arXiv subjects

Genki Ishigane

Publications and source records attributed to Genki Ishigane.

4 recordsLinked to original sources

arXiv2026

Mid-infrared photothermal imaging flow cytometry

Imaging flow cytometry (IFC) enables high-throughput single-cell analysis but largely relies on fluorescence labeling to obtain molecular specificity. Label-free vibrational imaging can provide intrinsic chemical contrast, yet coherent Raman-based methods interrogate only a limited axial volume, which restricts quantitative whole-cell analysis under flow. Mid-infrared photothermal (MIP) microscopy offers a promising route to overcome this limitation by combining linear mid-infrared (MIR) absorption-based chemical contrast with visible-light detection, allowing chemical imaging of a broader axial volume of each cell in a wide-field configuration. However, applying MIP microscopy to rapidly flowing cells has been difficult because conventional frame-sequential acquisition of MIR-ON and MIR-OFF images is highly susceptible to motion-induced subtraction artifacts. Here we demonstrate MIP-IFC, a label-free imaging flow cytometry platform based on single-shot nanosecond-dual-pulse MIP (SNAP-MIP) microscopy. SNAP-MIP encodes the MIR-ON and MIR-OFF states into separate holographic channels within a single camera exposure, reducing their temporal separation to 20 ns. This single-shot acquisition suppresses motion artifacts and increases the allowable sample velocity for artifact-free MIP imaging by five orders of magnitude compared with conventional frame-sequential MIP imaging. Leveraging this capability, MIP-IFC acquired chemical images at 500 frames per second and achieved a cellular event rate up to ~70 events s^-1. We demonstrate quantitative chemical discrimination of flowing microbeads and apply MIP-IFC to single-cell profiling of oleic-acid-induced lipid accumulation, adipocyte differentiation, and confluence-dependent cellular heterogeneity. These results establish MIP-IFC as a high-throughput, quantitative, label-free chemical imaging platform for single-cell phenotyping under flow.

physics.optics↗

arXiv2024

Label-free mid-infrared photothermal microscopy revisits intracellular thermal dynamics: what do fluorescent nanothermometers measure?

Fluorescent nanothermometry has revealed pronounced intracellular temperature heterogeneity, establishing the field of single-cell thermal biology. However, these observations have sparked a controversy known as the "10^5 gap issue", because heat conduction calculations in aqueous environments predict that such large temperature distributions cannot be sustained within cells. Here, we address this issue using label-free mid-infrared photothermal microscopy. This technique quantifies heat-induced temperature changes under local thermal equilibrium (LTE), in accordance with the conventional thermodynamic and statistical-mechanical definition of temperature, by detecting refractive index variations. From transient thermal decay measurements, we determined that intracellular thermal diffusivity corresponds to 93-94% that of water. This result indicates that intracellular heat conduction is essentially water-like and rules out the hypothesis that anomalously slow intracellular heat conduction underlies the 10^5 gap discrepancy. We then directly compared fluorescent nanothermometry with our label-free thermometry. Under seconds-long heating, the label-free method exhibited a rapid temperature response consistent with water-like heat conduction. In contrast, fluorescent nanothermometers showed not only a similarly fast response but also an additional slow variation that was absent in the label-free readout. This slow component cannot be explained by temperature changes defined under LTE, but instead likely reflects slower intracellular processes not governed by conductive heat transfer. These results suggest that the "10^5 gap issue" stems from comparing two fundamentally distinct physical quantities: the LTE-defined temperature and a slowly-varying, long-lived non-conductive signal detected by fluorescent nanothermometers.

physics.bio-ph↗

arXiv2023

Quantitative phase retrieval for Zernike phase-contrast microscopy

We present a single-image numerical phase retrieval method for Zernike phase-contrast microscopy (ZPM) that addresses halo and shade-off artifacts, as well as the weak phase condition, without requiring hardware modifications. By employing a rigorous physical model of ZPM and a gradient descent algorithm for its inversion, we achieve quantitative ZPM imaging. Our approach is experimentally validated using biological cells and its quantitative nature is confirmed through comparisons with digital holography observations.

physics.optics↗

arXiv2022

Mid-infrared photothermal single-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, 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 single-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 single-live-cell analysis.

physics.optics↗