Optotracing with EbbaBiolight

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 ^[3], β-glucans from S. cerevisiae and Chitin in C. albicans ^[4]. 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 (Em_max) when excited at or close to the excitation maximum (Ex_max). 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, Ex_max or Em_max 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. EbbaBiolight excitation- and emission spectra can be accessed here.

Table: EbbaBiolight spectral properties with maximum excitation (Ex_max) and emission (Em_max) 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 filter-sets
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	530 nm	680 nm	300 - 650 nm	660 - 800 nm	PI, mCherry, Cy3.5

Read More:

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