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., Benson, H. E., Watkinson, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+skin+permeation+methodology.+In+Transdermal+and+topical+drug+delivery:+principles+and+methodology.">In vitro skin permeation methodology. In Transdermal and topical drug delivery: principles and methodology.</a> Transdermal and topical drug delivery: principles and practice. , 85-108 (2012).</li><li>Shin, S. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation,+characterisation,+and+topical+delivery+of+terbinafine.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+permeation+test+(IVPT)+for+pharmacokinetic+assessment+of+topical+dermatological+formulations.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Franz+cell+diffusion+testing+andquantitative+confocal+Raman+spectroscopy:+In+vitro-in+vivo+correlation.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Topical+bioavailability+of+diclofenac+from+locally-acting,+dermatological+formulations.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stratum+corneum+sampling+to+assess+bioequivalence+between+topicalacyclovir+products.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dermal+delivery+of+niacinamide-in+vivo+studies.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Open+flow+microperfusion+as+a+dermal+pharmacokinetic+approach+to+evaluate+topical+bioequivalence.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluating+dermal+pharmacokinetics+and+pharmacodymanic+effect+of+soft+topical+PDE4+inhibitors:Open+flow+microperfusion+and+skin+biopsies.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Iontophoretic+current+and+intradermal+microdialysis+recovery+in+humans.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+adultatopic+dermatitis+on+topical+drug+penetration:+assessment+by+cutaneous+microdialysis+and+tape+stripping.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacokinetic+applications+of+cutaneous+microdialysis:+Continuous+intermittent+vs+continuous-only+sampling.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+local+bioavailability+of+metronidazole+from+topical+formulations+using+dermal+microdialysis:+Preliminary+study+in+a+Yucatan+mini-pig+model.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=L.Coherent+anti-Stokes+Raman+spectroscopy.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+imaging+of+tissue+in+vivo+with+video-rate+coherent+anti-Stokes+Raman+scattering+microscopy.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+imaging+depth+limit+of+stimulated+Raman+scattering+microscopy.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Label-free+quantification+of+pharmacokinetics+in+skin+with+stimulated+Raman+scattering+microscopy+and+deep+learning.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+skin+permeability+of+drugs+in+mice+and+human+cadaver+skin.">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., S&#248;rensen, J. A., Bagatolli, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Storage+conditions+of+skin+affect+tissue+structure+and+subsequent+in+vitro+percutaneous+penetration.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+frozen+human+epidermis+storage+duration+and+cryoprotectant+on+barrier+function+using+two+model+compounds.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+various+methods+of+cold+storage+of+skin+on+the+permeation+of+melatonin+and+nimesulide.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FDA+and+AAPS+report+of+the+workshop+on+principles+and+practices+of+in+vitro+percutaneous+penetration+studies:+relevance+to+bioavailability+and+bioequivalence.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guidance+document+for+the+conduct+of+skin+absorption+studies.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Test+no.+428:+Skin+absorption:+In+vitro+method.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Video-rate+molecular+imaging+in+vivo+with+stimulated+Raman+scattering.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+drug+delivery+to+skin+with+stimulated+Raman+scattering+microscopy.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multi-windowsparse+spectral+sampling+stimulated+Raman+scattering+microscopy.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+methods+for+the+assessment+of+topical+drug+bioavailability.">In vivo methods for the assessment of topical drug bioavailability.</a> Pharmaceutical Research. 25 (1), 87-103 (2008).</li><li>Alfonso-Garc&#305;a, A., Mittal, R., Lee, E. S., Potma, E. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+imaging+with+coherent+Raman+scattering+microscopy:+a+tutorial.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Longitudinal+monitoring+of+cancer+cell+subpopulations+in+monolayers,+3D+spheroids,+and+xenografts+using+the+photoconvertible+dye+DiR.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kohler+illumination:+a+simple+interpretation.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fundamentals+of+microscopy.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fiji:+an+open-source+platform+for+biological-image+analysis.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matplotlib:+A+2D+graphics+environment.">Matplotlib: A 2D graphics environment.</a> Computing in Science &amp; Engineering. 9 (3), 90-95 (2007).</li><li>Wickham, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> ggplot2: Elegant Graphics for Data Analysis. , (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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+R+packages:'Non-Compart'+and+'ncar'+for+noncompartmental+analysis+(NCA).">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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Tissue Preparation</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 
			https://www.fishersci.com/shop/products/fisherbrand-polyethylene-biohazard-autoclave-bags-without-sterilization-indicator-8/22044562?searchHijack=true&#38;searchTerm= 22044562&#38;searchType=RAPID&#38; matchedCatNo=22044562</td>
		</tr>
		<tr>
			<td>Cell Culture Buffers: Dulbecco&#39;s Phosphate-Buffered Salt Solution 1x</td>
			<td>Corning</td>
			<td>MT21030CV</td>
			<td>As refered to in text: PBS 
			https://www.fishersci.com/shop/products/corning-cellgro-cell-culture-buffers-dulbecco-s-phosphate-buffered-salt-solution-1x-8/MT21030CV?searchHijack=true&#38;searchTerm= 21-030-cv&#38;searchType= RAPID&#38;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 
			https://www.fishersci.com/shop/products/exel-international-disposable-scalpels-3/1484000?keyword=true</td>
		</tr>
		<tr>
			<td>High Precision 45&#176; Angle Broad Point Tweezers/Forceps</td>
			<td>Fisherbrand</td>
			<td>12-000-132</td>
			<td>As refered to in text: forceps 
			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 
			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 
			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 
			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 
			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 
			https://www.fishersci.com/shop/products/scissors-327/NC9411473?searchHijack=true&#38;searchTerm= RS-5915SC&#38;searchType=RAPID&#38; matchedCatNo=RS-5915SC</td>
		</tr>
		<tr>
			<td>Laser/microscope</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 
			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 
			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 
			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 
			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 
			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 &#8211; Precision Microscope Automation</td>
			<td>Prior Scientific Instruments</td>
			<td></td>
			<td>As refered to in text: stage controller 
			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 
			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 
			https://www.olympus-lifescience.com/en/objectives/uplsapo/</td>
		</tr>
		<tr>
			<td>Lipid/Drug Imaging</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;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 
			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 
			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 
			https://www.fishersci.com/shop/products/gilson-distritip-syringes-6/f164100g?keyword=true</td>
		</tr>
		<tr>
			<td>Data Analysis</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 
			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 
			https://jupyter.org/</td>
		</tr>
		<tr>
			<td>Rstudio</td>
			<td>Open-source</td>
			<td></td>
			<td>As refered to in text: Rstudio 
			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...

Watch this Scientific Journal Video about Visualizing and Quantifying Pharmaceutical Compounds within Skin using Coherent Raman Scattering Imaging at JoVE.com

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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Pharmacokinetics: Drug Distribution and Protein Binding

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2.7K 조회수.  Massachusetts General Hospital, Harvard Medical School. 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.

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

Accurate and Simple Measurement of the Pro-inflammatory Cytokine IL-1&beta; using a Whole Blood Stimulation Assay

Inflammatory processes resulting from the secretion of soluble mediators by immune cells, lead to various manifestations in skin, joints and other tissues as well as altered cytokine homeostasis. The innate immune system plays a crucial role in recognizing pathogens and other endogenous danger stimuli. One of the major cytokines released by innate immune cells is Interleukin (IL)-1. Therefore, we utilize a whole blood stimulation assay in order to measure the secretion of inflammatory cytokines and specifically of the pro-inflammatory cytokine IL-1&beta; 1, 2, 3.
Patients with genetic dysfunctions of the innate immune system causing autoinflammatory syndromes show an exaggerated release of mature IL-1&beta; upon stimulation with LPS alone. In order to evaluate the innate immune component of patients who present with inflammatory-associated pathologies, we use a specific immunoassay to detect cellular immune responses to pathogen-associated molecular patterns (PAMPs), such as the gram-negative bacterial endotoxin, lipopolysaccharide (LPS). These PAMPs are recognized by pathogen recognition receptors (PRRs), which are found on the cells of the innate immune system 4, 5, 6, 7. A primary signal, LPS, in conjunction with a secondary signal, ATP, is necessary for the activation of the inflammasome, a multiprotein complex that processes pro-IL-1&beta; to its mature, bioactive form 4, 5, 6, 8, 9, 10.
The whole blood assay requires minimal sample manipulation to assess cytokine production when compared to other methods that require labor intensive isolation and culturing of specific cell populations. This method differs from other whole blood stimulation assays; rather than diluting samples with a ratio of RPMI media, we perform a white blood cell count directly from diluted whole blood and therefore, stimulate a known number of white blood cells in culture 2. The results of this particular whole blood assay demonstrate a novel technique useful in elucidating patient cohorts presenting with autoinflammatory pathophysiologies.

Accurate and Simple Measurement of the Pro-inflammatory Cytokine IL-1&#946; using a Whole Blood Stimulation Assay

We describe a simple immunoassay to measure the production of pro-inflammatory cytokines, such as IL-1 beta production, in patients presenting with autoinflammatory phenotypes. By activating cells in whole blood cultures with pathogen-associated molecular patterns, specifically with lipopolysaccharide, cytokine secretion can be conveniently evaluated in whole blood supernatants.

전체 혈액 자극 분석을 사용하여 프로 염증성 시토킨 IL - 1β의 정확하고 간단한 측정

Inflammatory processes resulting from the secretion of soluble mediators by immune cells, lead to various manifestations in skin, joints and other tissues ...

연구

의학

Quantitative Visualization and Detection of Skin Cancer Using Dynamic Thermal Imaging

In 2010 approximately 68,720 melanomas will be diagnosed in the US alone, with around 8,650 resulting in death 1. To date, the only effective treatment for melanoma remains surgical excision, therefore, the key to extended survival is early detection 2,3. Considering the large numbers of patients diagnosed every year and the limitations in accessing specialized care quickly, the development of objective in vivo diagnostic instruments to aid the diagnosis is essential. New techniques to detect skin cancer, especially non-invasive diagnostic tools, are being explored in numerous laboratories. Along with the surgical methods, techniques such as digital photography, dermoscopy, multispectral imaging systems (MelaFind), laser-based systems (confocal scanning laser microscopy, laser doppler perfusion imaging, optical coherence tomography), ultrasound, magnetic resonance imaging, are being tested. Each technique offers unique advantages and disadvantages, many of which pose a compromise between effectiveness and accuracy versus ease of use and cost considerations. Details about these techniques and comparisons are available in the literature 4.
Infrared (IR) imaging was shown to be a useful method to diagnose the signs of certain diseases by measuring the local skin temperature. There is a large body of evidence showing that disease or deviation from normal functioning are accompanied by changes of the temperature of the body, which again affect the temperature of the skin 5,6. Accurate data about the temperature of the human body and skin can provide a wealth of information on the processes responsible for heat generation and thermoregulation, in particular the deviation from normal conditions, often caused by disease. However, IR imaging has not been widely recognized in medicine due to the premature use of the technology 7,8 several decades ago, when temperature measurement accuracy and the spatial resolution were inadequate and sophisticated image processing tools were unavailable. This situation changed dramatically in the late 1990s-2000s. Advances in IR instrumentation, implementation of digital image processing algorithms and dynamic IR imaging, which enables scientists to analyze not only the spatial, but also the temporal thermal behavior of the skin 9, allowed breakthroughs in the field.
In our research, we explore the feasibility of IR imaging, combined with theoretical and experimental studies, as a cost effective, non-invasive, in vivo optical measurement technique for tumor detection, with emphasis on the screening and early detection of melanoma 10-13. In this study, we show data obtained in a patient study in which patients that possess a pigmented lesion with a clinical indication for biopsy are selected for imaging. We compared the difference in thermal responses between healthy and malignant tissue and compared our data with biopsy results. We concluded that the increased metabolic activity of the melanoma lesion can be detected by dynamic infrared imaging.

We demonstrated that malignant pigmented lesions with increased metabolic activity generate quantifiable amounts of heat and the measurement of the transient thermal response of the skin to a cooling excitation allows quantitative identification of melanoma and other skin cancers (vs. non-proliferative nevi) at an early stage of the disease.

양적 시각화 및 동적 써멀 이미징을 사용하여 피부암의 감지

In 2010 approximately 68,720 melanomas will be diagnosed in the US alone, with around 8,650 resulting in death 1. To date, the only effective treatment ...

MALDI Imaging Mass Spectrometry of Neuropeptides in Parkinson's Disease

MALDI imaging mass spectrometry (IMS) is a powerful approach that facilitates the spatial analysis of molecular species in biological tissue samples2 (Fig.1). A 12 &mu;m thin tissue section is covered with a MALDI matrix, which facilitates desorption and ionization of intact peptides and proteins that can be detected with a mass analyzer, typically using a MALDI TOF/TOF mass spectrometer. Generally hundreds of peaks can be assessed in a single rat brain tissue section. In contrast to commonly used imaging techniques, this approach does not require prior knowledge of the molecules of interest and allows for unsupervised and comprehensive analysis of multiple molecular species while maintaining high molecular specificity and sensitivity2. Here we describe a MALDI IMS based approach for elucidating region-specific distribution profiles of neuropeptides in the rat brain of an animal model Parkinson's disease (PD). 
PD is a common neurodegenerative disease with a prevalence of 1% for people over 65 of age3,4. The most common symptomatic treatment is based on dopamine replacement using L-DOPA5. However this is accompanied by severe side effects including involuntary abnormal movements, termed L-DOPA-induced dyskinesias (LID)1,3,6. One of the most prominent molecular change in LID is an upregulation of the opioid precursor prodynorphin mRNA7. The dynorphin peptides modulate neurotransmission in brain areas that are essentially involved in movement control7,8. However, to date the exact opioid peptides that originate from processing of the neuropeptide precursor have not been characterized. Therefore, we utilized MALDI IMS in an animal model of experimental Parkinson's disease and L-DOPA induced dyskinesia. 
MALDI imaging mass spectrometry proved to be particularly advantageous with respect to neuropeptide characterization, since commonly used antibody based approaches targets known peptide sequences and previously observed post-translational modifications. By contrast MALDI IMS can unravel novel peptide processing products and thus reveal new molecular mechanisms of neuropeptide modulation of neuronal transmission. While the absolute amount of neuropeptides cannot be determined by MALDI IMS, the relative abundance of peptide ions can be delineated from the mass spectra, giving insights about changing levels in health and disease. In the examples presented here, the peak intensities of dynorphin B, alpha-neoendorphin and substance P were found to be significantly increased in the dorsolateral, but not the dorsomedial, striatum of animals with severe dyskinesia involving facial, trunk and orolingual muscles (Fig. 5). Furthermore, MALDI IMS revealed a correlation between dyskinesia severity and levels of des-tyrosine alpha-neoendorphin, representing a previously unknown mechanism of functional inactivation of dynorphins in the striatum as the removal of N-terminal tyrosine reduces the dynorphin's opioid-receptor binding capacity9. This is the first study on neuropeptide characterization in LID using MALDI IMS and the results highlight the potential of the technique for application in all fields of biomedical research.

Dopamine replacement pharmacotherapy using L-DOPA is the most commonly used symptomatic treatment of Parkinson&#x2019;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.

파킨슨병의 Neuropeptides의 든 이미징 질량 분광법

MALDI imaging mass spectrometry (IMS) is a powerful approach that facilitates the spatial analysis of molecular species in biological tissue samples2 ...

Three-dimensional Imaging of Nociceptive Intraepidermal Nerve Fibers in Human Skin Biopsies

A punch biopsy of the skin is commonly used to quantify intraepidermal nerve fiber densities (IENFD) for the diagnosis of peripheral polyneuropathy 1,2. At present, it is common practice to collect 3 mm skin biopsies from the distal leg (DL) and the proximal thigh (PT) for the evaluation of length-dependent polyneuropathies 3. However, due to the multidirectional nature of IENFs, it is challenging to examine overlapping nerve structures through the analysis of two-dimensional (2D) imaging. Alternatively, three-dimensional (3D) imaging could provide a better solution for this dilemma.
In the current report, we present methods for applying 3D imaging to study painful neuropathy (PN). In order to identify IENFs, skin samples are processed for immunofluorescent analysis of protein gene product 9.5 (PGP), a pan neuronal marker. At present, it is standard practice to diagnose small fiber neuropathies using IENFD determined by PGP immunohistochemistry using brightfield microscopy 4. In the current study, we applied double immunofluorescent analysis to identify total IENFD, using PGP, and nociceptive IENF, through the use of antibodies that recognize tropomyosin-receptor-kinase A (Trk A), the high affinity receptor for nerve growth factor 5. The advantages of co-staining IENF with PGP and Trk A antibodies benefits the study of PN by clearly staining PGP-positive, nociceptive fibers. These fluorescent signals can be quantified to determine nociceptive IENFD and morphological changes of IENF associated with PN. The fluorescent images are acquired by confocal microscopy and processed for 3D analysis. 3D-imaging provides rotational abilities to further analyze morphological changes associated with PN. Taken together, fluorescent co-staining, confocal imaging, and 3D analysis clearly benefit the study of PN.

In order to study the changes of nociceptive intraepidermal nerve fibers (IENFs) in painful neuropathies (PN), we developed protocols that could directly examine three-dimensional morphological changes observed in nociceptive IENFs. Three-dimensional analysis of IENFs has the potential to evaluate the morphological changes of IENF in PN.

인간의 피부 생검에서 통각 Intraepidermal 신경 섬유의 3 차원 영상

A punch biopsy of the skin is commonly used to quantify intraepidermal nerve fiber densities (IENFD) for the diagnosis of peripheral polyneuropathy 1,2. ...

Cerenkov Luminescence Imaging of Interscapular Brown Adipose Tissue

Brown adipose tissue (BAT), widely known as a &#8220;good fat&#8221; plays pivotal roles for thermogenesis in mammals. This special tissue is closely related to metabolism and energy expenditure, and its dysfunction is one important contributor for obesity and diabetes. Contrary to previous belief, recent PET/CT imaging studies indicated the BAT depots are still present in human adults. PET imaging clearly shows that BAT has considerably high uptake of 18F-FDG under certain conditions. In this video report, we demonstrate that Cerenkov luminescence imaging (CLI) with 18F-FDG can be used to optically image BAT in small animals. BAT activation is observed after intraperitoneal injection of norepinephrine (NE) and cold treatment, and depression of BAT is induced by long anesthesia. Using multiple-filter Cerenkov luminescence imaging, spectral unmixing and 3D imaging reconstruction are demonstrated. Our results suggest that CLI with 18F-FDG is a practical technique for imaging BAT in small animals, and this technique can be used as a cheap, fast, and alternative imaging tool for BAT research.

In this video report, we show the application of Cerenkov Luminescence Imaging (CLI) for interscapular brown adipose tissue in mice under activated and depressed conditions.

견갑골 갈색 지방 조직의 Cerenkov 발광 이미징

Brown adipose tissue (BAT), widely known as a &#8220;good fat&#8221; plays pivotal roles for thermogenesis in mammals. This special tissue is closely ...

Povidone Iodine Rectal Preparation at Time of Prostate Needle Biopsy is a Simple and Reproducible Means to Reduce Risk of Procedural Infection

Single institution and population-based studies highlight that infectious complications following transrectal ultrasound guided prostate needle biopsy (TRUS PNB) are increasing. Such infections are largely attributable to quinolone resistant microorganisms which colonize the rectal vault and are translocated into the bloodstream during the biopsy procedure. A povidone iodine rectal preparation (PIRP) at time of biopsy is a simple, reproducible method to reduce rectal microorganism colony counts and therefore resultant infections following TRUS PNB.
All patients are administered three days of oral antibiotic therapy prior to biopsy. The PIRP technique involves initially positioning the patient in the standard manner for a TRUS PNB. Following digital rectal examination, 15 ml&#160;of a 10% solution of commercially available povidone iodine is mixed with 5 ml&#160;of 1% lidocaine jelly to create slurry. A 4 cm x 4 cm sterile gauze is soaked in this slurry and then inserted into the rectal vault for 2 min after which it is removed. Thereafter, a disposable cotton gynecologic swab is used to paint both the perianal area and the rectal vault to a distance of 3 cm from the anus. The povidone iodine solution is then allowed to dry for 2 - 3 min prior to proceeding with standard transrectal ultrasonography and subsequent biopsy.
This PIRP technique has been in practice at our institution since March of 2012 with an associated reduction of post-biopsy infections from 4.3% to 0.6% (p = 0.02). The principal advantage of this prophylaxis regimen is its simplicity and reproducibility with use of an easily available, inexpensive agent to reduce infections. Furthermore, the technique avoids exposing patients to additional systemic antibiotics with potential further propagation of multi-drug resistant organisms. Usage of PIRP at TRUS PNB, however, is not applicable for patients with iodine or shellfish allergies.

Here, we present a protocol for use at the time of transrectal ultrasound guided prostate needle biopsy (TRUS PNB) that is a simple and cost-effective means to reduce infections following the procedure.

전립선 바늘 생검시 포비돈 요오드 직장 준비 절차 감염의 위험을 줄이기 위해 간단하고 재현 수단이다

Single institution and population-based studies highlight that infectious complications following transrectal ultrasound guided prostate needle biopsy ...

Combined Near-infrared Fluorescent Imaging and Micro-computed Tomography for Directly Visualizing Cerebral Thromboemboli

Direct thrombus imaging visualizes the root cause of thromboembolic infarction. Being able to image thrombus directly allows far better investigation of stroke than relying on indirect measurements, and will be a potent and robust vascular research tool. We use an optical imaging approach that labels thrombi with a molecular imaging thrombus marker&#160;&#8212; a Cy5.5 near-infrared fluorescent (NIRF) probe that is covalently linked to the fibrin strands of the thrombus by the fibrin-crosslinking enzymatic action of activated coagulation factor XIIIa during the process of clot maturation. A micro-computed tomography (microCT)-based approach uses thrombus-seeking gold nanoparticles (AuNPs) functionalized to target the major component of the clot: fibrin. This paper describes a detailed protocol for the combined in vivo microCT and ex vivo NIRF imaging of thromboemboli in a mouse model of embolic stroke. We show that in vivo microCT and fibrin-targeted glycol-chitosan AuNPs (fib-GC-AuNPs) can be used for visualizing both in situ thrombi and cerebral embolic thrombi. We also describe the use of in vivo microCT-based direct thrombus imaging to serially monitor the therapeutic effects of tissue plasminogen activator-mediated thrombolysis. After the last imaging session, we demonstrate by ex vivo NIRF imaging the extent and the distribution of residual thromboemboli in the brain. Finally, we describe quantitative image analyses of microCT and NIRF imaging data. The combined technique of direct thrombus imaging allows two independent methods of thrombus visualization to be compared: the area of thrombus-related fluorescent signal on ex vivo NIRF imaging vs. the volume of hyperdense microCT thrombi in vivo.

This protocol describes the application of combined near-infrared fluorescent (NIRF) imaging and micro-computed tomography (microCT) for visualizing cerebral thromboemboli. This technique allows the quantification of thrombus burden and evolution. The NIRF imaging technique visualizes fluorescently labeled thrombus in excised brain, while the microCT technique visualizes thrombus inside living animals using gold-nanoparticles.

직접 대뇌 Thromboemboli 시각화를위한 결합 된 근적외선 형광 이미징 및 마이크로 계산 된 단층 촬영

Direct thrombus imaging visualizes the root cause of thromboembolic infarction. Being able to image thrombus directly allows far better investigation of ...

Using Multi-fluorinated Bile Acids and In Vivo Magnetic Resonance Imaging to Measure Bile Acid Transport

Along with their traditional role as detergents that facilitate fat absorption, emerging literature indicates that bile acids are potent signaling molecules that affect multiple organs; they modulate gut motility and hormone production, and alter vascular tone, glucose metabolism, lipid metabolism, and energy utilization. Changes in fecal bile acids may alter the gut microbiome and promote colon pathology including cholerrheic diarrhea and colon cancer. Key regulators of fecal bile acid composition are the small intestinal Apical Sodium-dependent Bile Acid Transporter (ASBT) and fibroblast growth factor-19 (FGF19). Reduced expression and function of ASBT decreases intestinal bile acid up-take. Moreover, in vitro data suggest that some FDA-approved drugs inhibit ASBT function. Deficient FGF19 release increases hepatic bile acid synthesis and release into the intestines to levels that overwhelm ASBT. Either ASBT dysfunction or FGF19 deficiency increases fecal bile acids and may cause chronic diarrhea and promote colon neoplasia. Regrettably, tools to measure bile acid malabsorption and the actions of drugs on bile acid transport in vivo are limited. To understand the complex actions of bile acids, techniques are required that permit simultaneous monitoring of bile acids in the gut and metabolic tissues. This led us to conceive an innovative method to measure bile acid transport in live animals using a combination of proton (1H) and fluorine (19F) magnetic resonance imaging (MRI). Novel tracers for fluorine (19F)-based live animal MRI were created and tested, both in vitro and in vivo. Strengths of this approach include the lack of exposure to ionizing radiation and translational potential for clinical research and practice.

Using Multi-fluorinated Bile Acids and In Vivo Magnetic Resonance Imaging to Measure Bile Acid Transport

Tools to diagnose bile acid malabsorption and measure bile acid transport in vivo are limited. An innovative approach in live animals is described that utilizes combined proton (1H) plus fluorine (19F) magnetic resonance imaging; this novel methodology has translational potential to screen for bile acid malabsorption in clinical practice.

멀티 플루오르 담즙 산 및 사용<em&gt; 생체</em&gt; 자기 공명 영상은 측정하는 담즙 산 전송

Along with their traditional role as detergents that facilitate fat absorption, emerging literature indicates that bile acids are potent signaling ...

Quantitative Visualization of Leukocyte Infiltrate in a Murine Model of Fulminant Myocarditis by Light Sheet Microscopy

Light-sheet fluorescence microscopy (LSFM), in combination with chemical clearing protocols, has become the gold standard for analyzing fluorescently labelled structures in large biological specimens, and is down to cellular resolution. Meanwhile, the constant refinement of underlying protocols and the enhanced availability of specialized commercial systems enable us to investigate the microstructure of whole mouse organs and even allow for the characterization of cellular behavior in various live-cell imaging approaches. Here, we describe a protocol for the spatial whole-mount visualization and quantification of the CD45+ leukocyte population in inflamed mouse hearts. The method employs a transgenic mouse strain (CD11c.DTR)that has recently been shown to serve as a robust, inducible model for the study of the development of fulminant fatal myocarditis, characterized by lethal cardiac arrhythmias. This protocol includes myocarditis induction, intravital antibody-mediated cell staining, organ preparation, and LSFM with subsequent computer-assisted image post-processing. Although presented as a highly-adapted method for our particular scientific question, the protocol represents the blueprint of an easily adjustable system that can also target completely different fluorescent structures in other organs and even in other species.

Here, we describe a light-sheet microscopy approach to visualize the cardiac CD45+ leukocyte infiltrate in a murine model of aseptic fulminant myocarditis, which is induced by the intratracheal diphtheria toxin treatment of CD11c.DTR mice.

Light Sheet Microscopy에 의한 심근 성 심근염의 Murine 모델에서의 백혈구 침윤의 정량적 시각화

Light-sheet fluorescence microscopy (LSFM), in combination with chemical clearing protocols, has become the gold standard for analyzing fluorescently ...

Rapid Evaluation of Toxicity of Chemical Compounds Using Zebrafish Embryos

The zebrafish is a widely used vertebrate model organism for the disease and phenotype-based drug discovery. The zebrafish generates many offspring, has transparent embryos and rapid external development. Zebrafish embryos can, therefore, also be used for the rapid evaluation of toxicity of the drugs that are precious and available in small quantities. In the present article, a method for the efficient screening of the toxicity of chemical compounds using 1-5-day post fertilization embryos is described. The embryos are monitored by stereomicroscope to investigate the phenotypic defects caused by the exposure to different concentrations of compounds. Half-maximal lethal concentrations (LC50) of the compounds are also determined. The present study required 3-6 mg of an inhibitor compound, and the whole experiment takes about 8-10 h to be completed by an individual in a laboratory having basic facilities. The current protocol is suitable for testing any compound to identify intolerable toxic or off-target effects of the compound in the early phase of drug discovery and to detect subtle toxic effects that may be missed in the cell culture or other animal models. The method reduces procedural delays and costs of drug development.

Zebrafish embryos are used for evaluating the toxicity of chemical compounds. They develop externally and are sensitive to chemicals, allowing detection of subtle phenotypic changes. The experiment only requires a small amount of compound, which is directly added to the plate containing embryos, making the testing system efficient and cost-effective.

Zebrafish 배아를 이용한 화학 화합물의 독성에 대한 신속한 평가

The zebrafish is a widely used vertebrate model organism for the disease and phenotype-based drug discovery. The zebrafish generates many offspring, has ...