We thank J. Hagoort for reading and correcting the manuscript.

This protocol follows the guidelines of the local human research ethics committee.
1. Preparation
<ol>
	<li>Schedule the microcirculatory measurement so that it does not coincide with other procedures such as blood sampling. In term neonates it is best performed after feeding. This prevents agitation and will ease the measurement.</li>
	<li>Ensure that a nurse or a parent attends to support and comfort the neonate during the examination, using the principles of Newborn Individualized Develop...

<ol>
	<li>Groner, 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=Orthogonal+polarization+spectral+imaging:+a+new+method+for+study+of+the+microcirculation.">Orthogonal polarization spectral imaging: a new method for study of the microcirculation.</a> Nat Med. 5 (10), 1209-1212 (1999).</li><li>Goedhart, P. T., Khalilzada, M., Bezemer, R., Merza, J., Ince, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sidestream+Dark+Field+(SDF)+imaging:+a+novel+stroboscopic+LED+ring-based+imaging+modality+for+clinical+assessment+of+the+microcirculation.">Sidestream Dark Field (SDF) imaging: a novel stroboscopic LED ring-based imaging modality for clinical assessment of the microcirculation.</a> Opt Express. 15 (23), 15101-15114 (2007).</li><li>Sherman, H., Klausner, S., Cook, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incident+dark-field+illumination:+a+new+method+for+microcirculatory+study.">Incident dark-field illumination: a new method for microcirculatory study.</a> Angiology. 22 (5), 295-303 (1971).</li><li>Trzeciak, 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=Early+microcirculatory+perfusion+derangements+in+patients+with+severe+sepsis+and+septic+shock:+relationship+to+hemodynamics,+oxygen+transport,+and+survival.">Early microcirculatory perfusion derangements in patients with severe sepsis and septic shock: relationship to hemodynamics, oxygen transport, and survival.</a> Ann Emerg Med. 49 (1), 88-98 (2007).</li><li>Sakr, Y., Dubois, M. J., De Backer, D., Creteur, J., Vincent, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Persistent+microcirculatory+alterations+are+associated+with+organ+failure+and+death+in+patients+with+septic+shock.">Persistent microcirculatory alterations are associated with organ failure and death in patients with septic shock.</a> Crit Care Med. 32 (9), 1825-1831 (2004).</li><li>De Backer, 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=Microcirculatory+alterations+in+patients+with+severe+sepsis:+impact+of+time+of+assessment+and+relationship+with+outcome.">Microcirculatory alterations in patients with severe sepsis: impact of time of assessment and relationship with outcome.</a> Crit Care Med. 41 (3), 791-799 (2013).</li><li>Buijs, E. 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=Early+microcirculatory+impairment+during+therapeutic+hypothermia+is+associated+with+poor+outcome+in+post-cardiac+arrest+children:+A+prospective+observational+cohort+study.">Early microcirculatory impairment during therapeutic hypothermia is associated with poor outcome in post-cardiac arrest children: A prospective observational cohort study.</a> Resuscitation. , (2013).</li><li>Genzel-Boroviczeny, O., Christ, F., Glas, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+transfusion+increases+functional+capillary+density+in+the+skin+of+anemic+preterm+infants.">Blood transfusion increases functional capillary density in the skin of anemic preterm infants.</a> Pediatr Res. 56 (5), 751-755 (2004).</li><li>Ergenekon, E., 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=Peripheral+microcirculation+is+affected+during+therapeutic+hypothermia+in+newborns.">Peripheral microcirculation is affected during therapeutic hypothermia in newborns.</a> Arch Dis Child Fetal Neonatal Ed. 98 (2), F155-F157 (2013).</li><li>Schwepcke, A., Weber, F. D., Mormanova, Z., Cepissak, B., Genzel-Boroviczeny, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microcirculatory+mechanisms+in+postnatal+hypotension+affecting+premature+infants.">Microcirculatory mechanisms in postnatal hypotension affecting premature infants.</a> Pediatr Res. , (2013).</li><li>van Elteren, H. A., Ince, C., Tibboel, D., Reiss, I. K., de Jonge, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cutaneous+microcirculation+in+preterm+neonates:+comparison+between+sidestream+dark+field+(SDF)+and+incident+dark+field+(IDF)+imaging.">Cutaneous microcirculation in preterm neonates: comparison between sidestream dark field (SDF) and incident dark field (IDF) imaging.</a> J Clin Monit Comput. , (2015).</li><li>Als, 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=Individualized+Behavioral+and+Environmental+Care+for+the+Very-Low-Birth-Weight+Preterm+Infant+at+High-Risk+for+Bronchopulmonary+Dysplasia+-+Neonatal+Intensive-Care+Unit+and+Developmental+Outcome.">Individualized Behavioral and Environmental Care for the Very-Low-Birth-Weight Preterm Infant at High-Risk for Bronchopulmonary Dysplasia - Neonatal Intensive-Care Unit and Developmental Outcome.</a> Pediatrics. 78 (6), 1123-1132 (1986).</li><li>Massey, M. 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=The+microcirculation+image+quality+score:+development+and+preliminary+evaluation+of+a+proposed+approach+to+grading+quality+of+image+acquisition+for+bedside+videomicroscopy.">The microcirculation image quality score: development and preliminary evaluation of a proposed approach to grading quality of image acquisition for bedside videomicroscopy.</a> J Crit Care. 28 (6), 913-917 (2013).</li><li>Weidlich, 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=Changes+in+microcirculation+as+early+markers+for+infection+in+preterm+infants--an+observational+prospective+study.">Changes in microcirculation as early markers for infection in preterm infants--an observational prospective study.</a> Pediatr Res. 66 (4), 461-465 (2009).</li><li>De Backer, 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=How+to+evaluate+the+microcirculation:+report+of+a+round+table+conference.">How to evaluate the microcirculation: report of a round table conference.</a> Crit Care. 11 (5), R101 (2007).</li><li>Sallisalmi, M., Oksala, N., Pettila, V., Tenhunen, 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+sublingual+microcirculatory+blood+flow+in+the+critically+ill.">Evaluation of sublingual microcirculatory blood flow in the critically ill.</a> Acta Anaesthesiol Scand. 56 (3), 298-306 (2012).</li><li>van den Berg, V. 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=Reproducibility+of+microvascular+vessel+density+analysis+in+Sidestream+dark-field-derived+images+of+healthy+term+newborns.">Reproducibility of microvascular vessel density analysis in Sidestream dark-field-derived images of healthy term newborns.</a> Microcirculation. 22 (1), 37-43 (2015).</li></ol>

The authors have nothing to disclose.

In this manuscript we describe and demonstrate the approach for transcutaneous microcirculatory imaging in preterm neonates. Visualizing this method will help researchers overcome two of the biggest challenges in research: reproducibility and the time and labor intensive nature of learning new techniques. This technique can provide useful information of peripheral microcirculation in preterm infants in a non-invasive manner. Serial measurements can help clinicians evaluate the effects of therapeutic interventions. The mi...

Hemodynamic diagnostics in critically ill preterm neonates has always been difficult. Most diagnostic tools used in adults cannot be applied in these tiny preterm infants; and then there is a problem of the sensitivity of the outcome parameters. But most of all, these infants are so vulnerable, that the risks of diagnostic procedures do not outweigh the benefits. As a result, in the field of neonatology, hemodynamics has been neglected and therefore there is a lack of knowledge on this topic.
An interesting option for handling these problems might be visualizing the microcirculation. The introduction of handheld microscopes in the late 1990s made it possible to visualize the microcirculation in a non-invasive manner. Three generations of devices have been introduced: Orthogonal Polarization Spectral (OPS) imaging1, Sidestream Dark Field (SDF) imaging2, and Incident Dark Field (IDF) imaging3. They all use more or less the same technique in which green light with a specific wavelength (548nm) stroboscopic illuminates the microcirculation. The green light is absorbed by oxy- and deoxyhemoglobin and mostly reflected by the surrounding tissue. This property of green light therefore creates visible contrast. The reflected light passes a magnification lens and is projected on a camera sensor. Hereby it is possible to visualize the flowing red blood cells at a depth of approximately one millimeter of mucosal tissue or directly at solid organs. 
Over the past 15 years, the microcirculation has been mainly studied in adults, especially in patients with septic shock4-6. These observational studies found that persistent microcirculatory alterations were associated with organ failure and mortality. This observation cannot be extrapolated directly to (preterm) infants however, as in the adults the microcirculation was measured sublingually. High quality images of the sublingual microcirculation cannot be obtained in preterm infants because they are unable to cooperate. In term infants the buccal microcirculation has been the area of interest7. Fortunately, in preterm infants the thin skin allows transcutaneous microcirculatory imaging. This approach has been applied in neonatal studies focusing on blood transfusion8, therapeutic hypothermia9 and hypotension10.
In this manuscript we present our protocol for transcutaneous microcirculatory imaging using Incident Dark Field imaging in preterm neonates. We will focus on different strategies to acquire the highest quality images. Technical details and differences between the SDF and IDF devices can be found elsewhere11.

<table>
	<tbody>
		<tr>
			<td>Cytocam</td>
			<td>Braedius</td>
			<td>http://www.braedius.com/magnoliaPublic/braedius/products.html</td>
			<td>Other well known handheld microscopes to visualize the microcirculation are MicroScan (Microvision Medical) using SDF technique or the CytoScan (CytoMetrics) using OPS technique</td>
		</tr>
		<tr>
			<td>Disposable Lens Cover</td>
			<td>Glycocheck</td>
			<td>http://www.glycocheck.com/lenscovers.php</td>
			<td></td>
		</tr>
		<tr>
			<td>CCTools</td>
			<td>Braedius</td>
			<td>http://www.braedius.com/magnoliaPublic/braedius/products.html</td>
			<td>Another well known offline analysis programme is AVA (Microvision medical).&#160;</td>
		</tr>
	</tbody>
</table>

transcutaneous microcirculatory imaging in preterm neonates

Microcirculatory imaging (MI) is a relatively new research tool mainly used in the intensive care setting. MI provides a clear view of the smallest capillaries, arterioles and venules. The magnifying effect visualizes the flow pattern of erythrocytes through these vessels.
It's non-invasive character makes it suitable to apply in (preterm) neonates, even in cardiorespiratory unstable patients. In adults and children, MI is mainly performed sublingually, but this is not possible in preterm infants as these cannot cooperate and the size of the probe is problematic. In preterm infants, MI is therefore performed transcutaneously. Their thin skin makes it possible to obtain high quality images of peripheral microcirculation. 
In this manuscript we will demonstrate the method of transcutaneous MI in preterm infants. We will focus on the different techniques and provide tips to optimize image quality. The highlights of software settings, safety and offline analysis are also addressed.

Figures 1 and 2 show representative still images of high-quality MI videos. These examples demonstrate the difference in skin thickness in the same infants between day 1 (Figure 1) and day 28 (Figure 2) of postnatal age. On day 1, there is a bright illumination, adequate focus on the micro vessels and minimal presence of artefacts. On day 28 it is more difficult to find the right balance between focus on the micro vessels and artefacts du...

Watch this Scientific Journal Video about Transcutaneous Microcirculatory Imaging in Preterm Neonates at JoVE.com

Transcutaneous Microcirculatory Imaging in Preterm Neonates

Microcirculatory imaging (MI) is used to monitor peripheral perfusion in critically ill or preterm neonates. This manuscript and video demonstrates the optimal approach for obtaining high-quality images.

Microcirculatory imaging (MI) is a relatively new research tool mainly used in the intensive care setting. MI provides a clear view of the smallest ...

transcutaneous-microcirculatory-imaging-in-preterm-neonates

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Medicine

7.6K Views.  Erasmus MC-Sophia Children's Hospital. Microcirculatory imaging (MI) is used to monitor peripheral perfusion in critically ill or preterm neonates. This manuscript and video demonstrates the optimal approach for obtaining high-quality images.