The work wishes to acknowledge support from the National Natural Science Foundation of China (No. 82173976), National Key Research and Development Program (No. 2018YFC1707205), and State Key Laboratory of Innovative Drugs and High Energy Saving Pharmaceutical Equipment, Jiangxi University of Traditional Chinese Medicine (No. GZSYS202003).

1. Preparation of test sample
<ol>
	<li>Set up four different batches of Indigo Naturalis herbal powder for the test. Pass each sample through the seventh sieve and ninth sieve in turn and collect the sample between the seventh and ninth sieve5. 
	NOTE: The average aperture size of the seventh sieve is 125 &#181;m &#177; 5.8 &#181;m. The average aperture size of the ninth sieve is 75 &#181;m &#177; 4.1 &#181;m.</li>
	<li>Accurately weigh 0.2 g of the sample (Indigo Naturalis powder) on a weighing paper and set it aside.</li>
</ol>
2. Sample addition
<ol>
	<li>Build an iron support stand and put an iron ring with a 5 cm diameter funnel on it.</li>
	<li>Use a pipette to add 20 mL of pure water to the sample glass bottle (bottom diameter 2.6 cm, height 6 cm). Place the sample glass bottle directly under the funnel so that the lower edge of the funnel is flush with the mouth of the bottle. 
	NOTE: Clean the exterior of the sample glass vial with a clean, non-abrasive paper towel and inspect the glass surface for visible marks. If there are, change the glass bottle. Be careful not to spill when adding liquid.</li>
	<li>Release the sample at a height of 80 cm from the lower edge of the funnel so that it can slide freely along the funnel into the sample bottle.</li>
</ol>
3. Instrument operation
<ol>
	<li>Turn on the Turbiscan Lab instrument and warm it up for 30 min.</li>
	<li>Create the file. Click the Create file button in the top menu (or the New file function in the File menu) to create a new empty measurement file. Define its name and save location (by default, the data folder is located at: &#34;C:/users/admin/Formulaction/FAnalyser/Data&#34;.</li>
	<li>Click the Show Turbiscan Lab Temperature button in the top menu to set the instrument target temperature to 25 &#176;C. 
	NOTE: The instrument temperature is influenced by the room temperature, so be careful to adjust the ambient temperature.</li>
	<li>Click Program Scan in the top menu to enter the setup analysis program. Add the program to the list, and in the taskbar, add 30 s as a cycle and 21 scans to the analysis sequence. Select this analysis program for all subsequent measurements.</li>
	<li>Move the prepared sample vial into the measurement system. After setting up the program, click Start to start the measurement. 
	NOTE: Be careful not to shake the glass bottle when moving and only move it slightly.</li>
	<li>After data acquisition, click on the list of calculated parameters to automatically calculate TSI.</li>
</ol>

<ol>
	<li>Li, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+application+of+water+test+in+the+identification+of+traditional+Chinese+medicine.">The application of water test in the identification of traditional Chinese medicine.</a> Chinese Medicine Modern Distance Education of China. 19 (23), 153-155 (2021).</li><li>Ye, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+application+effect+of+water+test+method+in+identification+of+traditional+Chinese+medicine.">Analysis of application effect of water test method in identification of traditional Chinese medicine.</a> Heilongjiang Medicine Journal. 33 (02), 283-285 (2020).</li><li>Yang, Q. 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=From+natural+dye+to+herbal+medicine:+a+systematic+review+of+chemical+constituents,+pharmacological+effects+and+clinical+applications+of+indigo+naturalis.">From natural dye to herbal medicine: a systematic review of chemical constituents, pharmacological effects and clinical applications of indigo naturalis.</a> Chinese Medicine. 15 (1), 127 (2020).</li><li>Chen, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+application+value+of+water+test+method+in+the+identification+of+Chinese+medicine.">The application value of water test method in the identification of Chinese medicine.</a> Journal of Traditional Chinese Medicine Management. 30 (10), 133-134 (2022).</li><li>Liu, X. 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=Establishment+and+application+of+a+rapid+quality+inspection+method+for+Indigo+Naturalis+based+on+quantitative+portrayal+of+water+testing+process.">Establishment and application of a rapid quality inspection method for Indigo Naturalis based on quantitative portrayal of water testing process.</a> Acta Pharmacologica Sinica. 57 (11), 3411-3418 (2022).</li><li>Mengual, O., Meunier, G., Cayre, I., Puech, K., Snabre, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterisation+of+instability+of+concentrated+dispersions+by+a+new+optical+analyser:+the+TURBISCAN+MA+1000.">Characterisation of instability of concentrated dispersions by a new optical analyser: the TURBISCAN MA 1000.</a> Colloids and Surfaces A: Physicochemical and Engineering Aspects. 152 (1), 111-123 (1999).</li><li>Olatunji, O. N., Du, J., Hintz, W., Tomas, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+particle+sedimentation+analysis+in+sterically-stabilized+TiO2+particles+stability+assessment.">Application of particle sedimentation analysis in sterically-stabilized TiO2 particles stability assessment.</a> Advanced Powder Technology. 27 (4), 1325-1336 (2016).</li><li>Ferrentino, 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=Fining+of+red+wine+monitored+by+multiple+light+scattering.">Fining of red wine monitored by multiple light scattering.</a> Journal of Agricultural and Food Chemistry. 65 (27), 5523-5530 (2017).</li><li>Ramezani, M., Ferrentino, G., Morozova, K., Scampicchio, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+light+scattering+measurements+for+online+monitoring+of+milk+fermentation.">Multiple light scattering measurements for online monitoring of milk fermentation.</a> Foods. 10 (7), 1582 (2021).</li><li>Yang, H. B., 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=A+new+approach+to+evaluate+the+particle+growth+and+sedimentation+of+dispersed+polymer+microsphere+profile+control+system+based+on+multiple+light+scattering.">A new approach to evaluate the particle growth and sedimentation of dispersed polymer microsphere profile control system based on multiple light scattering.</a> Powder Technology. 315, 477-485 (2017).</li><li>Zhang, X. X., 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=Treatment+of+non-high-risk+acute+promyelocytic+leukemia+with+realgar-indigo+naturalis+formula+(RIF)+and+all-trans+retinoid+acid+(ATRA):+study+protocol+for+a+randomized+controlled+trial.">Treatment of non-high-risk acute promyelocytic leukemia with realgar-indigo naturalis formula (RIF) and all-trans retinoid acid (ATRA): study protocol for a randomized controlled trial.</a> Trials. 21 (1), 7 (2020).</li><li>Sugimoto, 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=Clinical+efficacy+and+safety+of+oral+Qing-Dai+in+patients+with+ulcerative+colitis:+a+single-center+open-label+prospective+study.">Clinical efficacy and safety of oral Qing-Dai in patients with ulcerative colitis: a single-center open-label prospective study.</a> Digestion. 93 (3), 193-201 (2016).</li><li>Lin, Y. K., 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=Clinical+assessment+of+patients+with+recalcitrant+psoriasis+in+a+randomized,+observer-blind,+vehicle-controlled+trial+using+indigo+naturalis.">Clinical assessment of patients with recalcitrant psoriasis in a randomized, observer-blind, vehicle-controlled trial using indigo naturalis.</a> Archives of Dermatology. 144 (11), 1457-1464 (2008).</li><li>Sun, Q., Leng, J., Tang, L., Wang, L., Fu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Comprehensive+review+of+the+chemistry,+pharmacokinetics,+pharmacology,+clinical+applications,+adverse+events,+and+quality+control+of+indigo+naturalis.">A Comprehensive review of the chemistry, pharmacokinetics, pharmacology, clinical applications, adverse events, and quality control of indigo naturalis.</a> Frontiers in Pharmacology. 12, 664022 (2021).</li><li>Yang, Y. 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=Investigation+and+analysis+of+the+commodity+quality+of+Indigo+Naturalis+herbs+in+Beijing+area.">Investigation and analysis of the commodity quality of Indigo Naturalis herbs in Beijing area.</a> Lishizhen Medicine and Materia Medica Research. 23 (07), 1787-1788 (2012).</li><li>Yao, Z. 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=Comparative+study+of+thirty-eight+batches+of+indigo+naturalis.">Comparative study of thirty-eight batches of indigo naturalis.</a> Journal of Chengdu University of TCM. 34 (02), 86-88 (2011).</li><li>Bai, Z., 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=Determination+of+indigo+and+indirubin+in+indigo+naturalis+by+HPLC.">Determination of indigo and indirubin in indigo naturalis by HPLC.</a> Modern Chinese Medicine. 12 (08), 27-29 (2010).</li></ol>

The authors have no conflicts of interest to disclose.

According to traditional Chinese medicine, Indigo Naturalis has the power to clear heat and detoxification, cool blood, eliminate spots, purge fire, and arrest convulsion. Based on randomized double-blind controlled clinical trials11,12,13, Indigo Naturalis is effective in the treatment of psoriasis, ulcerative colitis, and acute promyelocytic leukemia in addition to its traditional treatment of cough and phlegm, hemorrhagic sym...

In traditional Chinese medicine, during the course of disease treatment, the clinical effectiveness of medications and the safety of the course of treatment are directly impacted by the quality of Chinese herbal medicine. Utilizing cutting-edge identification technology, one can assess the efficacy of Chinese herbal medicine and guarantee user safety. The Chinese herbal medicine water test method refers to immersing the herbs in water or solvent, then quickly and precisely determining the medicine&#39;s authenticity by observing the changes in color, size, and shape1.
It was originally a good choice for the identification of Chinese medicine. However, the disadvantage of the traditional water test method is that the accuracy and sensitivity of distinguishing the authenticity of Chinese medicine are low because of the subjectivity of observation by the naked eye2. One of the key medicinal materials used in the water test method is Indigo Naturalis, considered an effective remedy for pediatric hyperthermia, psoriasis, leukemia, and ulcerative colitis3. The genuine Indigo Naturalis floats on the surface of water, and the water does not turn dark blue after shaking. However, the fake Indigo Naturalis has particles that sink, and the water will turn dark blue after shaking4. Its principle is due to hydrophobic and easily floatable indigo, indirubin, and other organic components of high-quality Indigo Naturalis. On the contrary, due to low organic matter, a large amount of lime, and heavy texture, some particles doped with fake Indigo Naturalis will sink quickly5. However, this method is only a simple qualitative identification, and it limits the rapid identification of the authenticity of Chinese herbal medicine and fails to reveal the changes of Indigo Naturalis in water.
Multiple light scattering technology is a technology that can measure multi-angle light signal scanning based on a laser passing through the sample. The incident light will be scattered when it penetrates the sample or encounters particles. If the scattered light penetrates through the sample, a transmission light signal is formed; If the sample concentration is high, the light will be reflected by the particles, forming a backscattering light signal. The changes in light intensity reflect the changes in particle concentration and particle size in liquid preparation6. The multiple light scattering instruments can quickly and accurately analyze phenomena such as emulsification, flocculation, precipitation, and rupture of suspension, emulsion, and foam liquid by multiple light scattering technology, as well as quantitatively analyze characteristics such as the rate of occurrence of the above phenomena.
Multiple light scattering technology has demonstrated significant benefits in particle stability monitoring7, red wine clarification8, and milk fermentation quality control9. Using this technology, the traditional water test method of Indigo Naturalis may be intuitive, quantitative, and scientific. Therefore, based on the principle of multiple light scattering technology, this study established a rapid evaluation method for the quality of Indigo Naturalis, taking the Turbiscan stability index (TSI) of the sample as the index of quality control (Figure 1).

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Analytical balance (1/10,000)</td>
			<td>Sartorious, Germany</td>
			<td>BSA224S&#160;</td>
			<td>www.sartorius.com.cn</td>
		</tr>
		<tr>
			<td>Funnel</td>
			<td>Chengdu Kelong Chemical Co. LTD</td>
			<td>Diameter 5 cm</td>
			<td>www.cdkelongchem.com</td>
		</tr>
		<tr>
			<td>Indigo Naturalis S1</td>
			<td>Xianyou, Fujian</td>
			<td>20210501</td>
			<td></td>
		</tr>
		<tr>
			<td>Indigo Naturalis S2</td>
			<td>Yaan, Sichuan</td>
			<td>20201102</td>
			<td></td>
		</tr>
		<tr>
			<td>Indigo Naturalis S3</td>
			<td>Xianyou, Fujian</td>
			<td>20161012</td>
			<td></td>
		</tr>
		<tr>
			<td>Indigo Naturalis S4</td>
			<td>Xianyou, Fujian</td>
			<td>20180305</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;Iron ring</td>
			<td>Chengdu Kelong Chemical Co. LTD</td>
			<td>/</td>
			<td>www.cdkelongchem.com</td>
		</tr>
		<tr>
			<td>Iron stand</td>
			<td>Chengdu Kelong Chemical Co. LTD</td>
			<td>/</td>
			<td>www.cdkelongchem.com</td>
		</tr>
		<tr>
			<td>Mili-Q ultra-pure water meter</td>
			<td>Milipore, USA</td>
			<td>Mili-Q</td>
			<td>www.merckmillipore.com</td>
		</tr>
		<tr>
			<td>Ninth sieve</td>
			<td>Chengdu Kelong Chemical Co. LTD</td>
			<td>Average aperture size 75 &#181;m</td>
			<td>www.cdkelongchem.com</td>
		</tr>
		<tr>
			<td>Sample bottle</td>
			<td>French Formulaction Company</td>
			<td>Bottom diameter 2.6 cm, height 6 cm</td>
			<td>www.formulaction.com</td>
		</tr>
		<tr>
			<td>Seventh sieve</td>
			<td>Chengdu Kelong Chemical Co. LTD</td>
			<td>Average aperture size 125 &#181;m</td>
			<td>www.cdkelongchem.com</td>
		</tr>
		<tr>
			<td>Turbisoft Lab multiple light scattering instrument</td>
			<td>French Formulaction Company</td>
			<td>Turbisoft Lab 2.3.1.125 Fanalyser 1.3.5</td>
			<td>www.formulaction.com</td>
		</tr>
		<tr>
			<td>Weighing paper</td>
			<td>Chengdu Kelong Chemical Co. LTD</td>
			<td>/</td>
			<td>www.cdkelongchem.com</td>
		</tr>
	</tbody>
</table>

fast inspection of quality of indigo naturalis by multiple light scattering

Quality control of Chinese herbal medicine is a crucial component of Chinese herbal medicine research and development. Faced with the challenges of modernization and internationalization of Chinese herbal medicine, it is urgent to establish thorough and effective procedures for quality identification of Chinese herbal medicine, and there is an urgent need for new analytical and testing techniques that are efficient, accurate, and environmentally friendly. 
Multiple light scattering is a cutting-edge and analytical method that can accurately and rapidly assess the quality of Chinese herbal medicine without altering the nature or state of the sample or using organic reagents. Indigo Naturalis is considered a good remedy for pediatric hyperthermia, psoriasis, leukemia, and ulcerative colitis. In this study, the process of addition of Indigo Naturalis powder in water was recorded precisely using a multiple light scattering instrument. 
The qualitative and quantitative measurements of the instrument can be used to accurately capture the overall trajectory and sinking behavior of Indigo Naturalis powder into water and to establish a rapid evaluation method for the quality of Indigo Naturalis with the transmission and backscattering spectrograms of the sample as qualitative indicators and stability index as a quantitative indicator. The analytical technique based on multiple light scattering provides a fast, accurate, green, and environmentally friendly method for the quality evaluation of Indigo Naturalis and supports the development and transformation of high-quality Indigo Naturalis.

Figure 2A-D correspond to S1, S2, S3, and S4 of Indigo Naturalis, respectively. A is of high-quality Indigo Naturalis, showing the same light transmittance at any height within 0-10 min, which is very stable. B is the common Indigo Naturalis, and its light transmittance fluctuates slightly with the change of time and is generally stable. C and D are fake and inferior products. There may be two conditions in the transmission spectrograms of fake Indigo Natura...

Watch this Scientific Journal Video about Fast Inspection of Quality of Indigo Naturalis by Multiple Light Scattering at JoVE.com

Fast Inspection of Quality of Indigo Naturalis by Multiple Light Scattering

Here, we present a quality evaluation method based on a multiple light scattering technique to evaluate the quality of Indigo Naturalis.

Quality control of Chinese herbal medicine is a crucial component of Chinese herbal medicine research and development. Faced with the challenges of ...

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340 Views.  Chengdu University of Traditional Chinese Medicine. Here, we present a quality evaluation method based on a multiple light scattering technique to evaluate the quality of Indigo Naturalis.

Video: Fast Inspection of Quality of Indigo Naturalis by Multiple Light Scattering

Fast and Accurate Exhaled Breath Ammonia Measurement

This exhaled breath ammonia method uses a fast and highly sensitive spectroscopic method known as quartz enhanced photoacoustic spectroscopy (QEPAS) that uses a quantum cascade based laser. The monitor is coupled to a sampler that measures mouth pressure and carbon dioxide. The system is temperature controlled and specifically designed to address the reactivity of this compound. The sampler provides immediate feedback to the subject and the technician on the quality of the breath effort. Together with the quick response time of the monitor, this system is capable of accurately measuring exhaled breath ammonia representative of deep lung systemic levels. 
Because the system is easy to use and produces real time results, it has enabled experiments to identify factors that influence measurements. For example, mouth rinse and oral pH reproducibly and significantly affect results and therefore must be controlled. Temperature and mode of breathing are other examples. As our understanding of these factors evolves, error is reduced, and clinical studies become more meaningful. This system is very reliable and individual measurements are inexpensive. 
The sampler is relatively inexpensive and quite portable, but the monitor is neither. This limits options for some clinical studies and provides rational for future innovations.

Ammonia is an important physiologic metabolite relevant to various disease and wellness states. It is also a difficult molecule to measure in breath, which demands particular precautions be taken to obtain accurate results. Not all factors influencing ammonia are known, but progress can be difficult without accounting for these factors.

This exhaled breath ammonia method uses a fast and highly sensitive spectroscopic method known as quartz enhanced photoacoustic spectroscopy (QEPAS) that ...

A Protocol for the Use of Remotely-Supervised Transcranial Direct Current Stimulation (tDCS) in Multiple Sclerosis (MS)

Transcranial direct current stimulation (tDCS) is a noninvasive brain stimulation technique that uses low amplitude direct currents to alter cortical excitability. With well-established safety and tolerability, tDCS has been found to have the potential to ameliorate symptoms such as depression and pain in a range of conditions as well as to enhance outcomes of cognitive and physical training. However, effects are cumulative, requiring treatments that can span weeks or months and frequent, repeated visits to the clinic. The cost in terms of time and travel is often prohibitive for many participants, and ultimately limits real-world access. 
Following guidelines for remote tDCS application, we propose a protocol that would allow remote (in-home) participation that uses specially-designed devices for supervised use with materials modified for patient use, and real-time monitoring through a telemedicine video conferencing platform. We have developed structured training procedures and clear, detailed instructional materials to allow for self- or proxy-administration while supervised remotely in real-time. The protocol is designed to have a series of checkpoints, addressing attendance and tolerability of the session, to be met in order to continue to the next step. The feasibility of this protocol was then piloted for clinical use in an open label study of remotely-supervised tDCS in multiple sclerosis (MS). This protocol can be widely used for clinical study of tDCS.

A Protocol for the Use of Remotely-Supervised Transcranial Direct Current Stimulation tDCS in Multiple Sclerosis MS

The goal of this pilot study is to describe a protocol for the remotely-supervised delivery of transcranial direct current stimulation (tDCS) so that the procedure maintains standards of in-clinic practice, including safety, reproducibility, and tolerability. The feasibility of this protocol was tested in participants with multiple sclerosis (MS).

Transcranial direct current stimulation (tDCS) is a noninvasive brain stimulation technique that uses low amplitude direct currents to alter cortical ...

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

3D Ultrasound Imaging: Fast and Cost-effective Morphometry of Musculoskeletal Tissue

The developmental goal of 3D ultrasound imaging (3DUS) is to engineer a modality to perform 3D morphological ultrasound analysis of human muscles. 3DUS images are constructed from calibrated freehand 2D B-mode ultrasound images, which are positioned into a voxel array. Ultrasound (US) imaging allows quantification of muscle size, fascicle length, and angle of pennation. These morphological variables are important determinants of muscle force and length range of force exertion. The presented protocol describes an approach to determine volume and fascicle length of m.&#160;vastus lateralis and m.&#160;gastrocnemius medialis. 3DUS facilitates standardization using 3D anatomical references. This approach provides a fast and cost-effective approach for quantifying 3D morphology in skeletal muscles. In healthcare and sports, information on the morphometry of muscles is very valuable in diagnostics and/or follow-up evaluations after treatment or training.

3D ultrasound imaging (3DUS) allows fast and cost-effective morphometry of musculoskeletal tissues. We present a protocol to measure muscle volume and fascicle length using 3DUS.

The developmental goal of 3D ultrasound imaging (3DUS) is to engineer a modality to perform 3D morphological ultrasound analysis of human muscles. 3DUS ...

Induction of Atherosclerotic Plaques Through Activation of Mineralocorticoid Receptors in Apolipoprotein E-deficient Mice

Atherosclerosis is due to a chronic inflammatory response affecting vascular endothelium and is promoted by several factors such as hypertension, dyslipidemia, and diabetes. To date, there is evidence to support a role for circulating aldosterone as a risk factor for the development of cardiovascular disease. Transgenic mouse models have been generated to study cellular and molecular processes leading to atherosclerosis. In this manuscript, we describe a protocol that takes advantage of continuous infusion of aldosterone in ApoE-/- mice and generates atherosclerotic plaques in the aortic root after 4 weeks of treatment. We, therefore, illustrate a method for quantification and characterization of atherosclerotic lesions at the aortic root level. The added value of aldosterone infusion is represented by the generation of atherosclerotic lesions rich in lipid and inflammatory cells after 4 weeks of treatment. We describe in detail the staining procedures to quantify lipid and macrophage content within the plaque. Notably, in this protocol, we perform heart tissue-embedding in OCT in order to preserve the antigenicity of cardiac tissue and facilitate detectability of antigens of interest. Analysis of the plaque phenotype represents a valid approach to study the pathophysiology of atherosclerosis development and to identify novel pharmacological targets for the development of anti-atherogenic drugs.

We describe a protocol to induce atherosclerosis in the aortic root of ApoE-/- mice fed with an atherogenic diet, through a continuous release of aldosterone. Methods to characterize plaque composition are also described.

Atherosclerosis is due to a chronic inflammatory response affecting vascular endothelium and is promoted by several factors such as hypertension, ...

Generation of Ventricular-Like HiPSC-Derived Cardiomyocytes and High-Quality Cell Preparations for Calcium Handling Characterization

Human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) provide a valuable human source for studying the basic science of calcium (Ca2+) handling and signaling pathways as well as high-throughput drug screening and toxicity assays. Herein, we provide a detailed description of the methodologies used to generate high-quality iPSC-CMs that can consistently reproduce molecular and functional characteristics across different cell lines. Additionally, a method is described to reliably assess their functional characterization through the evaluation of Ca2+ handling properties. Low oxygen (O2) conditions, lactate selection, and prolonged time in culture produce high-purity and high-quality ventricular-like cardiomyocytes. Similar to isolated adult rat cardiomyocytes (ARCMs), 3-month-old iPSC-CMs exhibit higher Ca2+ amplitude, faster rate of Ca2+ reuptake (decay-tau), and a positive lusitropic response to &#946;-adrenergic stimulation compared to day 30 iPSC-CMs. The strategy is technically simple, cost-effective, and reproducible. It provides a robust platform to model cardiac disease and for the large-scale drug screening to target Ca2+ handling proteins.

Here we describe and validate a method to consistently generate robust human induced pluripotent stem cell-derived cardiomyocytes and characterize their function. These techniques may help in developing mechanistic insight into signaling pathways, provide a platform for large-scale drug screening, and reliably model cardiac diseases.

Human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) provide a valuable human source for studying the basic science of calcium (Ca2+) ...

Using a Chemical Biopsy for Graft Quality Assessment

Kidney transplantation is a life-saving treatment for a large number of people with end-stage renal dysfunction worldwide. The procedure is associated with an increased survival rate and greater quality of patient&#8217;s life when compared to conventional dialysis. Regrettably, transplantology suffers from a lack of reliable methods for organ quality assessment. Standard diagnostic techniques are limited to macroscopic appearance inspection or invasive tissue biopsy, which do not provide comprehensive information about the graft. The proposed protocol aims to introduce solid phase microextraction (SPME) as an ideal analytical method for comprehensive metabolomics and lipidomic analysis of all low molecular compounds present in kidneys allocated for transplantation. The small size of the SPME probe enables performance of a chemical biopsy, which enables extraction of metabolites directly from the organ without any tissue collection. The minimum invasiveness of the method permits execution of multiple analyses over time: directly after organ harvesting, during its preservation, and immediately after revascularization at the recipient&#8217;s body. It is hypothesized that the combination of this novel sampling method with a high-resolution mass spectrometer will allow for discrimination of a set of characteristic compounds that could serve as biological markers of graft quality and indicators of possible development of organ dysfunction.

The protocol presents utilization of the chemical biopsy approach followed by comprehensive metabolomic and lipidomic analysis for quality assessment of kidney grafts allocated for transplantation.

Kidney transplantation is a life-saving treatment for a large number of people with end-stage renal dysfunction worldwide. The procedure is associated ...

Magnetic Resonance Imaging of Multiple Sclerosis at 7.0 Tesla

The overall goal of this article is to demonstrate a state-of-the-art ultrahigh field (UHF) magnetic resonance (MR) protocol of the brain at 7.0 Tesla in multiple sclerosis (MS) patients. MS is a chronic inflammatory, demyelinating, neurodegenerative disease that is characterized by white and gray matter lesions. Detection of spatially and temporally disseminated T2-hyperintense lesions by the use of MRI at 1.5 T and 3 T represents a crucial diagnostic tool in clinical practice to establish accurate diagnosis of MS based on the current version of the 2017 McDonald criteria. However, the differentiation of MS lesions from brain white matter lesions of other origins can sometimes be challenging due to their resembling morphology at lower magnetic field strengths (typically 3 T). Ultrahigh field MR (UHF-MR) benefits from increased signal-to-noise ratio and enhanced spatial resolution, both key to superior imaging for more accurate and definitive diagnoses of subtle lesions. Hence, MRI at 7.0 T has shown encouraging results to overcome the challenges of MS differential diagnosis by providing MS-specific neuroimaging markers (e.g., central vein sign, hypointense rim structures and differentiation of MS grey matter lesions). These markers and others can be identified by other MR contrasts other than T1 and T2 (T2*, phase, diffusion) and substantially improve the differentiation of MS lesions from those occurring in other neuroinflammatory conditions such as neuromyelitis optica and Susac syndrome. In this article, we describe our current technical approach to study cerebral white and grey matter lesions in MS patients at 7.0 T using different MR acquisition methods. The up-to-date protocol includes the preparation of the MR setup including the radio-frequency coils customized for UHF-MR, standardized screening, safety and interview procedures with MS patients, patient positioning in the MR scanner and acquisition of dedicated brain scans tailored for examining MS.

Here, we present a protocol to acquire magnetic resonance (MR) images of multiple sclerosis (MS) patient brains at 7.0 Tesla. The protocol includes preparation of the setup including the radio-frequency coils, standardized interview procedures with MS patients, subject positioning in the MR scanner and MR data acquisition.

The overall goal of this article is to demonstrate a state-of-the-art ultrahigh field (UHF) magnetic resonance (MR) protocol of the brain at 7.0 Tesla in ...

Quality-Controlled Sputum Analysis by Flow Cytometry

Sputum, widely used to study the cellular content and other microenvironmental features to understand the health of the lung, is traditionally analyzed using cytology-based methodologies. Its utility is limited because reading the slides is time-consuming and requires highly specialized personnel. Moreover, extensive debris and the presence of too many squamous epithelial cells (SECs), or cheek cells, often renders a sample inadequate for diagnosis. In contrast, flow cytometry allows for high-throughput phenotyping of cellular populations while simultaneously excluding debris and SECs.
The protocol presented here describes an efficient method to dissociate sputum into a single cell suspension, antibody stain and fix cellular populations, and acquire samples on a flow cytometric platform. A gating strategy that describes the exclusion of debris, dead cells (including SECs) and cell doublets is presented here. Further, this work also explains how to analyze viable, single sputum cells based on a cluster of differentiation (CD)45 positive and negative populations to characterize hematopoietic and epithelial lineage subsets. A quality control measure is also provided by identifying lung-specific macrophages as evidence that a sample is derived from the lung and is not saliva. Finally, it has been demonstrated that this method can be applied to different cytometric platforms by providing sputum profiles from the same patient analyzed on three flow cytometers; Navios EX, LSR II, and Lyric. Furthermore, this protocol can be modified to include additional cellular markers of interest. A method to analyze an entire sputum sample on a flow cytometric platform is presented here that makes sputum amenable for developing high-throughput diagnostics of lung disease.

This protocol describes an efficient method for dissociating sputum into a single cell suspension and the subsequent characterization of cellular subsets on standard flow cytometric platforms.

Sputum, widely used to study the cellular content and other microenvironmental features to understand the health of the lung, is traditionally analyzed ...

Interphase Fluorescence in situ Hybridization of Bone Marrow Smears of Multiple Myeloma

Fluorescence in situ hybridization (FISH) detection is an indispensable method in genetic risk stratification in multiple myeloma (MM), which is one of the most common hematological malignancies. The identifying characteristic of MM is accumulated malignant plasma cells in bone marrow. FISH reports for MM mainly focus on purified or identified clonal plasma cells, rather than all nucleated cells, by sorting with anti-CD138 magnetic beads or marking with cytoplasmic immunoglobulin light chain &#954; or &#955;. Bone marrow interphase nuclei are usually obtained from fresh bone marrow cells. However, satisfactory enrichment of plasma cell specimens requires large amounts of fresh heparin anti-coagulated bone marrow, which cannot be obtained in the case of difficult bone marrow extraction or a bone marrow dry tap. Herein, we establish a novel method to improve the success of FISH detection on stained or unstained bone marrow smears. Bone marrow smears are easier to obtain than anticoagulated bone marrow specimens.

Interphase Fluorescence in situ Hybridization of Bone Marrow Smears of Multiple Myeloma

Here, we present a protocol for improving the success of interphase fluorescence in situ hybridization detection on bone marrow smears from multiple myeloma patients.

Fluorescence in situ hybridization (FISH) detection is an indispensable method in genetic risk stratification in multiple myeloma (MM), which is one of ...

Fast Inspection of Quality of Indigo Naturalis by Multiple Light Scattering

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A Protocol for the Use of Remotely-Supervised Transcranial Direct Current Stimulation (tDCS) in Multiple Sclerosis (MS)

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3D Ultrasound Imaging: Fast and Cost-effective Morphometry of Musculoskeletal Tissue

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Generation of Ventricular-Like HiPSC-Derived Cardiomyocytes and High-Quality Cell Preparations for Calcium Handling Characterization

Using a Chemical Biopsy for Graft Quality Assessment

Magnetic Resonance Imaging of Multiple Sclerosis at 7.0 Tesla

Quality-Controlled Sputum Analysis by Flow Cytometry

Interphase Fluorescence in situ Hybridization of Bone Marrow Smears of Multiple Myeloma