A practical criterion for focusing of unstained cell samples using a digital holographic microscope

Abstract

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.

Accurate holographic cytometry using three-dimensional hydrodynamic focusing

“…The 3D focusing of cells in a single file not only allows imaging of all the cells in a single focal plane but also prevents cell-clustering observed in previous works on DHM-based IFC [21,22]. Additionally, we used a hologram domain criterion of least amplitude modulation of interference fringes [26], to ensure that the focus plane of the microscope objective lens nominally coincided with the hydrodynamic focus plane of our device. Therefore, image reconstruction using DHM in our experiments did not require numerical refocusing along the depth of the microchannel.…”

Standardization Techniques for Single-Shot Digital Holographic Microscopy

“…Computational refocus is also not possible in the bright-field mode as the recorded image does not contain any phase information. The focusing problem can be addressed in a clever manner by employing a focusing criterion directly in the hologram domain [21]. Unstained samples can be considered to be nearly pure phase objects in the focus plane, and this implies that the interference fringes in the focus-plane hologram will predominantly show phase modulation.…”

Automatic numerical focus plane estimation in digital holographic microscopy using calibration beads

“…Even if the reconstruction of the wave is possible anywhere in the object space in in-line holographic microscopy, it is important to find a criterion that can locate a refocusing plane in a reproducible and physical meaningful way, irrespective of the method used for the reconstruction. With traditional focusing criteria [8][9][10][11][12][13][14], this location is likely to vary depending on the density, spatial distribution, size, and transmission of the objects.…”