Improving axial resolution for holographic tracking of colloids and bacteria over a wide depth of field by optimizing different factors

Abstract

Improving the axial resolution for multiparticle three-dimensional (3D) holographic tracking is crucial but challenging. Here we study the impacts of incident light power, uniformity of the illumination as well as image pixel size on the axial tracking resolution for digital holographic microscopy (DHM). We demonstrate that the resolution highly depends on the image pixel size and the uniformity of the illumination. A 3D localization algorithm based on local-intensity-maxima searching and a Gaussian fit to the integrated intensity of the reconstructed lateral images along the axial direction proves a robust strategy to enhance the axial resolution for colloids and bacteria within a wide depth of field over several tens of micrometers.

Adaptive Escape of Pseudomonas aeruginosa by Application of Low-Amplitude Electric Pulses

“…Hydroxyl radicals and hydrogen bubbles have both been confirmed to influence bacteria activity. , Although the agar diffusion assay experiment confirmed that the effect of the pH (7.0–8.0) on bacterial growth activity is minimal, the influence of OH – on its motility cannot be ruled out. We failed to observe any microbubbles larger than 200 nm formed on the surfaces throughout the entire experiment under all the EP conditions because DHM has a resolution of 200 nm for 40× objective (NA = 0.6) . This suggests that the pressure of hydrogen gas generated by EP is less than the saturated vapor pressure in the solution so H 2 will be resolved once they are generated instead of forming bubbles.…”

“…We failed to observe any microbubbles larger than 200 nm formed on the surfaces throughout the entire experiment under all the EP conditions because DHM has a resolution of 200 nm for 40× objective (NA = 0.6). 26 This suggests that the pressure of hydrogen gas generated by EP is less than the saturated vapor pressure in the solution so H 2 will be resolved once they are generated instead of forming bubbles.…”

“…To fill the abovementioned knowledge gap, a unique optical technique, digital holographic microscopy (DHM), allowing real-time observation of the 3D behavior of swimming bacteria, 19,26,27 was utilized to track the 3D motions of PAO1 during and after EP application. DHM has been used to study the impact of dead bacteria on the 3D movement of live bacteria and the influence of rough surfaces on human sperm migration.…”

Role of Surface Coverage of Sessile Probiotics in Their Interplay with Pathogen Bacteria Investigated by Digital Holographic Microscopy

“…The DHM used in this study was based on an in-line configuration integrated with an inverted microscope (IX-83, Olympus) for the 3D dynamic tracking of planktonic ES, as described previously. − The chamber containing the ES suspension was illuminated in a defocused way using a collimated light-emitting diode (LED, λ = 505 nm, Thorlabs, USA). After equilibrium for 10 min, the focal plane was located 10 μm beneath the LGG bacterial layer of different densities.…”

Integration of polymeric membrane/dielectric sphere assemblies in microfluidic chips for enhanced-contrast imaging with low-magnification systems

“…3,16 For applications that target the detection of large objects (e.g., bacteria or cells) instead of small objects (e.g., biomolecule, nanoparticles or subcellular structures), a large depth-of-field is usually required. 18,19 Additionally, for applications that require long-term imaging of living microorganisms, sample handling and immobilization often constitute a significant challenge. Several microfluidic approaches have been proposed to facilitate such analyses, e.g., single-cell arraying 20 and single bacteria immobilization.…”