Single-shot two-frame interference microscopy for robust quantitative phase imaging of fixed and dynamic samples

Quantitative phase imaging, employing the refractive index as endogenous contrast agent, opens wide possibilities to perform high-contrast cell measurements enabling reliable diagnostics and assessable examination. This capability can be rather easily obtained in a common-path total-shear regime after introducing a grating or beam-splitter into a regular microscope layout with a (partially-)coherent light source. Although these solutions are attractive due to straightforward implementation, overall good stability (thanks to common-path configuration) and high contrast, they are limited in time-space-bandwidth product (TSBP) as multi-frame phase reconstruction is needed to ensure sufficient accuracy and robustness. Alternative single-shot Fourier transform based approaches do allow for dynamic imaging; however they need off-axis recording and thus limit detector bandwidth and can severely truncate object spectrum (phase lateral resolution), which stays virtually intact in the multi-frame approach. Additionally, low signal-to-noise ratio of recorded fringe pattern significantly adds to the error budget via low contrast, high noise and strong incoherent background. In this contribution we study an interesting way to bypass mentioned shortcomings by recording two out-of-phase interferograms simultaneously to subtract them and thus experimentally increase the signal-to-noise ratio of otherwise low-quality dimmed fringe patterns. Ideal subtraction should yield background-rejected and modulation-doubled pi-hologram, however in reality additional processing is needed. We will investigate three algorithms for such processing. Filtered interferogram is then analyzed employing Hilbert spiral transform, which is not sensitive to the carrier frequency like the Fourier transform and preserves object spectrum also in quasi on-axis configurations. Finally, sacrificing the field of view to record two interferograms at once, we gain unique feature of significantly increased TSBP enabling real-time investigation of broad-spectrum (highly detailed) transparent objects with enhanced phase resolution and signal-to-noise ratio. We corroborate the claims successfully analyzing prostate cancer cells and flowing microbeads otherwise measurable only in static regime using time-consuming phase-shifting. The technique has been validated utilizing 20x/0.46NA objective in a regular Olympus BX-60 upright microscope.