Visualization of the binding pattern of monoclonal antibodies against Klebsiella pneumoniae | SIMTRUM Photonics Store

Fondazione Toscana  Life  Sciences  (TLS)  is a no-profit organization based in Siena  (Italy) which supports research activities in  the Life Sciences, from basic research to  industrial application. Among their projects,  the Monoclonal Antibody Discovery (MAD)  Lab at TLS is focused on identification and  production of monoclonal antibodies(mAbs) as novel therapeutics against  bacteria and viruses. mAbs have various advantages when compared to standard  medications: for instance, their specificity, a  limited risk of resistance development, and  ability to work synergistically with antibiotics.  In addition, mAbs help the identification of vaccine targets and accelerate vaccine  development, thus representing excellent  tools to fight infectious diseases.

A relevant aspect of the mAbs research field is the validation of the binding efficiency of the antibody to the bacterial surface.  Here we show data of a 488-fluorophore-conjugated mAb isolated at TLS binding to Klebsiella pneumoniae expressing mCherry. Acquisitions were obtained with  SIMTRUM SpinDisk Advance confocal spinning disk system coupled with the SIM Basic Super-Resolution add on, Celesta laser  source (Lumencor) and sCMOS Prime BSI  Camera (Photometrics, 6.5 μm pixel size). We used 100x oil objective (Nikon, 1.45 NA) and performed Z-stack acquisitions along the bacterial volume (0.1 μm Z step size).

In Figure 1 we show the maximum intensity projection (MIP) of a Z stack of bacterial cells acquired with SIMTRUM SpinDisk Advance confocal spinning disk. To be more accurate in the  identification of the mAb binding pattern on  the bacterial surface, we analyzed the sample  with the SIM Basic Super-Resolution add on of SpinDisk Advance spinning disk and we visualize  bacteria as a 3D volume. The SIM Basic super resolution module is easily integrated into existing microscope systems to provide structured localization imaging of complex biological specimens using routine sample  preparation protocols. In Figure 2 we show a comparison between the confocal spinning disk and the super-resolution acquisition by displaying 3D volume views and highlighting cellular details appreciable only in the super-  resolved image, especially regarding the mAb  staining (green).

To demonstrate how SIMTRUM SIM Basic confers significant gains over traditional microscopy, we report a global comparison  of different microscopy methods ranging  from widefield, to confocal spinning disk  to super-resolution acquisitions (Figures  3-4). Of note, the monoclonal antibody 3D organization, the alternation of aggregates and even small, detailed protrusions are  detectable and certainly clearer in the super-  resolved image compared to widefield, original and deconvolved spinning disk data.  Overall, the screening of the binding quality and efficiency between antibodies and bacteria is an excellent biological example  suited for spinning disk microscopy. After selecting the promising antibodies, the possibility to easily switch to super-resolution microscopy inside the same set-up configuration helps researchers to deepen the data and make further biological considerations. SIMTRUM SpinDisk Advance spinning disk configuration with the SIM Basic module is the perfect solution  for every scientific need that requires  resolution enhancement at specific steps  of the experimental protocol, without the  need to move to a different microscope.