Background
Incident dark field (IDF) imaging is an important technique that allows real time visualisation of the microcirculation [
1]. Based upon the illumination of microvessels covered by a thin epithelial layer, it may be thought of as the successor to both orthogonal polarization spectroscopy (OPS) [
2], and more recently, sidestream dark field (SDF) imaging [
3]. Introduced in 2012, this third generation hand-held camera known as the Cytocam IDF video-microscope (Braedius Medical, Huizen, The Netherlands), was developed in an attempt to overcome many of the previous generations devices technical limitations [
1]. These included; i) the limitations imposed by analogue video cameras, ii) the inability to achieve automatic microcirculation analysis, iii) pressure-induced microcirculatory alterations (predominantly caused by the heavy weight of the devices (SDF camera weight 320 g), iv) the requirement for hand operated focussing, and v) poor quality of image acquisition [
4].
The Cytocam is a lightweight (120 g), fully digitalised pen-like device (length 220 mm, diameter 23 mm) that applies the principle of incident dark field microscopy introduced by Sherman and Cook in 1971 [
5]. Blood vessels <100 μm in diameter, and <1000 μm below the surface of an organ or mucosal surface, are visualised in a two-dimensional plane through the process of epi-illumination [
5]. Highly illuminating light emitting diodes (LEDs) enable suitable tissue penetration, and to avoid motion induced blurring secondary to fast moving erythrocytes [
6], a very short LED pulse time of two milliseconds is utilised. Image delineation is optimised using a 3.5 megapixel high-resolution sensor, an optical magnification factor of four times, and an optical resolution of more than 300 lines/mm - an improvement of 50 % over SDF devices. This is further enhanced with an effective field of view (FOV) almost three times as large as earlier devices (1.55 × 1.16 mm, FOV area = 1.79 mm
2), which may be magnified by a factor of 211 times on the display monitor [
1]. Improved focussing is achieved through an integrated distance measurement system, which through the means of a manually adjusting the piezo linear motor via the computer interface, can alter the sensor position in steps of two microns. This novel quantitative focusing mechanism results in an accurate and repeatable focus distance, without having to repeatedly adjust the focus depth for every subsequent measurement. Finally, the IDF video-microscope has the capabilities for direct microcirculation analysis where the images are recorded digitally and analysed automatically. Specialised software automatically detects and quantitatively assesses the vessels’ diameters, and the flow velocity of erythrocytes within visualised vessels. Previously analysis of SDF videos required their conversion from analogue to digital images, with subsequent off-line analysis using specialised image processing software [
7].
Although the IDF device should have significant superiority, in terms of image quality, over previous technologies this requires confirmation. We therefore set out to directly compare IDF and SDF images in a formalised manner.
Discussion
These results demonstrate for the first time, that the Cytocam IDF video-microscope is superior to the SDF video-microscope in terms of the quality of sublingual microcirculatory image acquisition.
High agreement between the two raters scoring values was demonstrated, and whilst it is evident from the Bland Altman plot that some individual variation existed between raters, neither individual demonstrated a trend in over- or under-estimating the score as the total values increased. Using the total score value to determine if an image was deemed suitable for analysis, (i.e. if given a total score ≥10 renders the video as unacceptable), there was 100 % exact agreement (95 % CI: 94 %; 100 %) between the two raters.
As to whether the IDF video-microscope was superior to the SDF video-microscope in terms of providing acceptable images for analysis, the median score of 7 given to the SDF images, as opposed to 1 for the IDF videos, indicates that the SDF camera is more prone to produce unacceptable results. In this instance, 100 % of the images obtained using the IDF video-microscope were judged to be acceptable for data analysis, as opposed to only 50 % of these data collected using the SDF device. Table
2 demonstrates how the individual components of the MIQS system were scored for both cameras. From this we are able to see which categories SDF scored particularly poorly for as compared to IDF. The IDF video-microscope did not receive any scores of 10 from either rater, however nearly a fifth of the SDF videos were scored as unacceptable for stability (17 %), pressure (20 %), and content (20 %), with focus scoring deficiently 13 % of the time. This indicates superior IDF image acquisition for multiple categories, as opposed to in only one area of data capture.
Although 60 films chosen at random from a large database of images were analysed (30 for each device), a weakness in this manuscript was that no power calculation was performed prior to commencing. This said, the strong statistical significance supports the belief that it was adequately powered. Additionally, as the MIQS still relies on observer input to grade images, it is thus subjective in its film assessment. Nevertheless, it is the most formal approach to image grading we have to date, and the high ICC supports its use.
Acknowledgements
The members of the Caudwell Xtreme Everest Research Group are as follows:
V. Ahuja, G. Aref-Adib, R. Burnham, A. Chisholm, K. Clarke, D. Coates, M. Coates, D. Cook, M. Cox, S. Dhillon, C. Dougall, P. Doyle, P. Duncan, M. Edsell, L. Edwards, L. Evans, P. Gardiner, M. Grocott, P. Gunning, N. Hart, J. Harrington, J. Harvey, C. Holloway, D. Howard, D. Hurlbut, C. Imray, C. Ince, M. Jonas, J. van der Kaaij, M. Khosravi, N. Kolfschoten, D. Levett, H. Luery, A. Luks, D. Martin, R. McMorrow, P. Meale, K. Mitchell, H. Montgomery, G. Morgan, J. Morgan, A. Murray, M. Mythen, S. Newman, M. O’Dwyer, J. Pate, T. Plant, M. Pun, P. Richards, A. Richardson, G. Rodway, J. Simpson, C. Stroud, M. Stroud, J. Stygal, B. Symons, P. Szawarski, A. Van Tulleken, C. Van Tulleken, A. Vercueil, L. Wandrag, M. Wilson, J. Windsor.
Scientific Advisory Group: B. Basnyat, C. Clarke, T. Hornbein, J. Milledge, J. West.
Members of the Xtreme Everest 2 Research Group are as follows:
S Abraham, T Adams, W Anseeuw, R Astin, B Basnyat, O Burdall, J Carroll, A Cobb, J Coppel, O Couppis, J Court, A Cumptsey, T Davies, S Dhillon, N Diamond, C Dougall, T Geliot, E Gilbert-Kawai, G Gilbert-Kawai, E Gnaiger, M Grocott, C Haldane, P Hennis, J Horscroft, D Howard, S Jack, B Jarvis, W Jenner, G Jones, J van der Kaaij, J Kenth, A Kotwica, R Kumar BC, J Lacey, V Laner, D Levett, D Martin, P Meale, K Mitchell, Z Mahomed, J Moonie, A Murray, M Mythen, P Mythen, K O’Brien, I. Ruggles-Brice, K Salmon, A Sheperdigian, T Smedley, B Symons, C Tomlinson, A Vercueil, L Wandrag, S Ward, A Wight, C Wilkinson, S Wythe.
Scientific Advisory Board: M Feelisch, E Gilbert-Kawai, M Grocott (chair), M Hanson, D Levett, D Martin, K Mitchell, H Montgomery, R Moon, A Murray, M Mythen, M Peters.
Competing interest
Can Ince has developed SDF imaging and is listed as inventor on related patents commercialized by MicroVision Medical (MVM) under a license from the Academic Medical Center (AMC). He has been a consultant for MVM in the past, but has not been involved with this company for more than 5 years now, except that he still holds shares. Braedius Medical, a company owned by a relative of Can Ince, has developed and designed a hand held microscope called CytoCam-IDF imaging. Dr Ince has no financial relation with Braedius Medical of any sort; he had never owned shares, or received consultancy or speaker fees from Braedius Medical. The authors declare that they had no competing interests.
Authors’ contributions
E G-K: Design of study, conduct of study, analysis of data, writing manuscript. JC: Conduct of study, analysis of data, writing manuscript. VB: Analysis of data, writing manuscript. CI: Design of study, writing manuscript. DM: Analysis of data, writing manuscript. All authors read and approved the final manuscript.