirefelt, S., Evans, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Label-free+quantification+of+pharmacokinetics+in+skin+with+stimulated+Raman+scattering+microscopy+and+deep+learning%5BTitle%5D)+AND+%22Journal+of+Investigative+Dermatology%22%5BJournal%5D)">Label-free quantification of pharmacokinetics in skin with stimulated Raman scattering microscopy and deep learning.</a> Journal of Investigative Dermatology. 141 (2), 395-403 (2021).</li>
<li>Ghosh, B., Reddy, L. H., Kulkarni, R. V., Khanam, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comparison+of+skin+permeability+of+drugs+in+mice+and+human+cadaver+skin%5BTitle%5D)+AND+%22Indian+Journal+of+Experimental+Biology%22%5BJournal%5D)">Comparison of skin permeability of drugs in mice and human cadaver skin.</a> Indian Journal of Experimental Biology. 38 (1), 42-45 (2000).</li>
<li>Nielsen, J. B., Plasencia, I., Sørensen, J. A., Bagatolli, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Storage+conditions+of+skin+affect+tissue+structure+and+subsequent+in+vitro+percutaneous+penetration%5BTitle%5D)+AND+%22Skin+Pharmacology+and+Physiology%22%5BJournal%5D)">Storage conditions of skin affect tissue structure and subsequent in vitro percutaneous penetration.</a> Skin Pharmacology and Physiology. 24 (2), 93-102 (2011).</li>
<li>Barbero, A. M., Frasch, H. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effect+of+frozen+human+epidermis+storage+duration+and+cryoprotectant+on+barrier+function+using+two+model+compounds%5BTitle%5D)+AND+%22Skin+Pharmacology+and+Physiology%22%5BJournal%5D)">Effect of frozen human epidermis storage duration and cryoprotectant on barrier function using two model compounds.</a> Skin Pharmacology and Physiology. 29 (1), 31-40 (2016).</li>
<li>Babu, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+influence+of+various+methods+of+cold+storage+of+skin+on+the+permeation+of+melatonin+and+nimesulide%5BTitle%5D)+AND+%22Journal+of+Controlled+Release%22%5BJournal%5D)">The influence of various methods of cold storage of skin on the permeation of melatonin and nimesulide.</a> Journal of Controlled Release. 86 (1), 49-57 (2003).</li>
<li>Skelly, J. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((FDA+and+AAPS+report+of+the+workshop+on+principles+and+practices+of+in+vitro+percutaneous+penetration+studies%3A+relevance+to+bioavailability+and+bioequivalence%5BTitle%5D)+AND+%22Pharmaceutical+Research%22%5BJournal%5D)">FDA and AAPS report of the workshop on principles and practices of in vitro percutaneous penetration studies: relevance to bioavailability and bioequivalence.</a> Pharmaceutical Research. 4 (3), 265-267 (1987).</li>
<li>OECD. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Guidance+document+for+the+conduct+of+skin+absorption+studies%5BTitle%5D)+AND+%22OECD%22%5BJournal%5D)">Guidance document for the conduct of skin absorption studies.</a> OECD. , (2004).</li>
<li>OECD. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Test+no.+428%3A+Skin+absorption%3A+In+vitro+method%5BTitle%5D)+AND+%22OECD%22%5BJournal%5D)">Test no. 428: Skin absorption: In vitro method.</a> OECD. , (2004).</li>
<li>Saar, B. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Video-rate+molecular+imaging+in+vivo+with+stimulated+Raman+scattering%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Video-rate molecular imaging in vivo with stimulated Raman scattering.</a> Science. 330 (6009), 1368-1370 (2010).</li>
<li>Saar, B. G., Contreras-Rojas, L. R., Xie, X. S., Guy, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Imaging+drug+delivery+to+skin+with+stimulated+Raman+scattering+microscopy%5BTitle%5D)+AND+%22Molecular+Pharmaceutics%22%5BJournal%5D)">Imaging drug delivery to skin with stimulated Raman scattering microscopy.</a> Molecular Pharmaceutics. 8 (3), 969-975 (2011).</li>
<li>Pence, I. J., Kuzma, B. A., Brinkmann, M., Hellwig, T., Evans, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Multi-windowsparse+spectral+sampling+stimulated+Raman+scattering+microscopy%5BTitle%5D)+AND+%22Biomedical+Optics+Express%22%5BJournal%5D)">Multi-windowsparse spectral sampling stimulated Raman scattering microscopy.</a> Biomedical Optics Express. 12 (10), 6095-6114 (2021).</li>
<li>Herkenne, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vivo+methods+for+the+assessment+of+topical+drug+bioavailability%5BTitle%5D)+AND+%22Pharmaceutical+Research%22%5BJournal%5D)">In vivo methods for the assessment of topical drug bioavailability.</a> Pharmaceutical Research. 25 (1), 87-103 (2008).</li>
<li>Alfonso-Garcıa, A., Mittal, R., Lee, E. S., Potma, E. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Biological+imaging+with+coherent+Raman+scattering+microscopy%3A+a+tutorial%5BTitle%5D)+AND+%22Journal+of+Biomedical+Optics%22%5BJournal%5D)">Biological imaging with coherent Raman scattering microscopy: a tutorial.</a> Journal of Biomedical Optics. 19 (7), 071407(2014).</li>
<li>Osseiran, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Longitudinal+monitoring+of+cancer+cell+subpopulations+in+monolayers%2C+3D+spheroids%2C+and+xenografts+using+the+photoconvertible+dye+DiR%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">Longitudinal monitoring of cancer cell subpopulations in monolayers, 3D spheroids, and xenografts using the photoconvertible dye DiR.</a> Scientific Reports. 9 (1), 1-10 (2019).</li>
<li>Evennett, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Kohler+illumination%3A+a+simple+interpretation%5BTitle%5D)+AND+%22Proceedings+of+the+Royal+Microscopical+Society%22%5BJournal%5D)">Kohler illumination: a simple interpretation.</a> Proceedings of the Royal Microscopical Society. 28 (4), 189-192 (1983).</li>
<li>Sanderson, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fundamentals+of+microscopy%5BTitle%5D)+AND+%22Current+Protocols+in+Mouse+Biology%22%5BJournal%5D)">Fundamentals of microscopy.</a> Current Protocols in Mouse Biology. 10 (2), 76(2020).</li>
<li>Schindelin, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fiji%3A+an+open-source+platform+for+biological-image+analysis%5BTitle%5D)+AND+%22Nature+Methods%22%5BJournal%5D)">Fiji: an open-source platform for biological-image analysis.</a> Nature Methods. 9 (7), 676-682 (2012).</li>
<li>Hunter, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Matplotlib%3A+A+2D+graphics+environment%5BTitle%5D)+AND+%22Computing+in+Science+%26+Engineering%22%5BJournal%5D)">Matplotlib: A 2D graphics environment.</a> Computing in Science & Engineering. 9 (3), 90-95 (2007).</li>
<li>Wickham, H. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aWickham%2C+H+%22ggplot2%3A+Elegant+Graphics+for+Data+Analysis%22">ggplot2: Elegant Graphics for Data Analysis</a>. , Springer-Verlag. New York. (2016).</li>
<li>Kim, H., Han, S., Cho, Y. S., Yoon, S. K., Bae, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Development+of+R+packages%3A%27Non-Compart%27+and+%27ncar%27+for+noncompartmental+analysis+%28NCA%29%5BTitle%5D)+AND+%22Translational+and+Clinical+Pharmacology%22%5BJournal%5D)">Development of R packages:'Non-Compart' and 'ncar' for noncompartmental analysis (NCA).</a> Translational and Clinical Pharmacology. 26 (1), 10-15 (2018).</li>
</ol>

CLE is an inventor on patents for CARS microscopy that have been licensed to multiple microscope manufacturers. All other authors have no conflicts of interest to disclose.

The evaluation of topical BA/BE is an area of research the requires a multifaceted approach as no single method can fully characterize in vivo cPK. This protocol presents a methodology for the evaluation of a topical drug product&#39;s BA/BE based on coherent Raman imaging. One of the first points that might be overlooked is how thin the skin samples must be, especially for quantitative transmission SRS imaging. If the skin is too thick (i.e., light cannot readily pass through), there is little to no si...

Methodologies to assess cPK after topical drug product application have expanded from classical in vitro permeation testing (IVPT) studies1,2,3,4,5 and tape-stripping6,7,8 to additional methodologies such as open-flow microperfusion or dermal microdialysis9,10,11,12,13,14. There are potentially various local sites of therapeutic action depending on the disease of interest. Hence, there may be a corresponding number of methodologies to assess the rate and extent to which an API gets to the intended local site of action. While each of the aforementioned methodologies has its advantages, the major disadvantage is the lack of microscale cPK information (i.e., the inability to visualize where the API goes and how it permeates).
One noninvasive methodology of interest to estimate topical BA and BE is CRI, which can be broken down into two imaging modalities: CARS and SRS microscopy. These coherent Raman methods enable chemically specific imaging of molecules via nonlinear Raman effects. In CRI, two laser pulse trains are focused and scanned within a sample; the difference in energy between the laser frequencies is set to target vibrational modes specific to the chemical structures of interest. As CRI processes are nonlinear, a signal is only generated at the microscope focus, allowing for three-dimensional pharmacokinetic tomographic imaging of the tissue. In the context of cPK, CARS has been used to obtain tissue structural information, such as the location of lipid-rich skin structures15. In contrast, SRS has been utilized to quantify molecular concentration as its signal is linear with concentration. For ex vivo skin specimens, it is advantageous to carry out CARS in the epi-direction16 and SRS in transmission mode17. Therefore, tissue samples that are thin will allow for SRS signal detection and quantification.
As a model tissue, the nude mouse ear presents several advantages with minor drawbacks. One advantage is that the tissue is already ~200-300 &#181;m in thickness and does not require further sample preparation. In addition, several skin stratifications are seen by axially focusing through one field of view (e.g., stratum corneum, sebaceous glands (SGs), adipocytes, and subcutaneous fat)16,18. This allows for preliminary preclinical estimation of cutaneous permeation pathways and topical BA estimates before moving to human skin samples. However, the nude mouse model presents limitations such as difficulty in extrapolation to in vivo scenarios due to differences in skin structure19. While the nude mouse ear is an excellent model to obtain preliminary results, the human skin model is the gold standard. Although there have been various commentaries on the suitability and applicability of frozen human skin to accurately recapitulate in vivo permeation kinetics20,21,22, the use of frozen human skin is an accepted method for the evaluation of in vitro&#160;API permeation kinetics23,24,25. This protocol visualizes various skin layers in mouse and human skin while quantifying API concentrations within lipid-rich and lipid-poor structures.
While CRI has been utilized across numerous fields to specifically visualize compounds within tissues, there have been limited efforts investigating the cPK of topically applied drug products. To evaluate the topical BA/BE of topical products using CRI, it is necessary to first have a standardized protocol in place to make accurate comparisons. Previous efforts using CRI for drug delivery to the skin have demonstrated variability within the data. As this is a relatively new application of CRI, establishing a protocol is critical to obtain reliable results18,26,27. This approach only targets one specific wavenumber in the biological silent region of the Raman spectrum. However, most APIs and inactive ingredients have Raman shifts within the fingerprint region. This has previously posed challenges due to the inherent signal arising from the tissue in the fingerprint region. Recent laser and computational advances have removed this barrier, which can also be utilized in combination with the approach presented here28. This approach presented here allows for the quantification of an API, which has a Raman shift in the silent region (2,000-2,300 cm-1). This is not limited to the physiochemical properties of the drug, which might be the case for some previously mentioned cPK monitoring methodologies29.
The protocol must reduce sample-to-sample variability in skin thickness for various preparations, as thick human skin samples will produce minimal signal after drug product application due to light scattering by the thick sample. A goal of this manuscript is to present a tissue preparation methodology that assures reproducible imaging standards. In addition, the CRI system is setup as described to reduce potential sources of error as well as minimize signal-to-noise.&#160;However, this paper will not discuss the guiding principles and technical merits of the CRI microscope as this has been previously covered30. Finally, the extensive data analysis procedure is explored to allow for interpretation of the results to determine an experiment&#39;s success or failure.

<table><tbody><tr><td>&lt;strong&gt;Tissue Preparation&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Autoclavable Biohazard Bags</td><td>FisherBrand</td><td>22-044562</td><td>As refered to in text: biohazard bags&lt;br/&gt; https://www.fishersci.com/shop/products/fisherbrand-polyethylene-biohazard-autoclave-bags-without-sterilization-indicator-8/22044562?searchHijack=true&amp;amp;searchTerm= 22044562&amp;amp;searchType=RAPID&amp;amp; matchedCatNo=22044562</td></tr><tr><td>Cell Culture Buffers: Dulbecco&#039;s Phosphate-Buffered Salt Solution 1x</td><td>Corning</td><td>MT21030CV</td><td>As refered to in text: PBS&lt;br/&gt; https://www.fishersci.com/shop/products/corning-cellgro-cell-culture-buffers-dulbecco-s-phosphate-buffered-salt-solution-1x-8/MT21030CV?searchHijack=true&amp;amp;searchTerm= 21-030-cv&amp;amp;searchType= RAPID&amp;amp;matchedCatNo=21-030-cv</td></tr><tr><td>Disposable Scalpels</td><td>Exel International</td><td>14-840-00</td><td>As refered to in text: scalpel&lt;br/&gt; https://www.fishersci.com/shop/products/exel-international-disposable-scalpels-3/1484000?keyword=true</td></tr><tr><td>High Precision 45&amp;deg; Angle Broad Point Tweezers/Forceps</td><td>Fisherbrand</td><td>12-000-132</td><td>As refered to in text: forceps&lt;br/&gt; https://www.fishersci.com/shop/products/high-precision-45-angle-broad-point-tweezers-forceps/12000132#?keyword=</td></tr><tr><td>Kimwipes Delicate Task Wipers, 1-Ply</td><td>Kimberly-Clark Professional Kimtech Science</td><td>06-666</td><td>As refered to in text: task wiper&lt;br/&gt; https://www.fishersci.com/shop/products/kimberly-clark-kimtech-science-kimwipes-delicate-task-wipers-7/06666</td></tr><tr><td>Parafilm M Laboratory Wrapping Film</td><td>Bemis</td><td>13-374-12</td><td>As refered to in text: parafilm&lt;br/&gt; https://www.fishersci.com/shop/products/curwood-parafilm-m-laboratory-wrapping-film-4/1337412</td></tr><tr><td>Petri Dish (35 mm x 10 mm)</td><td>Fisherbrand</td><td>FB0875711YZ</td><td>As refered to in text: small petri dish&lt;br/&gt; https://www.fishersci.com/shop/products/fisherbrand-petri-dishes-specialty-6/FB0875711YZ?keyword=true</td></tr><tr><td>Petri Dish (60 mm x 15 mm)</td><td>Fisherbrand</td><td>FB0875713A</td><td>As refered to in text: large petri dish&lt;br/&gt; https://www.fishersci.com/shop/products/fisherbrand-petri-dishes-clear-lid-12/FB0875713A?keyword=true</td></tr><tr><td>Surgical Scissors</td><td>Roboz</td><td>NC9411473</td><td>As refered to in text: scissors&lt;br/&gt; https://www.fishersci.com/shop/products/scissors-327/NC9411473?searchHijack=true&amp;amp;searchTerm= RS-5915SC&amp;amp;searchType=RAPID&amp;amp; matchedCatNo=RS-5915SC</td></tr><tr><td>&lt;strong&gt;Laser/microscope&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>650/60 nm BrightLine single-band bandpass filter</td><td>Semrock</td><td></td><td>As refered to in text: CARS filter - CH2 vibrations (645nm/60nm filter)</td></tr><tr><td>Control box IX2-UCB</td><td>Olympus</td><td></td><td>As refered to in text: Control Box</td></tr><tr><td>D700/30m</td><td>Chroma</td><td></td><td>As refered to in text: CARS filter - deuterated band&lt;br/&gt; https://www.chroma.com/products/parts/d700-30m</td></tr><tr><td>DeepSee Insight</td><td>Spectra-Physics</td><td></td><td>As refered to in text: Laser&lt;br/&gt; https://www.spectra-physics.com/f/insight-x3-tunable-laser</td></tr><tr><td>Digital Handheld Optical Power and Energy Meter Console</td><td>ThorLabs</td><td>PM100D</td><td>As refered to in text: power meter&lt;br/&gt; https://www.thorlabs.com/newgrouppage9.cfm?objectgroup_id=3341</td></tr><tr><td>Fluoview Software</td><td>Olympus</td><td></td><td>As refered to in text: Microscope Control software</td></tr><tr><td>Frosted Microscope Slides</td><td>FisherBrand</td><td></td><td>As refered to in text: microscope slides&lt;br/&gt; https://www.fishersci.com/shop/products/fisherbrand-frosted-microscope-slides-4/22265446</td></tr><tr><td>FV1000</td><td>Olympus</td><td></td><td>As refered to in text: Microscope</td></tr><tr><td>Incubation Chamber</td><td>Tokai Hit</td><td>GM-800</td><td>As refered to in text: incubation chamber</td></tr><tr><td>Integrating Sphere Photodiode Power Sensor</td><td>ThorLabs</td><td>S142C</td><td>As refered to in text: photodiode&lt;br/&gt; https://www.thorlabs.com/newgrouppage9.cfm?objectgroup_id=3341</td></tr><tr><td>Power supply FV31-PSU</td><td>Olympus</td><td></td><td>As refered to in text: Power Supply</td></tr><tr><td>Precision 4063, 80MHz Dual Channel Function Generator</td><td>BK Precision</td><td></td><td>As refered to in text: function generator</td></tr><tr><td>ProScan &amp;ndash; Precision Microscope Automation</td><td>Prior Scientific Instruments</td><td></td><td>As refered to in text: stage controller&lt;br/&gt; https://www.prior.com/microscope-automation/inverted-microscope-systems/proscan-linear-stage-highest-precision-microscope-automation</td></tr><tr><td>SecureSeal Imaging Spacers</td><td>Grace Biolabs</td><td>654004</td><td>As refered to in text: spacer&lt;br/&gt; https://gracebio.com/product/secureseal-imaging-spacers-654004/</td></tr><tr><td>SRS Detection Kit</td><td>APE</td><td></td><td>As refered to in text: SRS detector</td></tr><tr><td>UPLSAPO 20X NA:0.75</td><td>Olympus</td><td></td><td>As refered to in text: 20X Objective&lt;br/&gt; https://www.olympus-lifescience.com/en/objectives/uplsapo/</td></tr><tr><td>&lt;strong&gt;Lipid/Drug Imaging&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&amp;nbsp;35 mm Dish, No. 0 Uncoated Coverslip, 14 mm Glass Diameter</td><td>MatTek Corporation</td><td>NC9711297</td><td>As refered to in text: Glass bottom dish&lt;br/&gt; https://www.fishersci.com/shop/products/glass-bottom-mircrowell-dish/nc9711297</td></tr><tr><td>Cotton-tipped applicators</td><td>FisherBrand</td><td></td><td>As refered to in text: Cotton-tipped applicator</td></tr><tr><td>Distriman Postive Displacement Pipette</td><td>Gilson</td><td></td><td>As refered to in text: Postive Displacement Pipette&lt;br/&gt; https://www.fishersci.com/shop/products/gilson-distriman-positive-displacement-repetitive-pipette/F164001G#?keyword=</td></tr><tr><td>Distriman Postive Displacement Pipette Tips</td><td>Gilson</td><td></td><td>As refered to in text: Tips for pipette&lt;br/&gt; https://www.fishersci.com/shop/products/gilson-distritip-syringes-6/f164100g?keyword=true</td></tr><tr><td>&lt;strong&gt;Data Analysis&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>FIJI</td><td>Open-source</td><td></td><td>As refered to in text: FIJI/ImageJ&lt;br/&gt; https://imagej.net/software/fiji/</td></tr><tr><td>Jupyter-Lab</td><td>open-source</td><td></td><td>As refered to in text: JupyterLab&lt;br/&gt; https://jupyter.org/</td></tr><tr><td>Rstudio</td><td>Open-source</td><td></td><td>As refered to in text: Rstudio&lt;br/&gt; https://www.rstudio.com/</td></tr></tbody></table>

visualizing and quantifying pharmaceutical compounds within skin using coherent raman scattering imaging

Cutaneous pharmacokinetics (cPK) after topical formulation application has been a research area of particular interest for regulatory and drug development scientists to mechanistically understand topical bioavailability (BA). Semi-invasive techniques, such as tape-stripping, dermal microdialysis, or dermal open-flow microperfusion, all quantify macroscale cPK. While these techniques have provided vast cPK knowledge, the community lacks a mechanistic understanding of active pharmaceutical ingredient (API) penetration and permeation at the cellular level. 
One noninvasive approach to address microscale cPK is coherent Raman scattering imaging (CRI), which selectively targets intrinsic molecular vibrations without the need for extrinsic labels or chemical modification. CRI has two main methods-coherent anti-Stokes Raman scattering (CARS) and stimulated Raman scattering (SRS)-that enable sensitive and selective quantification of APIs or inactive ingredients. CARS is typically utilized to derive structural skin information or visualize chemical contrast. In contrast, the SRS signal, which is linear with molecular concentration, is used to quantify APIs or inactive ingredients within skin stratifications. 
Although mouse tissue has commonly been utilized for cPK with CRI, topical BA and bioequivalence (BE) must ultimately be assessed in human tissue before regulatory approval. This paper presents a methodology to prepare and image ex vivo skin to be used in quantitative pharmacokinetic CRI studies in the evaluation of topical BA and BE. This methodology enables reliable and reproducible API quantification within human and mouse skin over time. The concentrations within lipid-rich and lipid-poor compartments, as well as total API concentration over time are quantified; these are utilized for estimates of micro- and macroscale BA and, potentially, BE.

Imaging is considered successful if the tissue has not significantly moved in either axial (&#60;10 &#956;m) or lateral direction upon the completion of the experiment (Figure 4). This is an immediate indication if the SRS measurement for the API of interest is not representative of the initial depth, for which quantification is layer-specific. This is mitigated by imaging z-stacks for each XY position of interest, with the trade-off being the temporal resolution. If frozen skin is used in t...

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Visualizing and Quantifying Pharmaceutical Compounds within Skin using Coherent Raman Scattering Imaging

A coherent Raman scattering imaging methodology to visualize and quantify pharmaceutical compounds within the skin is described. This paper describes skin tissue preparation (human and mouse) and topical formulation application, image acquisition to quantify spatiotemporal concentration profiles, and preliminary pharmacokinetic analysis to assess topical drug delivery.

Cutaneous pharmacokinetics (cPK) after topical formulation application has been a research area of particular interest for regulatory and drug development ...

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Research

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Pharmacokinetics and Pharmacodynamics

Medicine

Unit 1

Pharmacokinetics: Drug Distribution and Protein Binding

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2.7K Views.  Massachusetts General Hospital, Harvard Medical School. Deze methodologie is belangrijk omdat het een reproduceerbare en nauwkeurige methode mogelijk maakt om topisch aangebrachte geneesmiddelen te kwantificeren met behulp van coherente Raman-beeldvorming. Andere methodologieën bieden omvangrijke cutane farmacokinetische informatie, terwijl deze methodologie zowel bulk- als microschaal cutane farmacokinetische informatie kan bieden, zoals de route van permeatie van een geneesmiddel. De visualisatie- en kwantificerings- en permeatieroutes zullen d...

Video: Visualizing and Quantifying Pharmaceutical Compounds within Skin using Coherent Raman Scattering Imaging

Coherent anti-Stokes Raman Scattering (CARS) Microscopy Visualizes Pharmaceutical Tablets During Dissolution

Traditional pharmaceutical dissolution tests determine the amount of drug dissolved over time by measuring drug content in the dissolution medium. This method provides little direct information about what is happening on the surface of the dissolving tablet. As the tablet surface composition and structure can change during dissolution, it is essential to monitor it during dissolution testing. In this work coherent anti-Stokes Raman scattering microscopy is used to image the surface of tablets during dissolution while UV absorption spectroscopy is simultaneously providing inline analysis of dissolved drug concentration for tablets containing a 50% mixture of theophylline anhydrate and ethyl cellulose. The measurements showed that in situ CARS microscopy is capable of imaging selectively theophylline in the presence of ethyl cellulose. Additionally, the theophylline anhydrate converted to theophylline monohydrate during dissolution, with needle-shaped crystals growing on the tablet surface during dissolution. The conversion of theophylline anhydrate to monohydrate, combined with reduced exposure of the drug to the flowing dissolution medium resulted in decreased dissolution rates. Our results show that in situ CARS microscopy combined with inline UV absorption spectroscopy is capable of monitoring pharmaceutical tablet dissolution and correlating surface changes with changes in dissolution rate.

Coherent anti-Stokes Raman Scattering CARS Microscopy Visualizes Pharmaceutical Tablets During Dissolution

Coherent anti-Stokes Raman scattering (CARS) microscopy is combined with an intrinsic flow-through dissolution setup to allow in situ and real-time visualization of the surface of pharmaceutical tablets undergoing dissolution. Using this custom-built setup, it is possible to correlate CARS videos with drug dissolution profiles recorded using inline UV absorption spectroscopy.

Traditional pharmaceutical dissolution tests determine the amount of drug dissolved over time by measuring drug content in the dissolution medium. This ...

Engineering

Combining Reflectance Confocal Microscopy with Optical Coherence Tomography for Noninvasive Diagnosis of Skin Cancers via Image Acquisition

Skin cancer is one of the most common cancers worldwide. Diagnosis relies on visual inspection and dermoscopy followed by biopsy for histopathological confirmation. While the sensitivity of dermoscopy is high, the lower specificity results in 70%-80% of the biopsies being diagnosed as benign lesions on histopathology (false positives on dermoscopy).
Reflectance confocal microscopy (RCM) and optical coherence tomography (OCT) imaging can noninvasively guide the diagnosis of skin cancers. RCM visualizes cellular morphology in en-face layers. It has doubled the diagnostic specificity for melanoma and pigmented keratinocytic skin cancers over dermoscopy, halving the number of biopsies of benign lesions. RCM acquired billing codes in the USA and is now being integrated into clinics.
However, limitations such as the shallow depth (~200 &#181;m) of imaging, poor contrast for nonpigmented skin lesions, and imaging in en-face layers result in relatively lower specificity for the detection of nonpigmented basal cell carcinoma (BCCs) &#8212; superficial BCCs contiguous with the basal cell layer and deeper infiltrative BCCs. In contrast, OCT lacks cellular resolution but images tissue in vertical planes down to a depth of ~1 mm, which allows the detection of both superficial and deeper subtypes of BCCs. Thus, both techniques are essentially complementary.
A &#34;multi-modal,&#34; combined RCM-OCT device simultaneously images skin lesions in both en-face and vertical modes. It is useful for the diagnosis and management of BCCs (nonsurgical treatment for superficial BCCs vs. surgical treatment for deeper lesions). A marked improvement in specificity is obtained for detecting small, nonpigmented BCCs over RCM alone. RCM and RCM-OCT devices are bringing a major paradigm shift in the diagnosis and management of skin cancers; however, their use is currently limited to academic tertiary care centers and some private clinics. This paper familiarizes clinicians with these devices and their applications, addressing translational barriers into routine clinical workflow.

Here, we describe protocols for acquiring good-quality images using novel, noninvasive imaging devices of reflectance confocal microscopy (RCM) and combined RCM and optical coherence tomography (OCT). We also familiarize clinicians with their clinical applications so that they can integrate the techniques into regular clinical workflows to improve patient care.

Skin cancer is one of the most common cancers worldwide. Diagnosis relies on visual inspection and dermoscopy followed by biopsy for histopathological ...

Cancer Research

Implementation of a Nonlinear Microscope Based on Stimulated Raman Scattering

Stimulated Raman scattering (SRS) microscopy uses near-infrared excitation light; therefore, it shares many multi-photon microscopic imaging properties. SRS imaging modality can be obtained using commercial laser-scanning microscopes by equipping with a non-descanned forward detector with proper bandpass filters and lock-in amplifier (LIA) detection scheme. A schematic layout of a typical SRS microscope includes the following: two pulsed laser beams, (i.e., the pump and probe directed in a scanning microscope), which must be overlapped in both space and time at the image plane, then focused by a microscope objective into the sample through two scanning mirrors (SMs), which raster the focal spot across an x-y plane. After interaction with the sample, transmitted output pulses are collected by an upper objective and measured by a forward detection system inserted in an inverted microscope. Pump pulses are removed by a stack of optical filters, whereas the probe pulses that are the result of the SRS process occurring in the focal volume of the specimen are measured by a photodiode (PD). The readout of the PD is demodulated by the LIA to extract the modulation depth. A two-dimensional (2D) image is obtained by synchronizing the forward detection unit with the microscope scanning unit. In this paper, the implementation of an SRS microscope is described and successfully demonstrated, as well as the reporting of label-free images of polystyrene beads with diameters of 3 &#181;m. It is worth noting that SRS microscopes are not commercially available, so in order to take advantage of these characteristics, the homemade construction is the only option. Since SRS microscopy is becoming popular in many fields, it is believed that this careful description of the SRS microscope implementation can be very useful for the scientific community.

In this manuscript, the implementation of a stimulated Raman scattering (SRS) microscope, obtained by the integration of an SRS experimental set-up with a laser scanning microscope, is described. The SRS microscope is based on two femtosecond (fs) laser sources, a Ti-Sapphire (Ti:Sa) and synchronized optical parametric oscillator (OPO).

Stimulated Raman scattering (SRS) microscopy uses near-infrared excitation light; therefore, it shares many multi-photon microscopic imaging properties. ...

Resolving Water, Proteins, and Lipids from In Vivo Confocal Raman Spectra of Stratum Corneum through a Chemometric Approach

Development of this in vivo confocal Raman spectroscopic method enables the direct measurement of water, proteins, and lipids with depth resolution in human subjects. This information is very important for skin-related diseases and characterizing skin care product performance. This protocol illustrates a method for confocal Raman spectra collection and the subsequent analysis of the spectral dataset leveraging chemometrics. The goal of this method is to establish a standard protocol for data collection and provide general guidance for data analysis. Preprocessing (e.g., removal of outlier spectra) is a critical step when processing large datasets from clinical studies. As an example, we provide guidance based on prior knowledge of a dataset to identify the types of outliers and develop specific strategies to remove them. A principal component analysis is performed, and the loading spectra are compared with spectra from reference materials to select the number of components used in the final multivariate curve resolution (MCR) analysis. This approach is successful for extracting meaningful information from a large spectral dataset.

Here, we present a protocol for collection of confocal Raman spectra from human subjects in clinical studies combined with chemometric approaches for spectral outlier removal and the subsequent extraction of key features.

Development of this in vivo confocal Raman spectroscopic method enables the direct measurement of water, proteins, and lipids with depth resolution in ...

Chemistry

Multiplex Chemical Imaging Based on Broadband Stimulated Raman Scattering Microscopy

Stimulated Raman scattering (SRS) microscopy is a nonlinear optical technique for label-free chemical imaging. This analytical tool delivers chemical maps at high speed, and high spatial resolution of thin samples by directly interrogating their molecular vibrations. In its standard implementation, SRS microscopy is narrowband and forms images with only a single vibrational frequency at a time. However, this approach not only hinders the chemical specificity of SRS but also neglects the wealth of information encoded within vibrational spectra. 
These limitations can be overcome by broadband SRS, an implementation capable of extracting a vibrational spectrum per pixel of the image in parallel. This delivers hyperspectral data that, when coupled with chemometric analysis, maximizes the amount of information retrieved from the specimen. Thus, broadband SRS improves the chemical specificity of the system, allowing the quantitative determination of the concentration of the different constituents of a sample. Here, we report a protocol for chemical imaging with broadband SRS microscopy, based on a home-built SRS microscope operating with a custom differential multichannel-lock-in amplifier detection. It discusses the sample preparation, alignment of the SRS apparatus, and chemometric analysis. By acquiring vibrational Raman spectra, the protocol illustrates how to identify different chemical species within a mixture, determining their relative concentrations.

We present a protocol to acquire chemical images with broadband stimulated Raman scattering (SRS) microscopy. Based on an SRS microscope that operates with differential multichannel-lock-in detection, the protocol describes the sample preparation, adjustment of the SRS apparatus, and chemometrics to disentangle different constituents of chemically heterogeneous samples.

Stimulated Raman scattering (SRS) microscopy is a nonlinear optical technique for label-free chemical imaging. This analytical tool delivers chemical maps ...

Direct Comparison of Hyperspectral Stimulated Raman Scattering and Coherent Anti-Stokes Raman Scattering Microscopy for Chemical Imaging

Stimulated Raman scattering (SRS) and coherent anti-Stokes Raman scattering (CARS) microscopy are the most widely used coherent Raman scattering imaging technologies. Hyperspectral SRS and CARS imaging offer Raman spectral information at every pixel, which enables better separation of different chemical compositions. Although both techniques require two excitation lasers, their signal detection schemes and spectral properties are quite different. The goal of this protocol is to perform both hyperspectral SRS and CARS imaging on a single platform and compare the two microscopy techniques for imaging different biological samples. The spectral focusing method is employed to acquire spectral information using femtosecond lasers. By using standard chemical samples, the sensitivity, spatial resolution, and spectral resolution of SRS and CARS in the same excitation conditions (i.e., power at the sample, pixel dwell time, objective lens, pulse energy) are compared. The imaging contrasts of CARS and SRS for biological samples are juxtaposed and compared. The direct comparison of CARS and SRS performances would allow for optimal selection of the modality for chemical imaging.

This paper directly compares the resolution, sensitivity, and imaging contrasts of stimulated Raman scattering (SRS) and coherent anti-Stokes Raman scattering (CARS) integrated into the same microscope platform. The results show that CARS has a better spatial resolution, SRS gives better contrasts and spectral resolution, and both methods have similar sensitivity.

Stimulated Raman scattering (SRS) and coherent anti-Stokes Raman scattering (CARS) microscopy are the most widely used coherent Raman scattering imaging ...

Differential Imaging of Biological Structures with Doubly-resonant Coherent Anti-stokes Raman Scattering (CARS)

Coherent Raman imaging techniques have seen a dramatic increase in activity over the past decade due to their promise to enable label-free optical imaging with high molecular specificity 1. The sensitivity of these techniques, however, is many orders of magnitude weaker than fluorescence, requiring milli-molar molecular concentrations 1,2. Here, we describe a technique that can enable the detection of weak or low concentrations of Raman-active molecules by amplifying their signal with that obtained from strong or abundant Raman scatterers. The interaction of short pulsed lasers in a biological sample generates a variety of coherent Raman scattering signals, each of which carry unique chemical information about the sample. Typically, only one of these signals, e.g. Coherent Anti-stokes Raman scattering (CARS), is used to generate an image while the others are discarded. However, when these other signals, including 3-color CARS and four-wave mixing (FWM), are collected and compared to the CARS signal, otherwise difficult to detect information can be extracted 3. For example, doubly-resonant CARS (DR-CARS) is the result of the constructive interference between two resonant signals 4. We demonstrate how tuning of the three lasers required to produce DR-CARS signals to the 2845 cm-1 CH stretch vibration in lipids and the 2120 cm-1 CD stretching vibration of a deuterated molecule (e.g. deuterated sugars, fatty acids, etc.) can be utilized to probe both Raman resonances simultaneously. Under these conditions, in addition to CARS signals from each resonance, a combined DR-CARS signal probing both is also generated. We demonstrate how detecting the difference between the DR-CARS signal and the amplifying signal from an abundant molecule's vibration can be used to enhance the sensitivity for the weaker signal. We further demonstrate that this approach even extends to applications where both signals are generated from different molecules, such that e.g. using the strong Raman signal of a solvent can enhance the weak Raman signal of a dilute solute.

Differential Imaging of Biological Structures with Doubly-resonant Coherent Anti-stokes Raman Scattering CARS

A combination of three single wavelength short-pulsed lasers is used to generate coherent anti-Stokes Raman scattering (CARS) and doubly-resonant CARS (DR-CARS). The difference between these signals provides enhanced sensitivity for otherwise difficult to detect coherent Raman signals, enabling imaging of weak Raman scatterers.

Coherent Raman imaging techniques have seen a dramatic increase in activity over the past decade due to their promise to enable label-free optical imaging ...

Biology

In vivo Imaging of Biological Tissues with Combined Two-Photon Fluorescence and Stimulated Raman Scattering Microscopy

Stimulated Raman scattering (SRS) microscopy enables label-free imaging of the biological tissues in its natural microenvironment based on intrinsic molecular vibration, thus providing a perfect tool for in vivo study of biological processes at subcellular resolution. By integrating two-photon excited fluorescence (TPEF) imaging into the SRS microscope, the dual-modal in vivo imaging of tissues can acquire critical biochemical and biophysical information from multiple perspectives which helps understand the dynamic processes involved in cellular metabolism, immune response and tissue remodeling, etc. In this video protocol, the setup of a TPEF-SRS microscope system as well as the in vivo imaging method of the animal spinal cord is introduced. The spinal cord, as part of the central nervous system, plays a critical role in the communication between the brain and peripheral nervous system. Myelin sheath, abundant in phospholipids, surrounds and insulates the axon to permit saltatory conduction of action potentials. In vivo imaging of myelin sheaths in the spinal cord is important to study the progression of neurodegenerative diseases and spinal cord injury. The protocol also describes animal preparation and in vivo TPEF-SRS imaging methods to acquire high-resolution biological images.

In vivo Imaging of Biological Tissues with Combined Two-Photon Fluorescence and Stimulated Raman Scattering Microscopy

Stimulated Raman scattering (SRS) microscopy allows label-free imaging of biomolecules based on their intrinsic vibration of specific chemical bonds. In this protocol, the instrumental setup of an integrated SRS and two-photon fluorescence microscope is described to visualize cellular structures in the spinal cord of live mice.

Stimulated Raman scattering (SRS) microscopy enables label-free imaging of the biological tissues in its natural microenvironment based on intrinsic ...

Bioengineering

Biomolecular Imaging of Cellular Uptake of Nanoparticles using Multimodal Nonlinear Optical Microscopy

Probing gold nanoparticles (AuNPs) in living systems is essential to reveal the interaction between AuNPs and biological tissues. Moreover, by integrating nonlinear optical signals such as stimulated Raman scattering (SRS), two-photon excited fluorescence (TPEF), and transient absorption (TA) into an imaging platform, it can be used to reveal biomolecular contrast of cellular structures and AuNPs in a multimodal manner. This article presents a multimodal nonlinear optical microscopy and applies it to perform chemically specific imaging of AuNPs in cancer cells. This imaging platform provides a novel approach for developing more efficient functionalized AuNPs and determining whether they are within vasculatures surrounding the tumor, pericellular, or cellular spaces.

This article presents the integration of a spectral-focusing module and a dual-output pulse laser, enabling rapid hyperspectral imaging of gold nanoparticles and cancer cells. This work aims to demonstrate the details of multimodal nonlinear optical techniques on a standard laser scanning microscope.

Probing gold nanoparticles (AuNPs) in living systems is essential to reveal the interaction between AuNPs and biological tissues. Moreover, by integrating ...

Implementation of a Coherent Anti-Stokes Raman Scattering (CARS) System on a Ti:Sapphire and OPO Laser Based Standard Laser Scanning Microscope

Laser scanning microscopes combining a femtosecond Ti:sapphire laser and an optical parametric oscillator (OPO) to duplicate the laser line have become available for biologists. These systems are primarily designed for multi-channel two-photon fluorescence microscopy. However, without any modification, complementary non-linear optical microscopy such as second-harmonic generation (SHG) or third harmonic generation (THG) can also be performed with this set-up, allowing label-free imaging of structured molecules or aqueous medium-lipid interfaces. These techniques are well suited for in-vivo observation, but are limited in chemical specificity. Chemically selective imaging can be obtained from inherent vibration signals based on Raman scattering. Confocal Raman microscopy provides 3D spatial resolution, but it requires high average power and long acquisition time. To overcome these difficulties, recent advances in laser technology have permitted the development of nonlinear optical vibrational microscopy, in particular coherent anti-Stokes Raman scattering (CARS). CARS microscopy has therefore emerged as a powerful tool for biological and live cell imaging, by chemically mapping lipids (via C-H stretch vibration), water (via O-H stretch vibrations), proteins or DNA. In this work, we describe the implementation of the CARS technique on a standard OPO-coupled multiphoton laser scanning microscope. It is based on the in-time synchronization of the two laser lines by adjusting the length of one of the laser beam path. We present a step-by-step implementation of this technique on an existing multiphoton system. A basic background in experimental optics is helpful and the presented system does not require expensive supplementary equipment. We also illustrate CARS imaging obtained on myelin sheaths of sciatic nerve of rodent, and we show that this imaging can be performed simultaneously with other nonlinear optical imaging, such as standard two-photon fluorescence technique and second-harmonic generation.

Implementation of a Coherent Anti-Stokes Raman Scattering CARS System on a Ti:Sapphire and OPO Laser Based Standard Laser Scanning Microscope

Coherent anti-Stokes Raman scattering (CARS) microscopy based on inherent vibration of molecule bonds permits label-free chemically selective live cell imaging. This work presents the implementation of a complementary microscopy technique on a standard multiphoton laser scanning microscope based on a femtosecond Ti:sapphire laser and an OPO laser.

Laser scanning microscopes combining a femtosecond Ti:sapphire laser and an optical parametric oscillator (OPO) to duplicate the laser line have become ...

Visualizing and Quantifying Pharmaceutical Compounds within Skin using Coherent Raman Scattering Imaging

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