LCI Light Microscopy Course: Noise Level versus Averaging Dataset

This dataset was created for the online microscopy course given by the Live Cell Imaging core facility, Karolinska Institutet, Sweden.

Images in this dataset were acquired with the goal of demonstrating the effect different levels of noise have on image analysis. 

To obtain the images labelled Nuclei, HeLa cells were seeded on a #1.5 coverslip and allowed to adhere for 24 hr. The cells were fixed with 4% PFA for 10 min., rinsed with PBS then labelled with the DNA dye DAPI (Molecular probes, F24630) at 300 nM for 5 min. The sample was then mounted on a glass slide using the Dako mounting medium (Merck, F4680) and allowed to harden for 24 hr before imaging.

The Nuclei sample was imaged using a Nikon Ti2 widefield microscope using a 20x/0.75 air objective, a Photometrics Kinetix sCMOS camera (pixel size 6.5 um, resulting in about 1.5x undersampling) and a Lumencore Celesta light source. The excitation laser was 405 nm and the emission filter 438/24 nm. The laser power and exposure time were adjusted to have no saturation in the images.

To obtain the images labelled Tissue,  a 16 μm cryostat section of mouse kidney stained with a combination of fluorescent dyes. Alexa Fluor 488 wheat germ agglutinin, a green-fluorescent lectin, was used to label elements of the glomeruli and convoluted tubules. The filamentous actin prevalent in glomeruli and the brush border were stained with red-fluorescent Alexa Fluor 568 phalloidin ((Molecular probes, F24630). Finally, the nuclei were counterstained with the blue-fluorescent DNA stain DAPI.The samples were mounted in ProLong antifade mounting medium (Molecular proves, P36965). 

The Tissue sample was imaged using a Nikon TiE widefield microscope a 20x/0.75 air objective, an Andor Zyla 4.2 camera (pixel size 6.45 um, rusulting in about 1.5x undersampling) and a Lumencore Spectra X light source. The laser power and exposure time were adjusted to have no saturation in the images. The excitation and emission wavelengths were as follows:

• DAPI: Ex: 395/25; Emission filter: 447/60;

• Alexa Fluor 488: Excitation filter: 475/28; Emission filter: 500LP;

Images named no_avg were acquired without averaging. Images named NNx_avg, were acquired with different (indicated by NN) levels of averaging.