Multiplexing Carbotrace and Antibodies
When trying to understand and build in a world reliant on polymers, plant tissue is at the forefront of green chemistry with the abundance provided by polysaccharides. Techniques for the analysis of structures, morphology and localisation of different carbohydrates provides deep insight in a variety of fields, making in depth imaging and visualising crucial to basic research.
When working with varied polysaccharides or other structural elements like proteins, researchers need to be able to distinguish components in complex environments such as within cells or in multicellular organisms reliably, which requires specificity.
Currently one of the most commonly used techniques are antibodies. Antibodies are proteins employed by the immune system of animals to bind and mark antigens which are alien to the host's body such as viruses or foreign bacteria. These proteins can be functionalised by the binding of a fluorescent marker. Antibodies however tend to be large and require a specific target to be effective, working best with proteins leaving polysaccharides largely under tagged.
Addressing this gap, Carbotrace can be used to target the native glycosidic linkages found in unbranched or moderately branched polysaccharides. Carbotrace are small structure-responsive fluorogenic molecules, highly fluorescent when binding and presenting a unique spectral fingerprint depending on polysaccharide configuration. This manifests in blue or redshift of the fluorescent spectrum. Using this technique several glucans can be imaged and identified in parallel.
A strong advantage is the seamless way that antibodies and Carbotrace can be paired or multiplexed when used on the same tissues. This provides in-depth localisation across a variety of polysaccharides and proteins. This versatility is possible by pairing different fluorescent channels, allowing further analysis with established methods with little disruption.
To exemplify this, Plachno and colleagues from the Jagellonian University, in Poland, investigated gland cell walls in Venus flytraps by multiplexing protein specific cell wall antibodies and Carbotrace 680 (see image). The researchers found specialised arabinogalactans and hemicellulose distributions within delicate in-grown regions that identified modulable composition in cell walls. In conclusion, multiplexing Carbotrace and Antibodies provides a simple method to build a deeper understanding into plant tissues by simultaneously exploring carbohydrates and proteins.
Image: Basal cell (Bc), stalk cell (Sc), secretory cells (white star) and glandular cells (green star) were all stained with Carbotrace 680 (in red) and LM15 antibody (in green) to identify cell wall ingrown composition. Scale bar: 10 µm. Image adapted from Figure 6B by Płachno, B. et al. (2026) International Journal of Molecular Sciences, 25(23), 13124. (CC BY 4.0).
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Monitoring heat-induced swelling of Starch granules
As starch is a thermo-sensitive material that undergoes drastic morphological changes when heated, it is even possible to monitor starch reorganization during heat-induced swelling. When freshly extracted potato starch granules marked with Carbotrace 680 are heated from 20 °C to 90 °C and imaged with a confocal microscope, it becomes obvious that no change in granule size and morphology occurs until the granules are heated to 60 °C. At temperatures higher than 60 °C swelling is observed which can be identified by growing starch granules, but also by a visible change in the fluorescence color and intensity produced by Carbotrace 680 (See image).
Fluorescence intensity (RFU) and color (determined by the wavelength at the excitation maximum, λ - Exmax) can be easily quantified using a fluorescence plate reader. This method greatly improves and simplifies the detection of heat-induced morphological changes in starch, since minor rearrangements in starch packing and swelling can be identified. While the RFU gives an indication on the onset of swelling, λ - Exmax identifies early, heat-induced starch rearrangement, long before morphological changes become visible.
Image: Freshly extracted potato starch granules marked with Carbotrace 680 are heated from 20 °C to 90 °C and the fluorescence intensity (RFU, circels) and color (λ - Exmax, triangles) are quantified using a fluorescence plate reader. The image is reproduced from Choong et al. 2019 (CC BY 4.0).
Detailed analysis of starch structure and morphology enabled by Carbotrace 680 can facilitate quality management of starch raw materials for use in food and pharmaceutical industries where starch reaction with water plays a pivotal role for texture and taste as well as for packing and release kinetics of pharmaceuticals.
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Carbotrace fluorescence spectra
We named our Carbotrace molecules after their peak emission wavelength when they are bound to their target. That means, when Carbotrace is bound to a target, it will emit fluorescence at peak emission indicated by the number associated with its name.
To view the excitation and emission spectra, please select your Carbotrace below :
Carbotrace 680 for monitoring starch
Detection of polysaccharides using Carbotrace 680 relies on regularly occurring units joined by glycosidic linkages. The specific type of glycosidic linkage and the occurrence of branches allow Carbotrace 680 to differentiate between different types of glucans. By means of its structure-responsive properties, Carbotrace 680 can differentiate between β(1-4) linked cellulose and α(1-4) configured amylose, but it can also detect differences between amylose and amylopectin, which contains α(1-6) linked branches.
Since starches from different sources often differ by the length of glucose chains or the amylose/amylopectin ratio, and the protein and fat content of the storage organs, Carbotrace 680 can distinguish between different source of starch like starch obtained from corn or potato.
Monitoring freshly obtained potato starch granules with Carbotrace 680 shows very fine details of the morphology of native starch granules at a sub-cellular scale. The movie shows Carbotrace 680 fluorescence when bound to potato starch granules while stepping through a stack of images collected on the confocal microscope. The most intense green fluorescence is observed on the exposed surface and the outer layer of granules, decreasing towards the core. Faint striations, likely corresponding to alternating layers in crystalline and amorphous lamina were observed in the larger granules. This pattern is likely attributed to differences in polysaccharide packing density in each lamina.
Movie: Carbotrace 680 fluorescence when bound to potato starch granules while stepping through a stack of images collected on the confocal microscope. The movie is reproduced from Choong et al. 2019 (CC BY 4.0)