Distinct Modulated Pupil Function System for Real-Time Imaging of Living Cells

<div><p>Optical microscopy is one of the most contributive tools for cell biology in the past decades. Many microscopic techniques with various functions have been developed to date, i.e., phase contrast microscopy, differential interference contrast (DIC) microscopy, confocal microscopy, two photon microscopy, superresolution microscopy, etc. However, person who is in charge of an experiment has to select one of the several microscopic techniques to achieve an experimental goal, which makes the biological assay time-consuming and expensive. To solve this problem, we have developed a microscopic system with various functions in one instrument based on the optical Fourier transformation with a lens system for detection while focusing on applicability and user-friendliness for biology. The present instrument can arbitrarily modulate the pupil function with a micro mirror array on the Fourier plane of the optical pathway for detection. We named the present instrument DiMPS (<u>Di</u>stinct optical <u>M</u>odulated <u>P</u>upil function <u>S</u>ystem). The DiMPS is compatible with conventional fluorescent probes and illumination equipment, and gives us a Fourier-filtered image, a pseudo-relief image, and a deep focus depth. Furthermore, DiMPS achieved a resolution enhancement (pseudo-superresolution) of 110 nm through the subtraction of two images whose pupil functions are independently modulated. In maximum, the spatial and temporal resolution was improved to 120 nm and 2 ms, respectively. Since the DiMPS is based on relay optics, it can be easily combined with another microscopic instrument such as confocal microscope, and provides a method for multi-color pseudo-superresolution. Thus, the DiMPS shows great promise as a flexible optical microscopy technique in biological research fields.</p> </div

Individual quantum dots observation with 2 ms temporal resolutions with the siDiMPS.

<p>(A) Four typical fluorescence images of QDs captured by a high speed CCD camera with a conventional microscope (upper) and the DiMPS (lower). One pixel, 64.5 nm. (B) Ten PSFs of fluorescence QD images acquired with the DiMPS with 2 ms temporal resolution. The gray line is the fitted Gaussian curve, which indicates the spatial resolution (122 nm). (C, D) Fluorescent images of E-cadherin-QD on the cell membrane acquired by conventional microscopy (C) and DiMPS (D). Scale bars, 1 µm. Inserts are magnifications of the red rectangles. Scale bars, 200 nm. Dotted cyan circles indicate individual QDs. (E) Time courses of the dissociation of the E-cadherin complex (upper) and kymograph of the cyan broken line showing dissociation (lower left) and association (lower right). Numbers in the panels indicate elapsed time (ms). Position scale bars, 200 nm; time scale bar, 100 ms. (F) Time course of the cross-sections (cyan broken line) for the upper panel in E. Red and blue lines are fitting results using double Gaussian functions and two single Gaussian functions used to make the fitted double Gaussian, respectively.</p

Resolution enhancement with the siDiMPS.

<p>(A, B, C) Pupil functions and fluorescence distributions of a φ100 nm bead with a non-masked (A), apodizing-masked (B), and superresolving-masked PSF (C). Upper panels, pupil functions; lower panels, PSF images in the longitudinal-transverse plane. (D) One-dimensional fluorescence intensity profiles of the non-masked (black), apodizing-masked (blue), and superresolving-masked PSF (red). Values indicate spatial resolution determined at FWHM. (E, F) Fluorescence distributions determined by subtracting an apodizing-masked PSF from a superresolving-masked one without (E), and with optimizing symmetry of PSF by adjusting lens L4 position along the light path (F). Left panels, PSF images in the longitudinal-transverse plane; middle and right panels, one-dimensional fluorescence intensity profile of each PSF along the transverse axis (middle) and longitudinal axis (right), respectively. Values indicate spatial resolution determined at FWHM. Red and gray lines are one dimensional fluorescence profiles of the non-masked (E) and extended PSF (F), respectively. All scale bars, 1 µm. (G) siDiMPS image of Alexa488-phalloidin stained actin bundles in a fixed cell. Top, non-masked. Middle, siDiMPS. Bottom, siDiMPS+deconvolution. Inserts, magnifications of the areas in yellow rectangles, respectively. Scale bars, 2 µm. The images were obtained with a 100 ms exposure time. (H) One-dimensional fluorescence intensity profiles of the yellow broken lines in G (black, conventional; green, siDiMPS; red, siDiMPS+deconvolution).</p

Biological applications of siDiMPS using green fluorescent protein.

<p>(A,B) Fluorescence images of localized E-cadherin-GFP in a living cell obtained by conventional microscopy (A) and siDiMPS (B). Scale bars, 1 µm. The exposure time for the camera was 30.28 ms. The images shown are the averages of 100 acquired images (total frame rate, 0.3 Hz). Inserts, magnifications of the areas in orange rectangles, respectively. Scale bars, 200 nm. (C) One-dimensional fluorescence intensity profiles inside the yellow rectangle in A (blue, conventional) and B (red, siDiMPS). Arrowheads indicate the periodic peaks of fluorescence intensity. (D) Histogram of the distance between two peaks within the periodic localization of E-cadherin-GFP. Arrowheads and values represent the peaks in the histogram. (E, F) Fluorescence images of autophagosomes labeled with GFP-LC3 in a living cell acquired by conventional microscopy (E) and siDiMPS (F). Scale bars, 1 µm. Inserts, the intensity profiles of the one-dimensional fluorescence intensity profile (yellow line) in each panel. (G) siDiMPS time lapse images of a small organelle labeled with GFP-LC3. Scale bar, 400 nm. The exposure time for the camera was 30.28 ms. Images E, F and G are averages of 10 acquired images (total frame rate, 3 Hz). (H, I) Three dimensional images of autophagosome acquired by conventional microscopy (H) and siDiMPS (I). Left and middle panels are XY-image and XZ-image, respectively. Right panels are one-dimensional fluorescence intensity profile along longitudinal axis (Z) at the center of the images. Scale bars, 1 µm. The exposure time for the camera was 100 ms.</p

Confocal and multi-color imaging with the DiMPS.

<p>(A) Comparison of a confocal image (left), a high-pass filtered image obtained with the DiMPS combined with a confocal unit (middle), and siDiMPS combined with a confocal unit (right). Top, microtubules. Middle, actin bundles. Bottom, merged images of microtubules and actin bundles. Scale bars, 1 µm. Insets are enlarged images of the dotted yellow rectangles. (B, C) One dimensional fluorescence intensity profiles of the cyan lines in (A). (B) Microtubule. (C) Actin bundles. Blue, Red, and green lines were obtained from confocal, confocal + DiMPS, and confocal + siDiMPS images, respectively.</p

Transmission observation with DiMPS.

<p>(A) Effect of Fourier-filter with DiMPS. Left panels, the pupil functions used. White indicates transmission and black indicates blocking. Middle panels, images of a KPL4 cell on transmitted light irradiation. Right panels, two-dimensional power spectra of the middle images. Yellow lines are the intensity profile of the center of the 2D power spectrum, that is, the power spectrum along X direction. Scale bar, 2 µm. (B) Comparison of normal (left upper), pseudo-relief (right), and high-pass filtered (left lower) images. Inserts, the pupil functions used. (<i>C</i>) One dimensional cross-section of the broken lines in <b>B</b>. Green, normal. Blue, high-pass. Red, pseudo-relief. Scale bars, 2 µm.</p

Basic of the DiMPS.

<p>(A) Photograph of the DiMPS. (B) Schematic drawing of the DiMPS. S, spatial mask; BS, polarizing beam splitter; LCM, liquid crystal micro mirror array; P, prism mirror; L, lens; FP, Fourier plane; CP, conjugate plane. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044028#s4" target="_blank">methods</a> for the optics construction. (C) Typical modulations of the pupil function. The pupil function is the polarization pattern in the LCM. Black part shows where the light is blocked. The PSF is represented by the fluorescence distribution of a φ100 nm bead with an emission peak at 515 nm (Invitrogen). Red lines show the one-dimensional fluorescence intensity profile of the PSF along the transverse and longitudinal axes. Gray lines are the one-dimensional fluorescence intensity profile of a normal PSF. Scale bars, 1 µm.</p

<i>Atg9a</i> deficiency causes axon-specific lesions including neuronal circuit dysgenesis

<p>Conditional knockout mice for <i>Atg9a</i>, specifically in brain tissue, were generated to understand the roles of ATG9A in the neural tissue cells. The mice were born normally, but half of them died within one wk, and none lived beyond 4 wk of age. SQSTM1/p62 and NBR1, receptor proteins for selective autophagy, together with ubiquitin, accumulated in <i>Atg9a</i>-deficient neurosoma at postnatal d 15 (P15), indicating an inhibition of autophagy, whereas these proteins were significantly decreased at P28, as evidenced by immunohistochemistry, electron microscopy and western blot. Conversely, degenerative changes such as spongiosis of nerve fiber tracts proceeded in axons and their terminals that were occupied with aberrant membrane structures and amorphous materials at P28, although no clear-cut degenerative change was detected in neuronal cell bodies. Different from autophagy, diffusion tensor magnetic resonance imaging and histological observations revealed <i>Atg9a</i>-deficiency-induced dysgenesis of the corpus callosum and anterior commissure. As for the neurite extensions of primary cultured neurons, the neurite outgrowth after 3 d culturing was significantly impaired in primary neurons from <i>atg9a</i>-KO mouse brains, but not in those from <i>atg7</i>-KO and <i>atg16l1</i>-KO brains. Moreover, this tendency was also confirmed in <i>Atg9a</i>-knockdown neurons under an <i>atg7</i>-KO background, indicating the role of ATG9A in the regulation of neurite outgrowth that is independent of autophagy. These results suggest that <i>Atg9a</i> deficiency causes progressive degeneration in the axons and their terminals, but not in neuronal cell bodies, where the degradations of SQSTM1/p62 and NBR1 were insufficiently suppressed. Moreover, the deletion of <i>Atg9a</i> impaired nerve fiber tract formation.</p

Impaired autophagy in macrophages promotes inflammatory eye disease

<p>Autophagy is critical for maintaining cellular homeostasis. Organs such as the eye and brain are immunologically privileged. Here, we demonstrate that autophagy is essential for maintaining ocular immune privilege. Deletion of multiple autophagy genes in macrophages leads to an inflammation-mediated eye disease called uveitis that can cause blindness. Loss of autophagy activates inflammasome-mediated IL1B secretion that increases disease severity. Inhibition of caspase activity by gene deletion or pharmacological means completely reverses the disease phenotype. Of interest, experimental uveitis was also increased in a model of Crohn disease, a systemic autoimmune disease in which patients often develop uveitis, offering a potential mechanistic link between macrophage autophagy and systemic disease. These findings directly implicate the homeostatic process of autophagy in blinding eye disease and identify novel pathways for therapeutic intervention in uveitis.</p