Dashan Dong

Structured illumination microscopy (SIM) has attained high spatiotemporal delineation of subcellular architecture, yet offers limited insight into chemical composition. We develop Chem-SIM, a structured-illumination fluorescence detected mid-infrared photothermal microscopy, for SIM-resolved chemical imaging of microorganisms and mammalian cells. Poisson maximum-likelihood demodulation and spectral normalization across wavenumber recover the weak IR-induced fluorescence intensity change under low photon budgets and convert the fluorescence intensity modulation to chemical fingerprints. Photothermal gating further rejects water backgrounds in aqueous samples, while the IR pump maintains cellular activity at near-physiological temperature. Chem-SIM preserves full vibrational fingerprints, achieves SIM-grade lateral resolution in a high-throughput camera-based format. Here, we show that this platform distinguishes stationary- from log-phase bacteria through chemical content mapping, reports deuterated fatty-acid incorporation in ovarian cancer cells, and resolves lipid-droplet dynamics in live cells, establishing a high-throughput route to high-resolution imaging of organelle chemistry, metabolism, and dynamics.

Metabolism unfolds within specific organelles in eukaryotic cells. Lysosomes are highly metabolically active organelles, and their metabolic states dynamically influence signal transduction, cellular homeostasis and organismal physiopathology. Despite the importance of lysosomal metabolism, a method for its in vivo measurement is currently lacking. Here we report a fluorescence-detected mid-infrared photothermal microscope (FILM) implemented with optical boxcar demodulation, artificial intelligence-assisted data denoising and spectral deconvolution, to map metabolic activity and composition of individual lysosomes in living cells and organisms. Using this method, we uncovered lipolysis and proteolysis heterogeneity across lysosomes within the same cell, as well as early-onset lysosomal dysfunction during organismal aging. In addition, we discovered organelle-level metabolic changes associated with diverse lysosomal storage diseases. This method holds the broad potential to profile metabolic fingerprints of individual organelles within their native context and quantitatively assess their dynamic changes under different physiological and pathological conditions, providing a high-resolution chemical cellular atlas.

Three-dimensional molecular imaging of living cells is essential for unraveling cellular metabolism and response to therapies. However, existing volumetric methods, including fluorescence microscopy and quantitative phase imaging, either require fluorescent labels or lack chemical specificity. Mid-infrared (mid-IR) photothermal microscopy provides label-free spectroscopic contrast with sub-micrometer resolution but is limited by slow acquisition rates, precluding 3D live-cell studies. Here, we present a photothermal relaxation intensity diffraction tomography (PRIDT) system that encodes mid-IR absorption-induced refractive index changes via a photothermal relaxation scheme and recovers it through intensity diffraction tomography. PRIDT achieves video-rate volumetric chemical imaging with up to 15 Hz per wavelength and offers lateral and axial resolutions of 264 nm and 1 . 12 µm, respectively, within a volumetric field of view of 50 × 50 × 10 µm ³ . We showcase high-speed PRIDT imaging of protein and lipid metabolism in ovarian cancer cells and lipid-droplet dynamics in live cells. PRIDT opens new avenues for rapid, quantitative, three-dimensional molecular imaging in living systems.

Forward and backward scattering provide complementary volumetric and interfacial information, yet conventional three-dimensional (3D) imaging typically accesses only one. We present a substrate-enhanced diffraction tomography approach that simultaneously recovers both channels under multi-angle epi-illumination. This geometry captures one forward- and two backward-scattering bands in axially symmetric Fourier regions, where their complementary coverage enables phase–absorption separation in a non-Hermitian spectrum. Explicit 3D transfer functions are derived for both channels, and an axial Kramers–Kronig relation is established to incorporate substrate-induced boundary conditions in a unified framework. Our results establish a label-free, high-resolution 3D imaging modality that surpasses the limits of existing methods.

Because of the lack of spatiotemporal characterization techniques, it has been of great challenge to investigate the crystallization of nanocrystals. With a high-speed structured illumination super-resolution fluorescence microscopy (SIM), we hereby report an in situ fluorescence imaging technique to monitor the crystallization of perovskite nanocrystals at single-particle level. By correlating the fluorescence intensity with particle size, we illustrate the coupled nucleation-and-growth of perovskite nanocrystals in polymer matrix. The temporal fluorescence intensity analysis of individual nanocrystals reveals the diffusion-controlled growth process with a fast growth at the beginning followed by a slow growth. The analysis of ensemble nanocrystals illustrates the evolution of nucleation rate with the change of precursor concentrations. We further analyze the Gibbs free energy fluctuation of couple nucleation-and-growth. The growth free energy dominates in the continuous nucleation of perovskite nanocrystals, which accounts for the narrow size distribution. In comparison with LaMer model, the coupled nucleation-and-growth provides an alternative model to fabricate narrow sized nanocrystals.

Chiral sum-frequency generation (SFG) has proven to be a versatile spectroscopic and imaging tool for probing chirality. However, due to polarization restriction, the conventional chiral SFG microscopes have mostly adopted noncollinear beam configurations, which only partially cover the aperture of microscope and strongly spoil the spatial resolution. In this study, we report the first experimental demonstration of collinear chiral SFG microscopy, which fundamentally supports diffraction-limited resolution. This advancement is attributed to the collinear focus of a radially polarized vectorial beam and a linearly polarized (LP) beam. The tightly focused vectorial beam has a very strong longitudinal component, which interacts with the LP beam and produces the chiral SFG. The collinear configuration can utilize the full aperture and thus push the spatial resolution close to the diffraction limit. This technique can potentially boost the understanding of chiral systems.

Light-sheet fluorescence microscopy (LSFM) has played an important role in bio-imaging due to its advantages of high photon efficiency, fast speed, and long-term imaging capabilities. The perpendicular layout between LSFM excitation and detection often limits the 3D resolutions as well as their isotropy. Here, we report on a reflective type light-sheet microscope with a mini-prism used as an optical path reflector. The conventional high NA objectives can be used both in excitation and detection with this design. Isotropic resolutions in 3D down to 300 nm could be achieved without deconvolution. The proposed method also enables easy transform of a conventional fluorescence microscope to high performance light-sheet microscopy.

Analytical modeling of optical field propagation in a scattering medium is essential for computational optics such as iterative imaging reconstructions and end-to-end photonic designs. Despite the existence of the beam propagation method framework and numerical electromagnetic simulation tools for scalar scattering processes, there has been a lack of an analytical model to include the full tensor nature of birefringent scattering. Here, we present an analytical tool with a polarization transfer function tensor to model the field propagation in a birefringent scattering medium by including a full set of field tensor elements and multiple-scattering characteristics. Birefringence-controlled field propagation experiments were successfully carried out to validate the proposed model. With the capability to solve the 3D reconstruction of the scattering potential tensor, we believe the proposed model will provide a more accurate and efficient forward analytical tool for iterative reconstruction of birefringence-related scattering processes.