This paper presents DMD-addressing based ptychographic phase microscopy (DA-PPM) and phase/fluorescence dual-modality imaging in DA-PPM. Compared with conventional ptychographic approaches, the imaging speed of DA-PPM is significantly enhanced by DMD based illumination selection, and is further enhanced by parallel illumination, i.e., lighting up parallelly multiple sub-areas in one shot. Furthermore, two-folds spatial resolution enhancement can be achieved in DA-PPM by incorporating structured illumination generated by DMD. Last but not least, phase/fluorescence dual-modality imaging will be performed in DA-PPM, providing for the same sample complementary information, including structural and functional information.
Conventional optical microscopy provides only intensity images, for which the contrast is induced by fluorescence or the absorption of the sample on the illumination light. Yet, the phase, polarization, and spectrum information of the sample is lost. Meanwhile, limited by design, conventional optical microscopy suffers from the conflict between spatial resolution and field of view (FOV). Modulated illuminations based computational microscopy (CM), which joints front-end optics and post-detection signal processing can, in general, extend the capability of conventional microscopy; for example, it allows the acquisition of the intensity, phase, polarization information, and enhance the spatial resolution within a large FOV. In this paper, modulated illumination based CM was exploited for implementation of phase imaging, resolution enhancement, dual-modality imaging. First, modulated illumination based CM provides quantitative amplitude and phase images, revealing the 3D shape and the inner structure of transparent or translucent samples in the absence of fluorescent labeling. Second, pupil-segmentation based CM measures the aberration of focus modulation microscopy (FMM). Hence, the resolution and SNR of FMM was enhanced after the aberration compensation. Third, phase and fluorescence dualmodality imaging was implemented in confocal laser scanning microscopy (CLSM) by extending the depth of field (DOF) of the CLSM system with a tunable acoustic gradient index of refraction (TAG) lens, providing complementary information (structural/functional) with pixel-to-pixel correspondence for the same sample. Furthermore, the combination of the two imaging modalities enables standalone determination of the refractive index of live cells.
Structured illumination microscopy (SIM) is a well-known super-resolution imaging technique, which exploits moiré patterns created when a sample is illuminated with periodic stripes. Conventional SIM often applies to fluorescent samples, or the samples which have absorption on illumination light. Here we report quantitative phase imaging of transparent samples with a SIM apparatus in transmittance-mode. For this purpose, two sets of fringe patterns, which have two orthogonal orientations and five phase-shifts for each orientation, were generated by a digital micro-mirror device (DMD) and projected on a sample. Under different fringe illuminations slightly-defocused images of the sample were recorded sequentially by a CCD camera, where the object waves along the ±1st orders of the illumination interfere with each other with a lateral shear in-between. The phase derivatives of the sample along the shear direction can be reconstructed from the phase-shifted intensity patterns. Eventually, the quantitative phase distribution of the sample was obtained by integrating the two phase derivatives. Furthermore, an iterative algorithm was used to enhance the resolution of the phase image, considering the structured illumination synthesizes a larger spectrum in the Fourier domain, similar to oblique illuminations in digital holography. This apparatus can also work in the conventional SIM mode, which images fluorescent samples in an in-focus manner. We believe such simple and versatile apparatus will be widely applied to biological imaging or industrial inspection.
Conventional transport-of-intensity equation (TIE) based phase imaging is performed in wide-field microscopes. In this paper, we present phase and fluorescence dual-modality imaging in a confocal laser scanning microscopy (CLSM) system. To perform phase imaging, the depth of field (DOF) of the CLSM system was extended by using a tunable acoustic gradient index of refraction (TAG) lens. Under transmitted illumination, a few intensity images of a sample at different defocusing distances were recorded. The phase image is reconstructed from these intensity images by using transport-of-intensity equation (TIE). Fluorescence image is obtained by 3D scan of the sample, providing a 3D sectioned fluorescence image. The obtained dual-modality images with pixel-to-pixel correspondence provide for the same sample complementary information (structural/functional), to extract complex biological parameters. We demonstrate the combination of the two imaging modalities enables standalone determination of the refractive index of live cells.