Enhanced quantitative phase imaging in self-interference digital holographic microscopy using an electrically focus tunable lens

Abstract

Self-interference digital holographic microscopy (DHM) has been found particular suitable for simplified quantitative phase imaging of living cells. However, a main drawback of the self-interference DHM principle are scattering patterns that are induced by the coherent nature of the laser light which affect the resolution for detection of optical path length changes. We present a simple and efficient technique for the reduction of coherent disturbances in quantitative phase images. Therefore, amplitude and phase of the sample illumination are modulated by an electrically focus tunable lens. The proposed method is in particular convenient with the self-interference DHM concept. Results from the characterization of the method show that a reduction of coherence induced disturbances up to 70 percent can be achieved. Finally, the performance for enhanced quantitative imaging of living cells is demonstrated.

Keywords: (090.1995) Digital holography; (110.0180) Microscopy; (110.1650) Coherence imaging; (120.5050) Phase measurement; (170.1530) Cell analysis.

Fig. 1

Self-interference DHM setup with modulated object illumination by an electrically focus tunable lens. SM, single mode fiber, delivering light from a frequency doubled Nd:YAG laser (λ = 532 nm); FL, focus tunable lens; BS1/BS2, non-polarizing beam splitters; CL, condenser lens; S, sample; OL, objective lens; TL, tube lens; RL, relay lens; M1/M2, mirrors.

Fig. 2

Amplitude images of an USAF 1951 test target coded to 256 gray levels, acquired with a 10x microscope lens (NA = 0.3). (a) Amplitude from a single hologram. (b) averaged amplitude reconstructed from a sequence of N = 15 holograms acquired under illumination modulation. (c), (d) magnified areas of the smallest test chart structures. (e) cross-sections through the magnified areas along the white lines in (c) and (d). The operation principle of the method, the underlying series of off-axis holograms and the corresponding series of single reconstructed amplitude images are illustrated in Media 1.

Fig. 3

(a), (c), (e) Signal to noise ratio (SNR) of the USAF test target structures in amplitude images for microscope lenses with different magnifications (10x: Zeiss 10x/ EC Plan-Neofluar, 40x: Zeiss 40x/0.6 Korr LD Plan-Neofluar, 63x: Zeiss 63x/0.75 Korr LD Plan-Neofluar); (b), (d), (f) phase noise σ and related OPL variations in the corresponding quantitative phase images; N: number of off-axis holograms that were recorded during modulated illumination with the FL. Data points and error bars represent the mean values for SNR and σ that were acquired in ten different areas of the averaged amplitude and phase images as well as the corresponding standard deviations. The solid curves in (b), (d), (f) present the theoretically expected phase noise decrease.

Fig. 4

(a) Representative segmented quantitative phase images of reconstructed holograms of human fibrosarcoma cells (HT-1080), acquired at different time points during a time-lapse experiment (Δt = 5 min, t_max = 11.5 h). Left column: phase images reconstructed from single holograms (N = 1). Right column: Averaged phase images retrieved from N = 15 holograms that were acquired during modulated illumination with the FL. Arrows mark differences of the detected cell covered surface. Cross-sections through a cell along the dashed lines are plotted in Fig. 5(a). (b) Corresponding numerically simulated differential interference contrast images calculated by the first derivative of the images in (a) in x-direction ( Media 2).

Fig. 5

(a) Illustration of spatial phase noise reduction by cross-sections through single (N = 1) and averaged (N = 15) quantitative phase distributions Δφ of a HT-1080 cell at t = 11.3 h (see dashed lines in in Fig. 4(a)). (b)-(d) Temporal development of cell growth parameters calculated from the segmented phase images in Fig. 4. (b) Area S_c covered by the cells, (c) Average phase contrast $\Delta \overline{\phi }$ in the area S_c and the corresponding average cell thickness $\overline{d}$. (d) Cellular dry mass DM in the area S_c.