He egfp filter set 38

Microscopy was performed using a Zeiss Axio Observer.Z1 inverted microscope equipped with an Axiocam 506 mono camera and Neofluar10x/0.3 Ph1/DICI (Hydroides co-cultures) or Apochromat 100×/1.4 Oil DICIII (bacteria only) objectives. The Zeiss HE eGFP filter set 38 was used to capture GFPoptim-1 expression and Zeiss HE mRFP filter set 63 was used to capture mRuby2 expression. For Nanoluciferase controls, images were captured using the same fluorescence exposure times as the gfp optim-1 and mRuby2 labeled strains of the same species.
Bacterial culture (2 µL) was added to freshly prepared 1% saltwater low-melt agarose (Apex catalog #20-103, Bioresearch products) pads on glass slides and coverslips were placed on top. Hydroides elegans were prepared in visualization chambers (Lab-Tek Chambered Coverglasses catalog #155411PK) with bacteria and imaged.

Entire biofilm colonies were analyzed using an Axio Zoom V16 stereomicroscope (Carl, Zeiss, Jena, Germany) equipped with a Zeiss CL 9000 LED light source and an AxioCam MRm monochrome camera (Carl Zeiss). Images were captured at ×5 magnification using both wide-field and fluorescence mode; for the latter, the HE eGFP filter set #38 (Carl Zeiss, excitation at 470/40 nm and emission at 525/50 nm) was used to image GFP fluorescence. Relative fluorescence mode values were calculated by subtracting the fluorescence mode of each biofilm colony from its respective (average) background fluorescence mode. For each growth day (B = 3), an average background fluorescence mode was calculated from a minimum of two WT biofilm colonies imaged with the GFP filter. Throughout the manuscript, relative fluorescence intensity mode values are referred to as “fluorescence intensity values” for simplicity.
The fluorescence of purified GFP (Merck, Darmstadt, Germany) mixed with solutions of 1.5 mM CuSO₄ or 0.5 mM ZnCl₂ was analyzed both at acidic and neutral pH using a Victor³ plate reader (PerkinElmer, MA, USA). Neutralization of the solutions containing GFP was performed using Tris buffer and NaOH.