No abstract

Based on truncated inverse filtering, a theory for deconvolution of complex fields is studied. The validity of the theory is verified by comparing with experimental data from digital holographic microscopy (DHM) using a high-NA system (NA=0.95). Comparison with standard intensity deconvolution reveals that only complex deconvolution deals correctly with coherent cross-talk. With improved image resolution, complex deconvolution is demonstrated to exceed the Rayleigh limit. Gain in resolution arises by accessing the objects complex field - containing the information encoded in the phase - and deconvolving it with the reconstructed complex transfer function (CTF). Synthetic (based on Debye theory modeled with experimental parameters of MO) and experimental amplitude point spread functions (APSF) are used for the CTF reconstruction and compared. Thus, the optical system used for microscopy is characterized quantitatively by its APSF. The role of noise is discussed in the context of complex field deconvolution. As further results, we demonstrate that complex deconvolution does not require any additional optics in the DHM setup while extending the limit of resolution with coherent illumination by a factor of at least 1.64.

The cell has a three-dimensional (3D) structure and its spatial arrangement is often very important to molecular mechanisms essential for life. In order to visualize 3D morphologies of cells, confocal laser imaging was developed. 1 The method is, however, only applicable to fluorescence-probed molecules, 2 which limits the observable number of molecules, and such artificial probing sometime perturbs normal molecular mechanisms. Cotte et al. applied holographic and tomographic irradiation to microscopy and finally innovated a threedimensional computed holographic and tomographic (HT) laser microscope. 3 The laser beam that penetrates the cell at an angle experiences a delay in the phase of its beam, which is magnified and overlayed with reference beam to make a holographic image. The holograms at various angles then deconvoluted by tomographic algorithms to create a precise 3D cell image. The 3D-HT microscope can visualize 3D morphological aspects by contrasting refractive indexes observed by the laser monochromatic wavelength, making staining unnecessary.We have developed live single-cell mass spectrometry, 4-7 in which the contents of a single cell, usually picoliter level or less, are sucked by a nanospray tip (a sort of glass capillary needle) and fed directly into a mass spectrometer after the addition of an ionization solvent to the rear end of the tip. In this method, the exact amount sucked is unclear because it is such a tiny volume. Furthermore, 3D spatial location and identity of the contents are also ambiguous. Through the combination of these two techniques, 3D-HT microscopy and live single-cell mass spectrometry, greater 3D spatial resolution (X-Y-axis 0.18 μm and Z-axis 0.33 μm) and improved quantitative single-cell analysis is expected. The first trial of this combination and its results are documented in this paper, and we think nextgeneration live single-cell mass spectrometry is quite promising.Human hepatocellular carcinoma cell line (HepG2) was cultured in Dulbecco's modified Eagle medium in addition to 10% fetal calf serum (FBS), 100 mg/mL penicillin, and 100 mg/mL streptomycin G in 35 mm glass bottom dishes at 37 C and 5% CO2. HepG2 cells were positioned under the HT laser microscope, and the HT scan took 2 s to acquire one 3D image. Figure 1 shows the schematic principle of the HT laser microscope (3D Cell Explorer, Nanolive, SA, Switzerland). Fig. 1 Schematic of live single-cell mass spectrometry with quantitation by holographic and tomographic laser microscopy. The laser beam is split into a reference beam (going down to the CCD camera) and an observation beam that irradiates the cell at 45 degree angle. A micromanipulator was setup next to microscope to allow precise suction with a nanospray tip. The sucked cellular matter was then blasted through the mass spectrometer.

Holo-tomographic microscopy (HTM) is a label-free non-phototoxic microscopy method reporting the fine changes of a cell's refractive indexes (RI) in 3D. By combining HTM with epifluorescence, we demonstrate that cellular organelles such as Lipid droplets and mitochondria show a specific RI signature that distinguishes them with high resolution and contrast. We further show that HTM allows to follow in unprecedented ways the dynamics of mitochondria, lipid droplets as well as that of endocytic structures in live cells over long period of time, which led us to observe to our knowledge for the first time a global organelle spinning occurring before mitosis.

We present a novel technique for three-dimensional (3D) image processing of complex fields. It consists in inverting the coherent image formation by filtering the complex spectrum with a realistic 3D coherent transfer function (CTF) of a high-NA digital holographic microscope. By combining scattering theory and signal processing, the method is demonstrated to yield the reconstruction of a scattering object field. Experimental reconstructions in phase and amplitude are presented under non-design imaging conditions. The suggested technique is best suited for an implementation in high-resolution diffraction tomography based on sample or illumination rotation.