We read with a great interest the paper published this month by Zötterman et al. [1]. We absolutely agree with the authors that Laser Speckle Contrast Imaging (LSCI) is a non-invasive and attractive tool for microvascular studies. LSCI allows easy clinical applications, provided that some care is taken not only about frame rate, number of frames/image, movement of the tissue, measuring distance and tissue curvature on the measured perfusion, but also environmental conditions. Indeed we previously reported that air movements, likely through the influence of micro-particles of dust in the air on the LSCI signal, might interfere with the results [2]. Since we did not standardize induced movements of our subjects, authors are to be congratulated for their standardized approach. Nevertheless their reading and analysis of our previous reports, detailed below, require some comments. First, it is not true that we only measured basal perfusion, we tested post occlusive reactive hyperemia (PORH)[3], a test largely used as a way of inducing microvascular reactivity [4]. Specifically, we showed that de-noising with the bilayer opaque adhesive (OBA) patch improved the reliability of PORH tests [5]. Second, the perfusion of skin was much more than, 2 to 3 times higher during movement compared to the immobile situation, for some experiment, it was by 100 times higher. What was found is that the effect of movements on the signal issued from skin was, with some of the tested patches, 2 to 3 times higher than their effects on the signal issued from the color patches tested. Third, this ratio was found almost equal to 1 with the use of the OBA patch. The characteristics of this OBA are of particular interest to allow light diffusion in the gel and block the diffusion of light to the skin with aluminum [5]. Forth, what we did is indeed a “point by point” subtraction on continuous recording of regions of interest. Nevertheless it must be kept in mind that each “point” is issued from a single image and that the whole image values may be corrected by the OBA value (by subtraction of mean OBA signal from each pixel). Then it is not true to claim that the technique is not applicable to the whole image. Last, the limit with this approach is not, as suggested by the authors, that movements may be present only during part of the measurement time and that the speed of motion can vary, since the OBA use perfectly fits these limitations. The first issue is rather the possibility that the movements might be heterogeneous on the image itself (with some areas moving and other areas not moving). The second issue is that, although the OBA has become a routine for de-noising of LSCI recordings [6] using the PeriCam PSI System (Perimed AB, Järfälla, Sweden), it is unknown whether the same OBA approach may apply similarly to other LSCI devices.

Samir HENNI;
Isabelle SIGNOLET
Anne HUMEAU-HEURTIER
Pierre ABRAHAM

REFERENCES
1 : :Zötterman J, Mirdell R, Horsten S, Farnebo S, Tesselaar E. Methodological concerns with laser speckle contrast imaging in clinical evaluation of microcirculation. PLoS One. 2017; 12(3):e0174703. doi: 10.1371/journal.pone.0174703. eCollection 2017.
2 : Mahé G, Durand S, Humeau A, Leftheriotis G, Rousseau P, Abraham P. Air movements interfere with laser speckle contrast imaging recordings. Lasers Med Sci. 2012; 27(5):1073-6. doi: 10.1007/s10103-011-1015-x. Epub 2011 Nov 1. PMID: 22041847 DOI: 10.1007/s10103-011-1015-x
3 : Mahé G, Rousseau P, Durand S, Bricq S, Leftheriotis G, Abraham P. Laser speckle contrast imaging accurately measures blood flow over moving skin surfaces. Microvasc Res. 2011; 81(2):183-8. doi: 10.1016/j.mvr.2010.11.013. Epub 2010 Dec 13. PMID: 21156183.
4 : de M Matheus AS, Clemente EL, de Lourdes Guimarães Rodrigues M, Torres Valença DC, Gomes MB. Assessment of microvascular endothelial function in type 1 diabetes using laser speckle contrast imaging. J Diabetes Complications. 2017; 31(4):753-757. doi: 10.1016/j.jdiacomp.2016.12.007. Epub 2017 Jan 3. PMID: 28089343
5 : Omarjee L, Signolet I, Humeau-Heutier A, Martin L, Henrion D, Abraham P. Optimisation of movement detection and artifact removal during laser speckle contrast imaging. Microvasc Res. 2015; 97:75-80. doi: 10.1016/j.mvr.2014.09.005. Epub 2014 Sep 28.PMID: 25261716
6 : Humeau-Heurtier A, Martin L, Bazeries P, Abraham P, Henni S. Laser Speckle Contrast Imaging of Skin Changes in Arteriovenous Malformation. Circ Cardiovasc Imaging. 2017; 10(3). pii: e005931. doi: 10.1161/CIRCIMAGING.116.005931. PMID: 28264869