Using DNA-PAINT Super-Resolution Microscopy
A recently published paper discusses how DNA-PAINT super-resolution microscopy is used in research involving spore-forming bacteria. The paper is entitled, “Comparing divisome organization between vegetative and sporulating Bacillus subtilis at the nanoscale using DNA-PAINT.“
Three filters from Chroma Technology are included in the microscope setup.
The paper’s Abstract and portions of the Setup section follow.
Spore-forming bacteria have two distinct division modes: sporulation and vegetative division. The placement of the foundational division machinery component (Z-ring) within the division plane is contingent on the division mode. However, investigating if and how division is performed differently between sporulating and vegetative cells remains challenging, particularly at the nanoscale. Here, we use DNA-PAINT super-resolution microscopy to compare the 3D assembly and distribution patterns of key division proteins SepF, ZapA, DivIVA, and FtsZ. We determine that ZapA and SepF placement within the division plane mimics that of the Z-ring in vegetative and sporulating cells. We find that DivIVA assemblies differ between vegetative and sporulating cells. Furthermore, we reveal that SepF assembles into ~50-nm arcs independent of division mode. We propose a nanoscale model in which symmetric or asymmetric placement of the Z-ring and early divisome proteins is a defining characteristic of vegetative or sporulating cells, respectively, and regulation of septal thickness differs between division modes.
DNA-PAINT Microscope Setup
DNA-PAINT imaging was carried out on an inverted microscope, applying an objective-type total internal reflection fluorescence (TIRF) configuration equipped with an oil-immersion objective. A 488-nm 200 mW or 561-nm 200 mW laser was used for excitation and was coupled into a single-mode fiber. The laser beam was passed through cleanup filters (Chroma Technology, ZET561/10) and coupled into the microscope objective using a beam splitter (Chroma Technology, ZT561rdc). Fluorescence light was spectrally filtered with an emission filter (Chroma Technology, ET600/50 m) and imaged with a scientific complementary metal-oxide semiconductor camera without further magnification, resulting in an effective pixel size of 130 nm after 2 × 2 binning. The camera readout sensitivity was set to 16-bit, and readout bandwidth to 540 MHz. Three-dimensional imaging was performed using a cylindrical lens in the detection path.
About Chroma Technology
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