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SummaryDigital holographic microscopy (DHM) is an important technique that may be used for quantitative phase imaging of unstained biological cell samples. Since the DHM technology is not commonly used in clinics or bioscience research labs, at present there is no well‐accepted focusing criterion for unstained samples that users can follow while recording image plane digital holograms of cells. The usual sharpness metrics that are useful for auto‐focusing of stained cells do not work well for unstained cells as there is no amplitude contrast. In this work, we report a practical method for estimating the best focus plane for unstained cells in the digital hologram domain. The method is based on an interesting observation that for the best focus plane the fringe pattern associated with individual unstained cells predominantly shows phase modulation effect in the form of bending of fringes and minimal amplitude modulation. This criterion when applied to unstained red blood cells shows that the central dip in the doughnut‐like phase profile of cells is maximal in this plane. The proposed methodology is helpful for standardizing the usage of DHM technology across different users and application development efforts.Lay DescriptionDigital holographic microscopy (DHM) is slowly but steadily becoming an important microscopy modality and gaining acceptability for basic bio‐science research as well as clinical usage. One of the important features of DHM is that it allows users to perform quantitative imaging of unstained transparent cells. Instead of using dyes or fluorescent labelling, DHM systems use quantitative phase as a contrast mechanism which depends on the natural refractive index variation within the cell samples. Since minimal wet lab processing is required in order to image cell samples with a DHM, cells can be imaged in their natural state. While DHM is gaining popularity among users, the imaging protocols across the labs or users need to be standardized in order to make sure that the same quantitative phase parameters are used for tasks such as quantitative phased based cell classification.One of the important operational tasks for any microscopy work is to focus the sample under study. While focusing comes naturally to users of brightfield microscopes based on image contrast, the focusing is not straightforward when samples are unstained so that they do not offer any amplitude contrast. When performing quantitative phase imaging, defocus can actually change the phase profile of the cell due to near‐zone (Fresnel) diffraction effects. So unless a standardized focusing methodology is used, it will be difficult for multiple DHM users (potentially at different sites) to agree on quantitative results out of their phase images. DHM literature has prior works which perform numerical focusing of recovered complex wave‐field in the hologram plane to find the best focus plane. However such methods are not user friendly and do not allow user the same focusing experience as in a brightfield microscope. The numerical focusing is therefore a reasonably good method for an optics researcher but not necessarily so for a microscopy technician looking at cell samples with a DHM system in a clinical setting.The present work provides a simple focusing criterion for unstained samples that works directly in the hologram domain. The technique is based on an interesting observation that the when an unstained cell sample is in the best‐focus plane, its corresponding hologram (or fringe pattern) predominantly shows phase modulation manifested by bending of fringes at the location of the cell. This criterion can be converted into a simple numerical method as we have used to find the best‐focus plane using a stack of through focus holograms. We believe that the technique can be used manually by visually observing the holograms or can be converted to an auto‐focus algorithm for a motorized DHM system.

Pendant droplets of water and paramagnetic solutions are studied in the presence of uniform and nonuniform magnetic fields produced by small permanent magnet arrays, both in static conditions and during dynamic pinch-off. Static measurements of the droplet shape are analysed in terms of an apparent surface tension γapp or an effective density ρeff. The change of surface tension of deionized water in a uniform field of 450 mT is insignificant, 0.19 {plus minus} 0.21 mNm-1. Measurements on droplets of compensated zero-susceptibility solutions of Cu2+, Mn2+ and Dy3+ where the shape is unaffected by any magnetic body force show changes of surface tension of about -1% in 500 mT. Magnetic field gradients of up to 100 T2m-1 deform the droplets and lead to changes of ρeff that are negative for diamagnetic solutions (buoyancy effect) and positive for paramagnetic solutions. The droplet profile of strongly-paramagnetic 0.1 Dy M DyCl3 solution is analysed, treating the nonuniform vertical field gradient as a spatial variation of gravity. The influence of Maxwell stress on droplet shape is discussed. In dynamic measurements, the droplet shape at pinch-off is recorded by high-speed photography and analysed in terms of a relative change of dynamic surface tension in the presence of a magnetic field. The surface-tension-dependent pre-factor of the scaling law that governs the pinch-off dynamics shows no difference for pure water or 0.11 M DyCl3 solutions in the field. The nonuniform field has no influence in the pinch-off region because the filament diameter is much less than the capillary length.