Phase/fluorescence dual-mode microscopy imaging based on structured light illumination

Abstract

Quantitative phase microscopy (QPM) is a label-free imaging technique often employed for long-term, high-contrast imaging of live bio-samples. Yet, QPM is not specific to a certain subcellular organelle. As a remedy, fluorescence microscopy can visualize specific subcellular organelles once labeled with fluorescent markers. In this paper, a high-resolution phase/fluorescence dual-modality microscopic imaging method based on structured illumination is proposed. In the dual-modality microscopic system, periodic stripes are generated by a digital micromirror array (DMD), and are used as the common illumination for both modalities. For QPM imaging, the holograms of the sample under structured illuminations of different orientations and phase shifts are recorded, from which a quantitative phase image with resolution enhancement can be reconstructed via a synthetic aperture procedure. Furthermore, a numerical approach is proposed to compensate for the environmental disturbances that often challenge aperture synthesis of phase imaging. This method determines each time the phase distortions caused by environmental disturbances using the spectrum of the 0th order of the structured illumination and subtracts it from the phase distributions of the waves along the 0th, and the ±1st diffraction orders. Resolution enhancement of QPM imaging is realized by synthesizing the spectra of all the waves along different diffraction orders of the structured illuminations of different orientations. With phase images, 3D shapes, inner structures, or refractive index distributions of transparent and translucent samples can be obtained. For fluorescence imaging, intensity images (Morie patterns) of the sample under different structured illuminations are recorded. The spectra along different diffraction orders are separated by using a phase shifting reconstruction algorithm, and are shifted to their original positions, forming a synthesized spectrum that is much larger than the spectra of raw intensity images (NA-limited spectra). An inverse Fourier transform on the synthesized spectrum yields a super-resolution fluorescence image of the sample. With the the reconstructed fluorescence images, specific subcellular organelles labeled with fluorescent markers can be visualized. The combination of quantitative phase microscopy and fluorescence microscopy can obtain multidimensional information about the sample. In this dual-mode imaging system, the spatial resolutions of quantitative phase imaging and fluorescence imaging are 840 nm and 440 nm, respectively. The proposed dual-mode microscopy imaging technique has been demonstrated for imaging fluorescent beads, fly wings, spring/rice leaves, mouse tail transection, and fluorescence-stained SiHa cells. We envisage that this method can be further applied to many fields, such as biomedicine, industry, and chemistry.

Pore-Scale Transport Dynamic Behavior of Microspheres and Their Mechanisms for Enhanced Oil Recovery

“…), the first four types of remaining oil can be divided into discontinuous remaining oil (sporadically distributed), and the fifth type is divided into continuous remaining oil (continuous distribution). Continuous remaining oil can be quantitatively identified through the mature MATLAB algorithm of our research group …”

“…Continuous remaining oil can be quantitatively identified through the mature MATLAB algorithm of our research group. 51 It should be noted here that the microspheres are colorless and transparent particles, and if they are dyed blue, then it is not conducive to the identification of the remaining oil. Therefore, in the microsphere flooding experiment, the unstained microsphere flooding and the stained microsphere flooding were carried out successively, which can realize the capture of the microsphere enhanced oil recovery and the migration and plugging phenomenon at the same time.…”