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Although biological soil crusts can be found in open landscapes worldwide, their species composition depends on soil properties such as texture and pH, on microclimate, and their respective developmental stage. In addition, local variations in water holding capacity and/or chemical properties of soils influence the formation of spatial patterns and different types of biocrusts on the landscape level. For the evaluation of biocrusts functions and their impact on soil carbon pools, the analysis of the interrelationship between photosynthetic activity and the variations of spatial distribution pattern and types of biocrust is indispensable. For this purpose, an image processing approach was applied that combined chlorophyll fluorescence analyses and multispectral BNDVI to comprehensively characterize the spatial patterns of photosynthetic hotspots in biological soil crusts. For image analysis, five biological soil crust samples with different ratios of substrate, mosses and lichens were collected on an inland dune system in Lieberose, dominated by the moss Polytrichum piliferum, and the lichens Cladonia fimbriata and C. coccifera. RGB-images of the biocrusts were taken with a standard consumer camera Nikon 5200, BNDVI images with a modified Canon S110 NIR camera and chlorophyll fluorescence images with a modular open FluorCAM FC 800-0/1010, respectively. BNDVI and F-v/F-m were nearly in the same range for all biocrust samples related to the total surface area. Although mosses showed higher BNDVI than lichens within the separate biocrust samples. F-0 and F-m increased with species coverage and with advancing biocrust development. Overlapping of BNDVI with F-0 and F-m images showed that not all crustal organisms contribute to BNDVI and chlorophyll fluorescence. The overlapping areas of BNDVI and F-0 ranged between 13% and 29%, that of BNDVI and F-m between 17% and 47%. Matching of RGB, BNDVI and CFI allows visualizing spatial pattern with high or low photosynthesis in biocrusts.