Name: detection and removal of tooth-colored composite resin using the fluorescence-aided identification technique
Uploaded: 2022-07-27T12:40:00
Description: Watch this Scientific Journal Video about Detection and Removal of Tooth-Colored Composite Resin Using the Fluorescence-Aided Identification Technique at JoVE.com

This study was supported by a research grant from the Swiss Dental Association (SSO Research Grant 292-16).

The teeth used in this study were part of a project approved by the local Ethics Committee (EKNZ UBE-15/111). The participants provided written informed consent, and all data were de-identified to protect patient confidentiality.
1. Detection of tooth colored composite resin material using FIT
<ol>
	<li>Darken the room (natural and artificial light).</li>
	<li>Wear clear or yellow-tinted safety glasses with UV protection.</li>
	<li>Use a fluorescence-inducing light source to...

<ol>
	<li>Meller, C., Connert, T., L&#246;st, C., ElAyouti, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reliability+of+a+Fluorescence-aided+Identification+Technique+(FIT)+for+detecting+tooth-colored+restorations:+an+ex+vivo+comparative+study.">Reliability of a Fluorescence-aided Identification Technique (FIT) for detecting tooth-colored restorations: an ex vivo comparative study.</a> Clinical Oral Investigations. 21 (1), 347-355 (2017).</li><li>Kiran, R., Chapman, J., Tennant, M., Forrest, A., Walsh, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence-aided+selective+removal+of+resin-based+composite+restorative+materials:+An+in+vitro+comparative+study.">Fluorescence-aided selective removal of resin-based composite restorative materials: An in vitro comparative study.</a> Journal of Esthetic and Restorative Dentistry. 32 (3), 310-316 (2020).</li><li>Meller, C., Klein, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+properties+of+commercial+composite+resin+restorative+materials+in+dentistry.">Fluorescence properties of commercial composite resin restorative materials in dentistry.</a> Dental Materials Journal. 31 (6), 916-923 (2012).</li><li>Uo, 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=Rare+earth+oxide-containing+fluorescent+glass+filler+for+composite+resin.">Rare earth oxide-containing fluorescent glass filler for composite resin.</a> Dental Materials Journal. 24 (1), 49-52 (2005).</li><li>Fondriest, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shade+matching+in+restorative+dentistry:+the+science+and+strategies.">Shade matching in restorative dentistry: the science and strategies.</a> International Journal of Periodontics and Restorative Dentistry. 23, 467-479 (2003).</li><li>Takahashi, M. 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=Fluorescence+intensity+of+resin+composites+and+dental+tissues+before+and+after+accelerated+aging:+a+comparative+study.">Fluorescence intensity of resin composites and dental tissues before and after accelerated aging: a comparative study.</a> Operative Dentistry. 33 (2), 189-195 (2008).</li><li>Klein, C., Wolff, D., Ohle, C. V., Meller, 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+fluorescence+of+resin-based+composites:+An+analysis+after+ten+years+of+aging.">The fluorescence of resin-based composites: An analysis after ten years of aging.</a> Dental Materials Journal. 40 (1), 94-100 (2020).</li><li>Lee, Y. K., Lu, H., Powers, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+opalescence+and+fluorescence+properties+of+resin+composites+after+accelerated+aging.">Changes in opalescence and fluorescence properties of resin composites after accelerated aging.</a> Dental Materials. 22 (7), 653-660 (2006).</li><li>Lee, Y. K., Lu, H., Powers, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+properties+of+four+esthetic+restorative+materials+after+accelerated+aging.">Optical properties of four esthetic restorative materials after accelerated aging.</a> American Journal of Dentistry. 19 (3), 155-158 (2006).</li><li>Dietschi, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Free-hand+composite+resin+restorations:+a+key+to+anterior+aesthetics.">Free-hand composite resin restorations: a key to anterior aesthetics.</a> Practical Periodontics and Aesthetic Dentistry. 7 (7), 15-25 (1995).</li><li>Bush, M. A., Hermanson, A. S., Yetto, R. J., Wieczkowski, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+ultraviolet+LED+illumination+for+composite+resin+removal:+an+in+vitro+study.">The use of ultraviolet LED illumination for composite resin removal: an in vitro study.</a> General Dentistry. 58 (5), 214-218 (2010).</li><li>Baelum, V., Fejerskov, O., Fejerskov, O., Nyvad, B., Kidd, E. 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=How+big+is+the+problem?+Epidemiological+features+of+dental+caries.">How big is the problem? Epidemiological features of dental caries.</a> Dental Caries-the Disease and its Clinical Management. 3rd edn. , 25 (2015).</li><li>Lynch, C. D., 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=Guidance+on+posterior+resin+composites:+Academy+of+Operative+Dentistry+-+European+Section.">Guidance on posterior resin composites: Academy of Operative Dentistry - European Section.</a> Journal of Denistry. 42 (4), 377-383 (2014).</li><li>Demarco, F. F., Corr&#234;a, M. B., Cenci, M. S., Moraes, R. R., Opdam, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Longevity+of+posterior+composite+restorations:+not+only+a+matter+of+materials.">Longevity of posterior composite restorations: not only a matter of materials.</a> Dental Materials Journal. 28 (1), 87-101 (2012).</li><li>Eltahlah, D., Lynch, C. D., Chadwick, B. L., Blum, I. R., Wilson, N. 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=An+update+on+the+reasons+for+placement+and+replacement+of+direct+restorations.">An update on the reasons for placement and replacement of direct restorations.</a> Journal of Dentistry. 72, 1-7 (2018).</li><li>Bonstein, T., Garlapo, D., Donarummo, J., Bush, P. J. <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+varied+repair+protocols+applied+to+aged+composite+resin.">Evaluation of varied repair protocols applied to aged composite resin.</a> Journal of Adhesive Dentistry. 7 (1), 41-49 (2005).</li><li>Crumpler, D. C., Bayne, S. C., Sockwell, S., Brunson, D., Roberson, T. 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An in vivo study in man.</a> Journal of Clinical Periodontology. 17 (3), 138-144 (1990).</li><li>Diangelis, A. 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=International+Association+of+Dental+Traumatology+guidelines+for+the+management+of+traumatic+dental+injuries:+1.+Fractures+and+luxations+of+permanent+teeth.">International Association of Dental Traumatology guidelines for the management of traumatic dental injuries: 1. Fractures and luxations of permanent teeth.</a> Dental Traumatology. 28 (1), 2-12 (2012).</li><li>Tani, K., Watari, F., Uo, M., Morita, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discrimination+between+composite+resin+and+teeth+using+fluorescence+properties.">Discrimination between composite resin and teeth using fluorescence properties.</a> Dental Materials Journal. 22 (4), 569-580 (2003).</li><li>Carson, D. O., Orihara, Y., Sorbie, J. L., Pounder, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+white+restorative+dental+materials+using+an+alternative+light+source.">Detection of white restorative dental materials using an alternative light source.</a> Forensic Science International. 88 (2), 163-168 (1997).</li><li>Kiran, R., Chapman, J., Tennant, M., Forrest, A., Walsh, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence-aided+selective+removal+of+resin-based+composite+restorative+materials:+An+in+vitro+comparative+study.">Fluorescence-aided selective removal of resin-based composite restorative materials: An in vitro comparative study.</a> Journal of Esthetic and Restorative Dentistry. 32 (3), 310-316 (2020).</li><li>Dettwiler, 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=Fluorescence-aided+composite+removal+in+directly+restored+permanent+posterior+teeth.">Fluorescence-aided composite removal in directly restored permanent posterior teeth.</a> Operative Dentistry. 45 (1), 62-70 (2020).</li><li>Dettwiler, 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=Evaluation+of+a+Fluorescence-aided+Identification+Technique+(FIT)+for+removal+of+composite+bonded+trauma+splints.">Evaluation of a Fluorescence-aided Identification Technique (FIT) for removal of composite bonded trauma splints.</a> Dental Traumatology. 34 (5), 353-359 (2018).</li><li>Schott, T. 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A., Almeida, L. F., Martins, L. P., Martins, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+adhesive+remnant+removal+and+enamel+damage+with+ultraviolet+light:+An+in-vitro+study.">Assessing adhesive remnant removal and enamel damage with ultraviolet light: An in-vitro study.</a> American Journal of Orthodontics and Dentofacial Orthopedics. 151 (2), 292-296 (2017).</li><li>Klein, 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=Minimally+invasive+removal+of+tooth-colored+restorations:+evaluation+of+a+novel+handpiece+using+the+fluorescence-aided+identification+technique+(FIT).">Minimally invasive removal of tooth-colored restorations: evaluation of a novel handpiece using the fluorescence-aided identification technique (FIT).</a> Clinical Oral Investigations. 28 (8), 2735-2743 (2019).</li><li>Leontiev, W., 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=Accuracy+of+the+fluorescence-aided+identification+technique+(FIT)+for+detecting+tooth-colored+restorations+utilizing+different+fluorescence-inducing+devices:+an+ex+vivo+comparative+study.">Accuracy of the fluorescence-aided identification technique (FIT) for detecting tooth-colored restorations utilizing different fluorescence-inducing devices: an ex vivo comparative study.</a> Clinical Oral Investigations. 25 (9), 5189-5196 (2021).</li><li>Eichenberger, M., Perrin, P., Neuhaus, K. 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The conventional illumination (for example by a dental unit lamp) is an unsatisfactory diagnostic tool for the identification of composite resin restorations. For superior diagnostics with conventional illumination, a magnification aid, drying, or even effortful cleaning of the teeth is necessary. Even under ideal circumstances, conventional illumination seems to be insufficient. A study showed that conventional illumination may lead to misdetection of composite resin restorations and sound tooth substance<sup class="xre...

The application of FIT facilitates the distinction of composite resin materials from sound tooth substance compared to conventional illumination, for example, by a dental unit lamp1,2. Fluorescence occurs when a material emits the light at a higher wavelength than it has been absorbed. As a result of this illumination, the material appears brighter than the tooth3. The maximum fluorescence of composite resin materials occurs when illuminated by a wavelength of 398 &#177; 5 nanometers3. Fluorescence in composite resin materials appears due to rare earth oxides added to the glass fillers, some of the main components of composite resins4,5. The addition of these fluorescent substances intends to adapt the optical properties of composite resins to the tooth structure to improve the esthetic properties of composite resins4,5. FIT is applicable on many composite resin materials as they show these fluorescence properties3. However, fluorescence decreases with the ageing of the composite resin materials6,7,8,9.
The distinguishment of composite resin materials from tooth structure with conventional illumination is a challenge since modern composite resin materials match the optical properties of tooth substance almost perfectly10,11. The misdiagnosis of composite resin results in inaccurate dental charts, false caries risk assessment, and inappropriate treatment planning11. Moreover, epidemiological data is falsified12.
Composite resin is the material of choice for direct restorations due to its straightforward handling, esthetic properties, and clinical performance13. Nevertheless, many composite restorations must be renewed due to secondary caries, fractures, or other reasons14,15. However, the removal of residual composite resin materials can be demanding under conventional light conditions. Even with the application of a magnification aid and the use of tactile probes or extensive drying of the teeth, composite residues are sometimes difficult to distinguish from sound tooth structure. Leftovers of composite remnants during the removal of the adhesive restoration lower the quality of further restorations and have an esthetic impairment due to possible discoloration of the margins1,16,17,18,19,20,21,22. On the contrary, an overpreparation due to misdiagnosis of composite resin versus tooth structure may result in unnecessary substance loss1,2.
In dental traumatology, fixation of the injured teeth using trauma splints is frequent and mandatory in many cases23. The trauma splints are usually fixed on the teeth using a flowable composite resin material. Incomplete removal of the composite resin material in this scenario may lead to the impairments described above. Since dental trauma occurs mostly in front teeth, an impairment of the esthetics and sufficient adhesion of further reconstructions are crucial. Therefore, the aim of the article is to demonstrate the application of the FIT method as an efficient and straightforward approach for detecting and removing composite resin materials.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Bonding Resin Remover, H22ALGK 016</td>
			<td>Komet Dental, Lemgo, Germany</td>
			<td></td>
			<td>Any other material/equpiment with the same function/purpose might be used.</td>
		</tr>
		<tr>
			<td>Cerec Omicam, Connect SW 5.1.3</td>
			<td>Dentsply Sirona, York, PA, USA</td>
			<td></td>
			<td>Any other material/equpiment with the same function/purpose might be used.</td>
		</tr>
		<tr>
			<td>Diamant bur</td>
			<td>Intensiv SA, Montagnola, Switzerland</td>
			<td></td>
			<td>Any other material/equpiment with the same function/purpose might be used.</td>
		</tr>
		<tr>
			<td>Mandrell</td>
			<td>3M, Saint Paul, MN, USA</td>
			<td></td>
			<td>Any other material/equpiment with the same function/purpose might be used.</td>
		</tr>
		<tr>
			<td>MASTERmatic</td>
			<td>KaVo Dental GmbH, Biberach, Germany</td>
			<td></td>
			<td>Any other material/equpiment with the same function/purpose might be used.</td>
		</tr>
		<tr>
			<td>Occlubrush</td>
			<td>Kerr, Orange, CA, USA</td>
			<td></td>
			<td>brush polishing system</td>
		</tr>
		<tr>
			<td>OraCheck Software, Version 5.0.0</td>
			<td>Cyfex AG, Zurich, Switzerland</td>
			<td></td>
			<td>Any other material/equpiment with the same function/purpose might be used.</td>
		</tr>
		<tr>
			<td>SIROInspect</td>
			<td>Dentsply Sirona, York, PA, USA</td>
			<td></td>
			<td>Any other material/equpiment with the same function/purpose might be used.</td>
		</tr>
		<tr>
			<td>Sof-Lex</td>
			<td>3M, Saint Paul, MN, USA</td>
			<td></td>
			<td>Contouring/polishing discs; any other material/equpiment with the same function/purpose might be used.</td>
		</tr>
	</tbody>
</table>

detection and removal of tooth-colored composite resin using the fluorescence-aided identification technique

The detection and removal of tooth-colored filling materials is a major challenge for every dentist. The Fluorescence-aided Identification Technique (FIT) is a noninvasive tool to facilitate the distinction of composite resin material from sound tooth substance. Compared to conventional illumination, FIT is a very accurate, reliable, and fast diagnostic method. When composite resin is illuminated with a wavelength of approximately 398 &plusmn; 5 nm, certain fluorescent components make the composite resin appear brighter than the tooth structure. Any fluorescence-inducing light source with the appropriate wavelength can be used for this method. Optimally, this technique is used without additional natural or artificial lighting. The application of FIT can be used for diagnostic purposes, for example, dental charts, and additionally for the complete and minimally invasive removal of composite resin restorations, bracket debonding, and trauma splint removal. The assessment of volumetric changes after composite removal can be provided by overlapping pre- and postoperative scans and subsequent calculation using suitable software.

Usage of the FIT method makes most composite resin materials appear brighter than sound tooth structure (Figure 2 and Figure 5). Therefore, FIT is applicable not only in the detection of composite resin material, but it also facilitates the removal of composite resin materials in general, and explicitly in posterior teeth, during orthodontic bracket debonding and in trauma splint removal1,2,<s...

Watch this Scientific Journal Video about Detection and Removal of Tooth-Colored Composite Resin Using the Fluorescence-Aided Identification Technique at JoVE.com

Detection and Removal of Tooth-Colored Composite Resin Using the Fluorescence-Aided Identification Technique

The Fluorescence-aided Identification Technique is a practicable, fast, and reliable approach for the differentiation of composite resin restorations from tooth substance, and facilitates the minimally invasive and complete removal of composite resin restorations and composite bonded trauma splints.

The detection and removal of tooth-colored filling materials is a major challenge for every dentist. The Fluorescence-aided Identification Technique (FIT) ...

The detection and removal of tooth-colored filling material is a challenge. The fluorescence-aided identification technique, FIT, is a diagnostic tool that facilitates the differentiation of composite resin from tooth substance. FIT enables the minimal invasive removal of composite resin restorations and composite-bonded trauma splints.It's a straightforward, fast, and reliable diagnostic approach. To begin, wear clear or yellow-tinted safety glasses with UV protection, then darken the room. Use a fluorescence-inducing light source to illuminate the tooth substance and tooth-colored composite resin restoration.For a pre-operative scan, start the intraoral scanning device and open the appropriate software For experimental evaluation purposes. Press Add New Patient to register the patient. Fill in the gaps such as last name, first name, date of birth, and patient ID, then press Add Case.Choose Jaw Scan and Impressions under the section Indications, then press Next. Darken the room and dry the field of operation to ease the scanning procedure. Start the intraoral scanner and perform a digital surface scan of the field of operation.To visualize the composite resin material, illuminate the tooth substance and tooth-colored composite resin restoration. Remove the composite resin material using a high-speed contra angle handpiece with diamond burs, a bonding resin remover, and polishing devices. Next, perform the post-operative scan for experimental volumetric assessment as per the steps demonstrated earlier for the pre-operative scan.For experimental volumetric assessment, select the patient and case, then open case and press Model. Then press Export to export the pre and post-operative scans as surface tessellation language in the highest resolution. Then open suitable software and press Recombine.Upload the pre and post-operative scans to the software by pressing Import. Next press Analysis to visualize the volumetric changes from pre to post-operative scans. Select the tooth sites where volumetric changes presumably occurred by choosing Region under the section Tools.Analyze the volumetric changes using the linear and volumetric measurement software tools Distances and Volume Analysis respectively. Press Distances under the section Tools for linear quantification of tooth substance loss and composite resin remnants in color. Use the color bar on the left to quantify linear volumetric changes.Additionally, locate the cursor on the relevant tooth sites and observe the exact cursor distance in the box on the left. The representative tooth model with several composite resin restorations and trauma splint fixed with composite resin is shown here. The usage of the fluorescence-aided identification technique made most composite resin materials brighter than sound tooth structures.A tooth model after trauma splint removal under conventional illumination and using the fluorescence-aided identification technique is shown here. A volumetric assessment showed quantification of the composite remnants and tooth substance loss. The discrepancy of the pre and post-operative scan revealed composite remnants and substance loss in teeth 13, 12, and 11.In contrast, teeth 21, 22, and 23 did not show any surface changes after trauma splint removal. Moreover, the composite remnants were undetected under conventional light illumination and became visible using the fluorescence-aided identification technique. Any fluorescence-inducing light source is applicable for the FIT method.Darken the room to avoid interference with other light sources. FIT is applicable not only for the detection and removal of composite resin restorations and trauma splints. It can also be applied in bracket debonding and dental forensics.

detection-removal-tooth-colored-composite-resin-using-fluorescence

Research

JoVE Journal

Medicine

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.

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 ...

Identification and Isolation of Slow-Dividing Cells in Human Glioblastoma Using Carboxy Fluorescein Succinimidyl Ester (CFSE)

Tumor heterogeneity represents a fundamental feature supporting tumor robustness and presents a central obstacle to the development of therapeutic strategies1. To overcome the issue of tumor heterogeneity, it is essential to develop assays and tools enabling phenotypic, (epi)genetic and functional identification and characterization of tumor subpopulations that drive specific disease pathologies and represent clinically relevant targets. It is now well established that tumors exhibit distinct sub-fractions of cells with different frequencies of cell division, and that the functional criteria of being slow cycling is positively associated with tumor formation ability in several cancers including those of the brain, breast, skin and pancreas as well as leukemia2-8. The fluorescent dye carboxyfluorescein succinimidyl ester (CFSE) has been used for tracking the division frequency of cells in vitro and in vivo in blood-borne tumors and solid tumors such as glioblastoma2,7,8. The cell-permeant non-fluorescent pro-drug of CFSE is converted by intracellular esterases into a fluorescent compound, which is retained within cells by covalently binding to proteins through reaction of its succinimidyl moiety with intracellular amine groups to form stable amide bonds9. The fluorescent dye is equally distributed between daughter cells upon divisions, leading to the halving of the fluorescence intensity with every cell division. This enables tracking of cell cycle frequency up to eight to ten rounds of division10. CFSE retention capacity was used with brain tumor cells to identify and isolate a slow cycling subpopulation (top 5% dye-retaining cells) demonstrated to be enriched in cancer stem cell activity2. 
This protocol describes the technique of staining cells with CFSE and the isolation of individual populations within a culture of human glioblastoma (GBM)-derived cells possessing differing division rates using flow cytometry2. The technique has served to identify and isolate a brain tumor slow-cycling population of cells by virtue of their ability to retain the CFSE labeling.

Identification and Isolation of Slow-Dividing Cells in Human Glioblastoma Using Carboxy Fluorescein Succinimidyl Ester CFSE

This video protocol demonstrates the application of the fluorescent dye carboxyfluorescein succinimidyl ester (CFSE) for the identification and separation of different sub-populations of cells in human glioblastoma based on frequency of cell division.

Tumor heterogeneity represents a fundamental feature supporting tumor robustness and presents a central obstacle to the development of therapeutic ...

Clinical Testing and Spinal Cord Removal in a Mouse Model for Amyotrophic Lateral Sclerosis (ALS)

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder resulting in progressive degeneration of motoneurons. Peak of onset is around 60 years for the sporadic disease and around 50 years for the familial disease. Due to its progressive course, 50% of the patients die within 30 months of symptom onset. In order to evaluate novel treatment options for this disease, genetic mouse models of ALS have been generated based on human familial mutations in the SOD gene, such as the SOD1 (G93A) mutation. Most important aspects that have to be evaluated in the model are overall survival, clinical course and motor function. Here, we demonstrate the clinical evaluation, show the conduction of two behavioural motor tests and provide quantitative scoring systems for all parameters. Because an in depth analysis of the ALS mouse model usually requires an immunohistochemical examination of the spinal cord, we demonstrate its preparation in detail applying the dorsal laminectomy method. Exemplary histological findings are demonstrated. The comprehensive application of the depicted examination methods in studies on the mouse model of ALS will enable the researcher to reliably test future therapeutic options which can provide a basis for later human clinical trials.

Clinical Testing and Spinal Cord Removal in a Mouse Model for Amyotrophic Lateral Sclerosis ALS

A mouse model for amyotrophic lateral sclerosis (ALS) is examined clinically and behaviorally. As a prerequisite for an accompanying immunohistological analysis the preparation of the spinal cord is depicted in detail.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder resulting in progressive degeneration of motoneurons. Peak of onset is around 60 ...

Cerenkov Luminescence Imaging (CLI) for Cancer Therapy Monitoring

In molecular imaging, positron emission tomography (PET) and optical imaging (OI) are two of the most important and thus most widely used modalities1-3. PET is characterized by its excellent sensitivity and quantification ability while OI is notable for non-radiation, relative low cost, short scanning time, high throughput, and wide availability to basic researchers. However, both modalities have their shortcomings as well. PET suffers from poor spatial resolution and high cost, while OI is mostly limited to preclinical applications because of its limited tissue penetration along with prominent scattering optical signals through the thickness of living tissues.
 Recently a bridge between PET and OI has emerged with the discovery of Cerenkov Luminescence Imaging (CLI)4-6. CLI is a new imaging modality that harnesses Cerenkov Radiation (CR) to image radionuclides with OI instruments. Russian Nobel laureate Alekseyevich Cerenkov and his colleagues originally discovered CR in 1934. It is a form of electromagnetic radiation emitted when a charged particle travels at a superluminal speed in a dielectric medium7,8. The charged particle, whether positron or electron, perturbs the electromagnetic field of the medium by displacing the electrons in its atoms. After passing of the disruption photons are emitted as the displaced electrons return to the ground state. For instance, one 18F decay was estimated to produce an average of 3 photons in water5. 
 Since its emergence, CLI has been investigated for its use in a variety of preclinical applications including in vivo tumor imaging, reporter gene imaging, radiotracer development, multimodality imaging, among others4,5,9,10,11. The most important reason why CLI has enjoyed much success so far is that this new technology takes advantage of the low cost and wide availability of OI to image radionuclides, which used to be imaged only by more expensive and less available nuclear imaging modalities such as PET.
 Here, we present the method of using CLI to monitor cancer drug therapy. Our group has recently investigated this new application and validated its feasibility by a proof-of-concept study12. We demonstrated that CLI and PET exhibited excellent correlations across different tumor xenografts and imaging probes. This is consistent with the overarching principle of CR that CLI essentially visualizes the same radionuclides as PET. We selected Bevacizumab (Avastin; Genentech/Roche) as our therapeutic agent because it is a well-known angiogenesis inhibitor13,14. Maturation of this technology in the near future can be envisioned to have a significant impact on preclinical drug development, screening, as well as therapy monitoring of patients receiving treatments.

Cerenkov Luminescence Imaging CLI for Cancer Therapy Monitoring

Use of Cerenkov Luminescence Imaging (CLI) for monitoring preclinical cancer treatment is described here. This method takes advantage of Cerenkov Radiation (CR) and optical imaging (OI) to visualize radiolabeled probes and thus provides an alternative to PET in preclinical therapeutic monitoring and drug screening.

In  molecular imaging, positron emission tomography (PET) and optical imaging (OI)  are two of the most important and thus most widely used modalities1-3. ...

Identification of Disease-related Spatial Covariance Patterns using Neuroimaging Data

The scaled subprofile model (SSM)1-4 is a multivariate PCA-based algorithm that identifies major sources of variation in patient and control group brain image data while rejecting lesser components (Figure 1). Applied directly to voxel-by-voxel covariance data of steady-state multimodality images, an entire group image set can be reduced to a few significant linearly independent covariance patterns and corresponding subject scores. Each pattern, termed a group invariant subprofile (GIS), is an orthogonal principal component that represents a spatially distributed network of functionally interrelated brain regions. Large global mean scalar effects that can obscure smaller network-specific contributions are removed by the inherent logarithmic conversion and mean centering of the data2,5,6. Subjects express each of these patterns to a variable degree represented by a simple scalar score that can correlate with independent clinical or psychometric descriptors7,8. Using logistic regression analysis of subject scores (i.e. pattern expression values), linear coefficients can be derived to combine multiple principal components into single disease-related spatial covariance patterns, i.e. composite networks with improved discrimination of patients from healthy control subjects5,6. Cross-validation within the derivation set can be performed using bootstrap resampling techniques9. Forward validation is easily confirmed by direct score evaluation of the derived patterns in prospective datasets10. Once validated, disease-related patterns can be used to score individual patients with respect to a fixed reference sample, often the set of healthy subjects that was used (with the disease group) in the original pattern derivation11. These standardized values can in turn be used to assist in differential diagnosis12,13 and to assess disease progression and treatment effects at the network level7,14-16. We present an example of the application of this methodology to FDG PET data of Parkinson's Disease patients and normal controls using our in-house software to derive a characteristic covariance pattern biomarker of disease.

Multivariate techniques including principal component analysis (PCA) have been used to identify signature patterns of regional change in functional brain images. We have developed an algorithm to identify reproducible network biomarkers for the diagnosis of neurodegenerative disorders, assessment of disease progression, and objective evaluation of treatment effects in patient populations.

The scaled subprofile model (SSM)1-4 is a multivariate PCA-based algorithm that identifies major sources of variation in patient and control group brain ...

Orthotopic Hind Limb Transplantation in the Mouse

In vivo animal model systems, and in particular mouse models, have evolved into powerful and versatile scientific tools indispensable to basic and translational research in the field of transplantation medicine. A vast array of reagents is available exclusively in this setting, including mono- and polyclonal antibodies for both diagnostic and interventional applications. In addition, a vast number of genotyped, inbred, transgenic, and knock out strains allow detailed investigation of the individual contributions of humoral and cellular components to the complex interplay of an immune response and make the mouse the gold standard for immunological research. 
Vascularized Composite Allotransplantation (VCA) delineates a novel field of transplantation using allografts to replace "like with like" in patients suffering traumatic or congenital tissue loss. This surgical methodological protocol shows the use of a non-suture cuff technique for super-microvascular anastomosis in an orthotopic mouse hind limb transplantation model. The model specifically allows for comparison between established paradigms in solid organ transplantation with a novel form of transplants consisting of various different tissue components. Uniquely, this model allows for the transplantation of a viable vascularized bone marrow compartment and niche that have the potential to exert a beneficial effect on the balance of immune acceptance and rejection. This technique provides a tool to investigate alloantigen recognition and allograft rejection and acceptance, as well as enables the pursuit of functional nerve regeneration studies to further advance this novel field of transplantation.

This novel model for orthotopic hind limb transplantation in the mouse, applying a non-suture cuff technique for super-microvascular anastomosis, provides a powerful tool for in&#160;vivo mechanistic immunological research related to vascularized composite allotransplantation (VCA).

In vivo animal model systems, and in particular mouse models, have evolved into powerful and versatile scientific tools indispensable to basic and ...

Mouse Model for Pancreas Transplantation Using a Modified Cuff Technique

Mouse models have several advantages in transplantation research, including easy handling, a variety of genetically well-defined strains, and the availability of the widest range of molecular probes and reagents to perform in vivo as well as in vitro studies. Based on our experience with various murine transplantation models, we developed a heterotopic pancreas transplantation model in mice with the intent to analyze mechanisms underlying severe ischemia reperfusion injury-associated early graft damage. In contrast to previously described techniques using suture techniques, herein we describe a new procedure using a non-suture cuff technique. 
In recent years, we have performed more than 300 pancreas transplantations in mice with an overall success rate of &gt;90%, a success rate never described before in mouse pancreas transplantation. The backbone of this non-suture cuff technique for graft revascularization consists of two major steps: (I) pulling the recipient vessel over a polyethylene/polyamide cuff and fixing it with a circumferential ligature, and (II) placing the donor vessel over the everted recipient vessel and fixing it with a second circumferential ligature. The resultant continuity of the endothelial layer results in less thrombogenic lesions with high patency rates and, finally, high success rates.
In this model, arterial anastomosis is achieved by pulling the abdominal aorta of the donor graft over the everted common carotid artery of the recipient animal. Venous drainage of the graft is achieved by pulling the portal vein of the graft over the everted external jugular vein of the recipient. This manuscript provides details and crucial steps of the organ recovery and organ implantation procedures, which will allow researchers with microsurgical skills to perform the transplantation successfully in their laboratories.

Among abdominal solid organ transplantation, pancreatic grafts are prone to develop severe ischemia reperfusion injury-associated graft damage, leading eventually to early graft loss. This protocol describes a model of murine pancreas transplantation using a non-suture cuff technique, ideally suited for analyzing these early, deleterious damages.

Mouse models have several advantages in transplantation research, including easy handling, a variety of genetically well-defined strains, and the ...

Assessment of Sarcoplasmic Reticulum Calcium Reserve and Intracellular Diastolic Calcium Removal in Isolated Ventricular Cardiomyocytes

Intracellular calcium recycling plays a critical role in regulation of systolic and diastolic function in cardiomyocytes. Cardiac sarcoplasmic reticulum (SR) serves as a Ca2+ reservoir for contraction, which reuptakes intracellular Ca2+ during relaxation. The SR Ca2+ reserve available for beats is determinate for cardiac contractibility, and the removal of intracellular Ca2+ is critical for cardiac diastolic function. Under some pathophysiological conditions, such as diabetes and heart failure, impaired calcium clearance and SR Ca2+ store in cardiomyocytes may be involved in the progress of cardiac dysfunction. Here, we describe a protocol to evaluate SRCa2+ reserve and diastolic Ca2+ removal. Briefly, a single cardiomyocyte was enzymatically isolated, and the intracellular Ca2+ fluorescence indicated by Fura-2 was recorded by a calcium imaging system. To employ caffeine for inducing total SR Ca2+ release, we preset an automatic perfusion switch program by interlinking the stimulation system and the perfusion system. Then, the mono-exponential curve fitting was used for analyzing decay time constants of calcium transients and caffeine-induced calcium pulses. Accordingly, the contribution of the SR Ca2+-ATPase (SERCA) and Na+-Ca2+ exchanger (NCX) to diastolic calcium removal was evaluated.

Intracellular calcium recycling plays a critical role in regulation of systolic and diastolic function in cardiomyocytes. Here, we describe a protocol to evaluate sarcoplasmic reticulum Ca2+ reserve and diastolic calcium removal function in cardiomyocytes by a calcium imaging system.

Intracellular calcium recycling plays a critical role in regulation of systolic and diastolic function in cardiomyocytes. Cardiac sarcoplasmic reticulum ...

Laparoscopic Pancreatoduodenectomy for Pancreatic Cancer Using In-Situ No-Touch Isolation Technique

Laparoscopic pancreatoduodenectomy (LPD) is a standard radical operation for pancreatic head malignant tumors by now. Due to the complex laparoscopic resection and reconstruction techniques, it is difficult to perform LPD for patients with locally advanced pancreatic head cancer after neoadjuvant therapy. Our team initiates LPD using the in-situ No-Touch isolation technique. The innovation and optimization of this modified No-Touch isolation technique emphasize exploring the distal section of superior mesenteric vein&#160;(SMV) and the left side of the superior mesenteric artery (SMA) prior to evaluating the resectability by subcolonic mesenteric approach, which is an ideal exploring approach. After that, we use the median-anterior, and left-posterior of SMA approaches to cut off the blood flow of the pancreatic head to make the tumor isolated intact, then move and dissect the tumor. It is a process fitting the surgical principle of tumor-free. This article aims to demonstrate the feasibility and safety of performing LPD using the in-situ No-Touch isolation technique, which might elevate the R0 resection rate. It is an oncological ideal operation process.

Laparoscopic Pancreatoduodenectomy for Pancreatic Cancer Using In-Situ No-Touch Isolation Technique

No-Touch isolation procedures might prevent the dissemination of cancer cells from the primary tumor. However, these techniques are not widely accepted in laparoscopic pancreatoduodenectomy (LPD) by now. We herein present in-situ No-Touch isolation LPD with partial resection and reconstruction of the superior mesenteric vein (SMV) for pancreatic cancer after neoadjuvant therapy.

Laparoscopic pancreatoduodenectomy (LPD) is a standard radical operation for pancreatic head malignant tumors by now. Due to the complex laparoscopic ...

Hickman Catheter for Long-Term Vascular Access in Swine

Hickman Catheter Use for Long-Term Vascular Access in a Preclinical Swine Model

Central venous catheters (CVCs) are invaluable devices in large animal research as they facilitate a wide range of medical applications, including blood monitoring and reliable intravenous fluid and drug administration. Specifically, the tunneled multi-lumen Hickman catheter (HC) is commonly used in swine models due to its lower extrication and complication rates. Despite fewer complications relative to other CVCs, HC-related morbidity presents a significant challenge, as it can significantly delay or otherwise negatively impact ongoing studies. The proper insertion and maintenance of HCs is paramount in preventing these complications, but there is no consensus on best practices. The purpose of this protocol is to comprehensively describe an approach for the insertion and maintenance of a tunneled HC in swine that mitigates HC-related complications and morbidity. The use of these techniques in &gt;100 swine has resulted in complication-free patent lines up to 8 months and no catheter-related mortality or infection of the ventral surgical site. This protocol offers a method to optimize the lifespan of the HC and guidance for approaching issues during use.

Author Spotlight: Hickman Catheter Use for Long-Term Vascular Access in a Preclinical Swine Model  

A reliable and reproducible approach for the insertion and maintenance of a tunneled Hickman catheter for long-term vascular access in swine is described. Placement of a central venous catheter allows for convenient daily sampling of whole blood from awake animals and intravenous administration of medication and fluids.

Central venous catheters (CVCs) are invaluable devices in large animal research as they facilitate a wide range of medical applications, including blood ...