Multispectral in-line hologram reconstruction with aberration compensation applied to Gram-stained bacteria microscopy

Abstract

In multispectral digital in-line holographic microscopy (DIHM), aberrations of the optical system affect the repeatability of the reconstruction of transmittance, phase and morphology of the objects of interest. Here we address this issue first by model fitting calibration using transparent beads inserted in the sample. This step estimates the aberrations of the optical system as a function of the lateral position in the field of view and at each wavelength. Second, we use a regularized inverse problem approach (IPA) to reconstruct the transmittance and phase of objects of interest. Our method accounts for shift-variant chromatic and geometrical aberrations in the forward model. The multi-wavelength holograms are jointly reconstructed by favouring the colocalization of the object edges. The method is applied to the case of bacteria imaging in Gram-stained blood smears. It shows our methodology evaluates aberrations with good repeatability. This improves the repeatability of the reconstructions and delivers more contrasted spectral signatures in transmittance and phase, which could benefit applications of microscopy, such as the analysis and classification of stained bacteria.

Figure 1

Experimental principle of our study: Left: experimental configuration of DIHM with plane wave illumination and defocused imaging of both calibration beads and biological objects of interest. Center: Example of a conventional color image of a Gram-stained blood smear showing red blood cells, Gram positive and Gram negative bacteria and calibration beads. Right: Corresponding diffraction patterns of these objects illustrated on the multispectral hologram stack.

Figure 2

Examples of the pupil function estimated for the whole set of wavelengths. For easier visualization of the aberrations, the radial components (defocus and spherical aberrations) and non-radial components (tilt, coma, astigmatism, trefoil and quadrafoil) are presented separately at the bottom of the figure. In the top-left inset, ${\nu }_x$ and ${\nu }_y$ are the spatial frequencies in Fourier space.

Figure 3

Left: Illustration of the evolution of the estimated ATFs in the field of view at $\lambda =532\text{nm}$. Right: Further details on the location (slide 1, 2 or 3) of the beads used in the ATF estimation.

Figure 4

Illustration of transmittance and OPD color reconstructions of a sample containing two types of bacteria (Gram positive Staphylococcus aureus and Gram negative Escherichia coli). (A) Full field. (B) Regularized reconstructions reconstructed independently at each $\lambda$, with no correction of aberrations and at a fixed focus estimated at $431\text{nm}$. (C) Multi-wavelength joint reconstructions with aberration correction and with the colocalization prior.

Figure 5

Illustration of the wavelength dependent lateral shifts and shape distortions of a bacterium (Escherichia coli) on uncorrected reconstructions (A) compared with our aberration correction methodology (B).

Figure 6

Illustrations of the spectral signatures in transmittance and OPD for 2 Gram positive and 2 Gram negative bacteria. (A) Uncorrected, independent reconstructions at each wavelength with a fixed focus evaluated at 431 nm. (B) Uncorrected, independent reconstructions with estimation of the focus at each wavelength. (C) Aberration corrected method with aberration corrected estimation of the focus at each wavelength and aberration corrected multi-wavelength joint reconstructions using the colocalization regularization. Bottom-right:  Typical white light images of the 4 species studied here.

Figure 7

Schematic illustration of our DIHM regularized reconstruction methodology with the calibration of aberrations (A) and aberration corrected multispectral reconstructions (B).

Figure 8

Illustration of the home-made automated DIHM setup used in this work with 8 wavelength partially coherent illumination: (a) fibered 8-LED light source, (b) filter wheel, (c) sample holder, (d) apochromatic objective, (e) beamsplitter T70/R30, (f) defocused monochrome camera, (g) incoherent light, (h) PIFOC piezo focus scanner, (i) in-focus color camera.