Single-image phase retrieval for off-the-shelf Zernike phase-contrast microscopes.

Quantitative phase imaging (QPI), such as digital holography, is considered a promising tool in the field of life science due to its noninvasive and quantitative visualization capabilities without the need for fluorescence labeling. However, the popularity of QPI systems is limited due to the cost and complexity of their hardware. In contrast, Zernike phase-contrast microscopy (ZPM) has been widely used in practical scenarios but has not been categorized as QPI, owing to halo and shade-off artifacts and the weak phase condition. Here, we present a single-image phase retrieval method for ZPM that addresses these issues without requiring hardware modifications. By employing a rigorous physical model of ZPM and a gradient descent algorithm for its inversion, we achieve single-shot QPI with an off-the-shelf ZPM system. Our approach is validated in simulations and experiments, demonstrating QPI of a polymer microbead and biological cells. The quantitative nature of our method for single-cell imaging is confirmed through comparisons with observations from an established QPI technique conducted through digital holography. This study paves the way for transforming non-QPI ZPM systems into QPI systems.

Label-free mid-infrared photothermal live-cell imaging beyond video rate.

Advancement in mid-infrared (MIR) technology has led to promising biomedical applications of MIR spectroscopy, such as liquid biopsy or breath diagnosis. On the contrary, MIR microscopy has been rarely used for live biological samples in an aqueous environment due to the lack of spatial resolution and the large water absorption background. Recently, mid-infrared photothermal (MIP) imaging has proven to be applicable to 2D and 3D single-cell imaging with high spatial resolution inherited from visible light. However, the maximum measurement rate has been limited to several frames s-1, limiting its range of use. Here, we develop a significantly improved wide-field MIP quantitative phase microscope with two orders-of-magnitude higher signal-to-noise ratio than previous MIP imaging techniques and demonstrate live-cell imaging beyond video rate. We first derive optimal system design by numerically simulating thermal conduction following the photothermal effect. Then, we develop the designed system with a homemade nanosecond MIR optical parametric oscillator and a high full-well-capacity image sensor. Our high-speed and high-spatial-resolution MIR microscope has great potential to become a new tool for life science, in particular for live-cell analysis.

Thermo-optical measurements using quantitative phase microscopy.

Quantitative phase microscopy (QPM) literally images the quantitative phase shift associated with image contrast, where the phase shift can be altered by laser heating. In this study, the thermal conductivity and thermo-optic coefficient (TOC) of a transparent substrate are simultaneously determined by measuring the phase difference induced by an external heating laser using a QPM setup. The substrates are coated with a 50-nm-thick titanium nitride film to photothermally generate heat. Then, the phase difference is semi-analytically modeled based on the heat transfer and thermo-optic effect to simultaneously extract the thermal conductivity and TOC. The measured thermal conductivity and TOC agree reasonably well, indicating the potential for measuring the thermal conductivities and TOCs of other transparent substrates. The concise setup and simple modeling differentiate the advantages of our method from other techniques.

Adaptive dynamic range shift (ADRIFT) quantitative phase imaging.

Quantitative phase imaging (QPI) with its high-contrast images of optical phase delay (OPD) maps is often used for label-free single-cell analysis. Contrary to other imaging methods, sensitivity improvement has not been intensively explored because conventional QPI is sensitive enough to observe the surface roughness of a substrate that restricts the minimum measurable OPD. However, emerging QPI techniques that utilize, for example, differential image analysis of consecutive temporal frames, such as mid-infrared photothermal QPI, mitigate the minimum OPD limit by decoupling the static OPD contribution and allow measurement of much smaller OPDs. Here, we propose and demonstrate supersensitive QPI with an expanded dynamic range. It is enabled by adaptive dynamic range shift through a combination of wavefront shaping and dark-field QPI techniques. As a proof-of-concept demonstration, we show dynamic range expansion (sensitivity improvement) of QPI by a factor of 6.6 and its utility in improving the sensitivity of mid-infrared photothermal QPI. This technique can also be applied for wide-field scattering imaging of dynamically changing nanoscale objects inside and outside a biological cell without losing global cellular morphological image information.

Quantitative phase imaging with molecular vibrational sensitivity.

Quantitative phase imaging (QPI) quantifies the sample-specific optical-phase-delay enabling objective studies of optically transparent specimens such as biological samples but lacks chemical sensitivity, limiting its application to a morphology-based diagnosis. We present wide-field molecular vibrational (MV) microscopy realized in the framework of QPI utilizing a mid-infrared (MIR) photothermal effect. Our technique provides MIR spectroscopic performance comparable to that of a conventional infrared spectrometer in the molecular fingerprint region of 1450-1640 cm-1 and realizes wide-field molecular imaging of a silica-polystyrene bead mixture over a 100 µm×100 µm area at 1 frame per second with the spatial resolution of 430 nm and 2-3 orders of magnitude lower fluence of ∼10 pJ/µm2 compared to other high-speed label-free molecular imaging methods, reducing photodamages to the sample. With a high-energy MIR pulse source, our technique could enable high-speed, label-free, simultaneous, and in situ acquisition of quantitative morphology and MV contrast, providing new insights for studies of optically transparent complex dynamics.

Molecular contrast on phase-contrast microscope.

An optical microscope enables image-based findings and diagnosis on microscopic targets, which is indispensable in many scientific, industrial and medical settings. A standard benchtop microscope platform, equipped with e.g., bright-field and phase-contrast modes, is of importance and convenience for various users because the wide-field and label-free properties allow for morphological imaging without the need for specific sample preparation. However, these microscopes never have capability of acquiring molecular contrast in a label-free manner. Here, we develop a simple add-on optical unit, comprising of an amplitude-modulated mid-infrared semiconductor laser, that is attached to a standard microscope platform to deliver the additional molecular contrast of the specimen on top of its conventional microscopic image, based on the principle of photothermal effect. We attach this unit, termed molecular-contrast unit, to a standard phase-contrast microscope, and demonstrate high-speed label-free molecular-contrast phase-contrast imaging of silica-polystyrene microbeads mixture and molecular-vibrational spectroscopic imaging of HeLa cells. Our simple molecular-contrast unit can empower existing standard microscopes and deliver a convenient accessibility to the molecular world.