Optotracing with EbbaBiolight

EbbaBiolight Fluorescence Detection:

EbbaBiolight fluorescent tracer molecules are optotracers. Unlike conventional fluorescent dyes, optotracers bind promiscuously to a range of targets with repetitive motifs. EbbaBiolight has been shown to bind to curli and cellulose in Salmonella extracellular matrix [1,2], peptidoglycan and lipoteichoic acids in the cell envelope of Staphylococci [2], β-glucans from S. cerevisiae and Chitin in C. albicans [3]. Upon binding, the fluorescence intensity of the optotracer increases. This property makes it possible to use EbbaBiolight for live fluorescent tracking of microorganisms, without the need to wash away unbound molecules. It is possible to read out fluorescence intensity at the emission maximum (Emmax) when excited at or close to the excitation maximum (Exmax). This is useful for microscopy or fluorescence spectroscopy when straight-forward data analysis is required. Yet, due to the unique properties of the optotracers, a unique optical fingerprint is produced reflecting the specific nature of the target (sample composition) and environment (pH, osmolarity, polarity of the medium). This means that depending on the specific properties of the sample, Exmax or Emmax can shift, or the appearance of double peaks or shoulders might indicate binding to multiple targets. We therefore recommend acquiring fluorescence excitation and emission spectra whenever possible within experimental limitations.

Table: EbbaBiolight spectral properties with maximum excitation (Exmax) and emission (Emmax) when bound and recommended range for acquisition of excitation- and emission spectra as well as recommended filter sets for microscopy.
Exmax Emmax Excitation spectrum (detect at Emmax) Emission spectrum (excite at Exmax) Recommended
EbbaBiolight 480 420 nm 480 nm 300 - 450 nm 450 - 800 nm DAPI
EbbaBiolight 520 460 nm 520 nm 300 - 490 nm 490 - 800 nm FITC, GFP
EbbaBiolight 540 480 nm 540 nm 300 - 510 nm 510 - 800 nm FITC, GFP, YFP
EbbaBiolight 630 520 nm 630 nm 300 - 600 nm 550 - 800 nm PI, Cy3, TxRed, mCherry, Cy3.5
EbbaBiolight 680 630 nm 680 nm 300 - 650 nm 660 - 800 nm PI, mCherry, Cy3.5

Access EbbaBiolight spectra

EbbaBiolight excitation- and emission spectra can be accessed here.


  1. Choong FX et al. (2016) Real-Time optotracing of curli and cellulose in live Salmonella biofilms using luminescent oligothiophenes. npj Biofilms and Microbiomes, 2, 16024
  2. Choong FX et al. (2021) A semi high-throughput method for real-time monitoring of curli producing Salmonella biofilms on air-solid interfaces. Biofilm, 3, 100060
  3. Butina K. et al. (2020) Optotracing for selective fluorescence-based detection, visualization and quantification of live S. aureus in real-time. npj Biofilms and Microbiomes, 6(1), 35
  4. Kärkkäinen, E. et al. (2022) Optotracing for live selective fluorescence-based detection of Candida albicans biofilms. Frontiers in Cellular and Infection Microbiology, 12, 2235-2988

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