Quantitative phase imaging by gradient retardance optical microscopy

Abstract

Quantitative phase imaging (QPI) has become a vital tool in bioimaging, offering precise measurements of wavefront distortion and, thus, of key cellular metabolism metrics, such as dry mass and density. However, only a few QPI applications have been demonstrated in optically thick specimens, where scattering increases background and reduces contrast. Building upon the concept of structured illumination interferometry, we introduce Gradient Retardance Optical Microscopy (GROM) for QPI of both thin and thick samples. GROM transforms any standard Differential Interference Contrast (DIC) microscope into a QPI platform by incorporating a liquid crystal retarder into the illumination path, enabling independent phase-shifting of the DIC microscope's sheared beams. GROM greatly simplifies related configurations, reduces costs, and eradicates energy losses in parallel imaging modalities, such as fluorescence. We successfully tested GROM on a diverse range of specimens, from microbes and red blood cells to optically thick (~ 300 μm) plant roots without fixation or clearing.

Figure 1

(a) GROM illustration combining a liquid–crystal retarder (LC) with a standard differential interference contrast (DIC) microscope that includes polarizers (P1 and P2), Wollaston prisms (WP1 and WP2), condenser (CD), sample (SS) and objective (OL). The optical path illustrates the e- and o-waves generated at WP1 and recombining at WP2. (b) An example 3D phase image of 500 nm polystyrene particles immersed in water; scale bar represents the phase delay in radians, which, in this case, depends on the local particle size; the image is composed of a z-stack of 12 layers, each separated by 200 nm.

Figure 2

(a) Gradient-phase (∇_xΦ) and (b) reconstructed quantitative-phase (ΔΦ) images at 40 × of ~ 1 μm polystyrene particles embedded in immersion oil, with the corresponding 2D maps of an individual particle highlighted by the square in (c,d), respectively. (d) These data denote a peak phase value of 0.65–0.7 rad, in agreement with the expected value of 0.68 rad.

Figure 3

Full-width half maximum (FWHM) of polystyrene particles on a glass coverslip at (a) 10× magnification (1 μm diameter particles), (b) 20× magnification (1 μm diameter), and (c) 40× magnification (200 nm diameter) denoting the planar resolution; blue thick lines depict the experimentally determined mean values and the blue shaded areas the 95% confidence intervals, while the legend notes the average (± s.e.) of n = 20 observations.

Figure 4

GROM images of red blood cells (a), Escherichia coli (b), and Yarrowia lipolytica (c). All calibration bars are in radians.

Figure 5

Gradient (a) and quantitative (b) phase images of a live Medicago truncatula root tip using GROM. The corresponding SLIM image of the same root segment is displayed in (c). The red square at the root tip in (b) indicates the location of starch granules. (d) Select z-sections of the image presented in (b); inset displays the different z-planes relative to the central plane at 0 μm.

Figure 6

GROM image of a live Medicago truncatula root; the image represents a single plane from a 3D stack; scale bar represents the image contrast in refractive index units. At its thickest part (white rectangle), the root has a diameter of 325 μm; inset plots the radially averaged phase (ΔΦ) from the left to the right.