Fluorescent thermometry has reported noteworthy intracellular temperature gradients, suggesting the possibility of thermal regulation of cellular functions. However, heat conduction calculations assuming an aqueous intracellular environment contradict these findings. To resolve this contradiction, we visualized intracellular heat conduction using label-free mid-infrared photothermal optical diffraction tomography and obtained thermal diffusivity similar to that of water. Furthermore, we found a discrepancy in heat-induced temperature changes measured with label-free and fluorescent thermometry. This suggests that fluorescent thermometers may sense other factors, including the system’s internal energy, in addition to the temperature at a local equilibrium state.
Quantitative phase microscopy (QPM), which quantifies the complex amplitude of forward-scattered (FS) light induced by specimens, enables the visualization of the macroscopic refractive index distribution in transparent samples and has been used for various applications such as dry mass analysis and cell cycle estimation. Conversely, interferometric scattering microscopy (iSCAT), which captures backward-scattered (BS) light, offers higher sensitivity compared to QPM and can effectively detect nanoparticles such as individual proteins and viruses, with the drawback of being unable to provide quantitative information on macrostructure. These microscopy techniques provide complementary information and have undergone independent advancements. In this study, we present bidirectional quantitative scattering microscopy (bidirectional QSM) for wide dynamic range imaging by simultaneously performing FS and BS measurements with a single image sensor using a digital holographic technique. This approach enables quantitative measurement of macrostructures as well as high sensitivity to nanoparticles. Moreover, digital holography facilitates the acquisition of complex amplitude FS and BS images, which allows simultaneous computational z-axis focusing, enabling subsequent quantitative correlation analysis between them. To provide a practical demonstration, time-lapse imaging of a cell is performed to characterize temporal changes in the state of cellular vital activity.
Vibrational spectroscopy is widely used in various fields where non-invasive molecular diagnosis is required, but conventional infrared and Raman spectroscopy techniques suffer from low measurement speed and therefore are used mainly for measuring static samples. Recent advancement of nonlinear frequency generation and conversion techniques allow us to develop sophisticated high-speed measurement techniques that make possible to measure dynamic phenomena with high temporal resolutions. In this talk, I introduce newly developed infrared absorption and Raman scattering spectroscopy techniques enabled by light sources with nonlinear optical techniques, including phase-controlled Fourier-transform infrared spectroscopy, rapid-scan Fourier-transform coherent Raman scattering spectroscopy, complementary vibrational spectroscopy and time-stretch infrared spectroscopy.
We develop a chemically-selective quantitative phase imaging technique based on mid-infrared photothermal effect, with which we can simultaneously measure a morphologically sensitive quantitative phase image and a molecularly sensitive mid-infrared photothermal phase image. We show that the wide-field label-free dual-modal microscopy technique can be implemented with digital holography (2D) and optical diffraction tomography (3D). We also demonstrate a new technique to improve the sensitivity of the mid-infrared photothermal phase imaging via a dynamic-range expanded quantitative phase imaging, called adaptive dynamic range shift quantitative phase imaging (ADRIFT-QPI).
Label-free optical imaging is valuable for studying fragile biological phenomena where chemical and/or optical damages associated with exogenous labelling of biomolecules are not wanted. Molecular vibrational (MVI) and quantitative phase imaging (QPI) are the two most-established label-free imaging methods that provide biochemical and morphological information of the sample, respectively. While these methods have pioneered numerous important biological analyses along their intensive technological development over the past twenty years, their inherent limitations are still left unresolved. In this contribution, we present a unified imaging scheme that bridges the technological gap between MVI and QPI, achieving simultaneous and in-situ integration of the two complementary label-free contrasts using the midinfrared (MIR) photothermal effect. Our method is a super-resolution MIR imaging where vibrational resonances induced by wide-field MIR excitation and the resulting photothermal RI changes are detected and localized with the spatial resolution determined by a visible-light-based QPI system. We demonstrate applicability of this method, termed MV-sensitive QPI (MV-QPI), to live-cell imaging. Our MV-QPI method could allow for quantitative mapping of subcellular biomolecular distributions within the global cellular morphology in a label-free and damage-less manner, providing more comprehensive pictures of complex and fragile biological activities.
We present a coherent Raman scattering (CRS) spectroscopy technique achieving a CRS spectral acquisition rate of 50,000 spectra/second over a Raman spectral region of 200 - 1430 cm-1 with a resolution of 4.2 cm-1. This ultrafast, broadband and high-resolution CRS spectroscopic performance is realized by a polygonal Fourier-domain delay line serving as an ultra-rapid optical-path-length scanner in a broadband Fourier-transform coherent anti-Stokes Raman scattering (CARS) spectroscopy platform. We present a theoretical description of the technique and demonstrate continuous, ultrafast, broadband, and high-resolution CARS spectroscopy on a liquid toluene sample using our proof-of-concept setup.
Particle analysis is an effective method in analytical chemistry for sizing and counting microparticles such as emulsions, colloids, and biological cells. However, conventional methods for particle analysis, which fall into two extreme categories, have severe limitations. Sieving and Coulter counting are capable of analyzing particles with high throughput, but due to their lack of detailed information such as morphological and chemical characteristics, they can only provide statistical results with low specificity. On the other hand, CCD or CMOS image sensors can be used to analyze individual microparticles with high content, but due to their slow charge download, the frame rate (hence, the throughput) is significantly limited. Here by integrating a time-stretch optical microscope with a three-color fluorescent analyzer on top of an inertial-focusing microfluidic device, we demonstrate an optofluidic particle analyzer with a sub-micrometer spatial resolution down to 780 nm and a high throughput of 10,000 particles/s. In addition to its morphological specificity, the particle analyzer provides chemical specificity to identify chemical expressions of particles via fluorescence detection. Our results indicate that we can identify different species of microparticles with high specificity without sacrificing throughput. Our method holds promise for high-precision statistical particle analysis in chemical industry and pharmaceutics.
The invention of the optical frequency comb technique has revolutionized the field of precision spectroscopy, providing a way to measure the absolute frequency of any optical transition. Since, frequency combs have become common equipment for frequency metrology. In the last decade, novel applications for the optical frequency comb have been demonstrated beyond its original purpose. Broadband molecular spectroscopy is one of those. One such technique of molecular spectroscopy with frequency combs, dual-comb Fourier transform spectroscopy provides short measurement times with resolution and accuracy. Two laser frequency combs with slightly different repetition frequencies generate pairs of pulses with a linearly-scanned delay between pulses in a pair. The system without moving parts mimics a fast scanning Fourier transform interferometer. The measurement speed may be several orders of magnitude faster than that of a Michelson-based Fourier transform spectrometer, which opens up new opportunities for broadband molecular spectroscopy. Recently, dual-comb spectroscopy has been extended to nonlinear phenomena. A broadband Raman spectrum of molecular fingerprints may be measured within a few tens of microseconds with coherent Raman dual-comb spectroscopy. Raster scanning the sample leads to hyperspectral images. This rapid and broadband label-free vibrational spectroscopy and imaging technique might provide new diagnostic methods in a variety of scientific and industrial fields.
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