This work is financially supported by Young Investigator program (NRF-2015R1C1A1A02036464), Engineering Research Center program (NRF-2015R1A5A1037668) and Global Frontier program (CAMM-2014M3A6B3063708), M.K., S.S., I.K. acknowledge the Global Ph.D. Fellowships (NRF-2017H1A2A1043204, NRF-2017H1A2A1043322, NRF-2016H1A2A1906519) through the National Research Foundation of Korea (NRF) grant funded by the Ministry of Science, ICT and Future Planning (MSIP) of Korean government.

1. Substrate Preparation
<ol>
	<li>Obtain highly refined quartz wafer. For the fabrication reported here, use a wafer with a 500 &#181;m thickness.</li>
	<li>Spin-coat the quartz wafer with a positive photoresist at 2,000 rpm and bake for 60 s at 90 &#176;C. 
	NOTE: The positive photoresist layer is coated to prevent damage during the subsequent cutting step.</li>
	<li>Use a dicing machine to cut the wafer with photoresist into small pieces 20 x 20 mm2 in size.</li>
	<li>Blow using a compressed nitrogen gun to remove particulates resulting from the cutting step.</li>
	<li>Place it in an ultrasonic bath in de-ionized (DI) water for 5 min at 45 &#176;C. Remove the photoresist layer using an ultrasonic bath in acetone for 5 min at 45 &#176;C. Clean the substrate using two ultrasonic baths, acetone and isopropyl alcohol, each for 5 min at 45 &#176;C.</li>
	<li>Dry the substrate with a compressed nitrogen gun.</li>
</ol>
2. Etching the Mask Pattern
<ol>
	<li>Load the clean quartz substrates into a high-vacuum electron beam evaporation system. Ensure that substrate rotation is enabled.</li>
	<li>Deposit the chromium layer with a deposition rate of 2 &#197;/s. 
	NOTE: A layer at least 100 nm-thick should be deposited for the etching mask to prevent pinholes made from deposition.</li>
	<li>Press the vent button to vent the chamber and mount a sample on the focused ion beam (FIB) holder using conducting copper tape.</li>
	<li>Load the FIB holder into the FIB chamber.</li>
	<li>Close the chamber door and press the pump button to evacuate the chamber.</li>
	<li>Select &#34;beam on&#34; under the beam control tab and set the ion beam current (7.7 pA) and acceleration voltage (30 kV) for FIB mode.</li>
	<li>Turn on the ion beam system.</li>
	<li>Select &#34;beam on&#34; under the beam control tab to turn on the electron beam and focus the image with low magnification using software.</li>
	<li>Set the working distance (WD) at 4 mm under the navigation tab in scanning electron microscope (SEM) mode.</li>
	<li>Set the tilt angle of the holder to 52&#176; and take the SEM images at different magnifications before hole array mask pattern fabrication.</li>
	<li>Under the patterning tab, choose the patterning region and make a 50 nm hole array on the chromium layer. 
	NOTE: There are simple patterning tools accessible under the patterning tab. More complex geometry and exposure control can be achieved by importing bitmaps or generating scripts.</li>
	<li>After finishing, turn off the electron beam and ion beam systems and cool down the system.</li>
	<li>Press the vent button and vent the chamber with nitrogen gas. Take the holder out of the chamber.</li>
	<li>Close the chamber door and evacuate the chamber by pressing the pump button.</li>
</ol>
3. Wet-etching Process and Removal of the Mask Layer
<ol>
	<li>Put the patterned substrate into 1:10 buffered oxide etchant for 5 min. 
	NOTE: The quartz is selectively and isotropically wet-etched by the etchant and forms a spherical shape. The shape of the lens can be obtained with the etching mask, and the diameter is precisely controlled by the etching time. A better spherical shape can be formed with a smaller pattern diameter. A 1.5 &#181;m-diameter hemisphere can be obtained within 5 min.</li>
	<li>Put the patterned substrate into DI water to clean the buffered oxide etchant (5 min, two times). 
	NOTE: Buffered oxide etchant can be dangerous, so be careful when using this etchant.</li>
	<li>Dry the sample with compressed nitrogen gas.</li>
	<li>Put the patterned substrate into CR-7 chromium etchant to remove the chromium mask layer. 
	NOTE: After removing the chromium layer, a spherical patterned substrate 1.5 &#181;m in diameter can be obtained.</li>
	<li>Put the patterned substrate into DI water to clean it (5 min).</li>
</ol>
4. Multilayer Deposition and Nano-sized Object Inscription
NOTE: A pair of layers are deposited on the spherical quartz substrate. Here, Ag and TiO2 are used as the deposition materials. Ag and TiO2 are deposited alternately at a thickness of 15 nm.
<ol>
	<li>Press the vent button of the electron beam evaporation system and wait until the vent is over.</li>
	<li>Load the patterned substrate into a high-vacuum electron beam evaporation system after the vent.</li>
	<li>Close the chamber door and evacuate the chamber to a vacuum degree of 10-7 Torr by pressing the pump button. 
	NOTE: The vacuum condition should be kept at 10-7 Torr to reduce the scattering from the surface roughness.</li>
	<li>Deposit the Ag layer with a growth rate of 1 &#197;/s and deposit a 15 nm-thick Ag layer.</li>
	<li>After the deposition of the Ag layer, cool down the substrate for 5 min.</li>
	<li>Change the pocket of the electron beam evaporation system by choosing another crucible and deposit the TiO2 layer with a growth rate of 1 &#197;/s. Deposit a 15 nm-thick TiO2 layer. 
	NOTE: During the deposition process, the film growth rate is kept low to maintain the surface roughness uniformity.</li>
	<li>After the deposition of the TiO2 layer, cool down the substrate for 5 min.</li>
	<li>Repeat steps 4.4 - 4.7 for tens of cycles to deposit a multilayer of Ag and TiO2. 
	NOTE: At this point, the hyperlens fabrication is over. The next step is for making an arbitrary sub-diffraction-limited feature for testing the hyperlens imaging ability. Nanometer-sized apertures and slits are inscribed by FIB milling.</li>
	<li>Change the pocket of the electron beam evaporation system and deposit the chromium layer at a thickness of 50 nm.</li>
	<li>After the deposition of a Cr layer, turn off the electron beam evaporation system. Press the vent button and vent the chamber by introducing nitrogen gas.</li>
	<li>After the vent, open the chamber door and take the mount holder out of the chamber. Strip off the fabricated hyperlens device.</li>
	<li>Close the chamber door and evacuate the chamber by pressing the pump button.</li>
	<li>Mount the hyperlens deposited with chromium into the FIB milling system and pattern a nano-sized structure, per the manufacturer&#39;s instructions.</li>
</ol>
5. Setting Up the Imaging System and Imaging Procedure
<ol>
	<li>Place a conventional transmission-type optical microscope on the optical table. 
	NOTE: Here, an inverted optical microscope was used as the main body.</li>
	<li>Connect a white-light source to the microscope illumination path using an adapter.</li>
	<li>Place an optical bandpass filter centered at 410 nm. 
	NOTE: The bandpass filter selectively penetrates the specific wavelength of light; here, 410 nm light is illuminated on the sample. A hyperlens consisting of Ag and TiO2 has high performance at a 410 nm wavelength. The simulation result (Figure 2c) shows the performance of the hyperlens, which satisfies the hyperbolic dispersion relation at 410 nm light.</li>
	<li>Select a high-magnification oil-immersion objective lens. Use a high-quality CCD camera to obtain the images. 
	NOTE: This optical setting just puts the bandpass filter into the light illumination path to sort out the 410 nm wavelength light. A specific wavelength of light can be illuminated on the sample without using white light, but in a normal laboratory, optical microscopes may have a white-light source for the observation of samples through bright-field or fluorescence imaging.</li>
	<li>Place a drop of immersion oil on the objective lens. Place a hyperlens on the sample stage and capture images. 
	NOTE: The inscribed nano-sized objects on the inner surface of the hyperlens can be illuminated with 410 nm light. With the hyperlens, the nano-sized objects will be magnified and be captured by the objective lens and imaged by CCD camera.</li>
</ol>

<ol>
	<li>Abbe, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beitr&#228;ge+zur+Theorie+des+Mikroskops+und+der+mikroskopischen+Wahrnehmung.">Beitr&#228;ge zur Theorie des Mikroskops und der mikroskopischen Wahrnehmung.</a> Archiv f&#252;r mikroskopische Anatomie. 9 (1), 413-418 (1873).</li><li>D&#252;rig, U., Pohl, D. W., Rohner, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Near-field+optical-scanning+microscopy.">Near-field optical-scanning microscopy.</a> J Appl Phys. 59 (10), 3318-3327 (1986).</li><li>Pendry, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Negative+Refraction+Makes+a+Perfect+Lens.">Negative Refraction Makes a Perfect Lens.</a> Phys Rev Lett. 85 (18), 3966-3969 (2000).</li><li>Fang, N., Liu, Z., Yen, T. -. J., Zhang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regenerating+evanescent+waves+from+a+silver+superlens.">Regenerating evanescent waves from a silver superlens.</a> Opt Express. 11 (7), 682-687 (2003).</li><li>Fang, N., Lee, H., Sun, C., Zhang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sub-Diffraction-Limited+Optical+Imaging+with+a+Silver+Superlens.">Sub-Diffraction-Limited Optical Imaging with a Silver Superlens.</a> Science. 308 (5721), 534-537 (2005).</li><li>Melville, D. O. S., Blaikie, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Super-resolution+imaging+through+a+planar+silver+layer.">Super-resolution imaging through a planar silver layer.</a> Opt Express. 13 (6), 2127-2134 (2005).</li><li>Taubner, T., Korobkin, D., Urzhumov, Y., Shvets, G., Hillenbrand, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Near-Field+Microscopy+Through+a+SiC+Superlens.">Near-Field Microscopy Through a SiC Superlens.</a> Science. 313 (5793), 1595-1595 (2006).</li><li>Jacob, Z., Alekseyev, L. V., Narimanov, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+Hyperlens:+Far-field+imaging+beyond+the+diffraction+limit.">Optical Hyperlens: Far-field imaging beyond the diffraction limit.</a> Opt Express. 14 (18), 8247-8256 (2006).</li><li>Lee, H., Liu, Z., Xiong, Y., Sun, C., Zhang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+optical+hyperlens+for+imaging+below+the+diffraction+limit.">Development of optical hyperlens for imaging below the diffraction limit.</a> Opt Express. 15 (24), 15886-15891 (2007).</li><li>Liu, Z., Lee, H., Xiong, Y., Sun, C., Zhang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Far-Field+Optical+Hyperlens+Magnifying+Sub-Diffraction-Limited+Objects.">Far-Field Optical Hyperlens Magnifying Sub-Diffraction-Limited Objects.</a> Science. 315 (5819), 1686-1686 (2007).</li><li>Kim, M., Rho, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metamaterials+and+imaging.">Metamaterials and imaging.</a> Nano Converg. 2 (1), 22 (2015).</li><li>Byun, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Demonstration+of+nanoimprinted+hyperlens+array+for+high-throughput+sub-diffraction+imaging.">Demonstration of nanoimprinted hyperlens array for high-throughput sub-diffraction imaging.</a> Sci Rep. 7, 46314 (2017).</li><li>Shekhar, P., Atkinson, J., Jacob, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyperbolic+metamaterials:+fundamentals+and+applications.">Hyperbolic metamaterials: fundamentals and applications.</a> Nano Converg. 1 (1), 14 (2014).</li><li>Liu, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sub-diffraction+demagnification+imaging+lithography+by+hyperlens+with+plasmonic+reflector+layer.">Sub-diffraction demagnification imaging lithography by hyperlens with plasmonic reflector layer.</a> RSC Advances. 6 (98), 95973-95978 (2016).</li><li>Sun, J., Xu, T., Litchinitser, N. 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The authors declare that they have no competing financial interests.

The fabrication of a hyperlens includes three major steps: defining hemispherical geometry into the quartz substrate through a wet-etching process, stacking the metal and dielectric multilayer using an electron beam evaporation system, and inscribing the object on the Cr layer. The most important step is the second, since it can significantly affect quality of the hyperlens. In the thin-film deposition process, there are two conditions that require special care for a clear super-resolved image. Stacking the multilayer co...

A desire to observe biomolecules in living cells led to the invention of microscopy, and the advent of microscopy propagated the revolution of various fields, such as biology, pathology, and material science, over last few centuries. However, further advancement of research has been restricted by diffraction, which limits the resolution of conventional microscopes to about half of the wavelength1. Therefore, super-resolution imaging to overcome the diffraction limit has been an interesting research area in recent decades.
As the diffraction limit is attributed to the loss of the evanescent waves that contain sub-wavelength information on objects, early studies have been conducted to keep evanescent waves from fading away or to recover them2,3. The effort to overcome the diffraction limit was first reported with near-field scanning optical microscopy, which collects the evanescent field in close proximity to the object before it is dissipated2. However, as scanning the whole image region and reconstructing it takes a long time, it cannot be applied to real-time imaging. Although another approach based on the &#34;superlens,&#34; which amplifies evanescent waves, provides the possibility of real-time imaging, sub-wavelength imaging is only capable in the near-field region and cannot reach far beyond the objects4,5,6,7.
Recently, the hyperlens has emerged as a novel approach to real-time far-field optical imaging8,9,10,11,12. The hyperlens, which is made of highly anisotropic hyperbolic metamaterials13, exhibits a flat hyperbolic dispersion so that it supports high spatial information with the same phase velocity. Furthermore, due to the momentum conservation law, the high transverse wavevector is gradually compressed as the wave goes through the cylindrical geometry. This magnified information thus can be detected by a conventional microscope in the far-field region. This is of particular importance to real-time far-field imaging, as it does not require any point-by-point scanning or image reconstruction. Moreover, the hyperlens can be used for applications other than imaging, including nanolithography. Light that passes through the hyperlens in the reverse direction will be focused onto a sub-diffraction area due to the time-reversal symmetry14,15,16.
Here, we report on a spherical hyperlens that magnifies two-dimensional information at the visible frequency. Unlike conventional cylindrical geometry, the spherical hyperlens magnifies objects in two lateral dimensions, facilitating practical imaging applications. The fabrication method and imaging setup with the hyperlens are presented in detail for the reproduction of a high-quality hyperlens. A sub-wavelength object is inscribed on the hyperlens for the sake of proving its super-resolving power. It is confirmed that small features of inscribed objects are magnified by the hyperlens. Thus, clearly resolved images are obtained in the far-field region in real time. This new type of spherical hyperlens, with its ease of integration with conventional microscopy, provides the possibility of practical imaging applications, leading to the dawn of a new era in biology, pathology, and general nanoscience.

<table><tbody><tr><td>Focused Ion Beam milling machine</td><td>FEI</td><td>Helios Nanolab G3 CX</td><td></td></tr><tr><td>E-beam evaporation system</td><td>Korea Vacuum Tech</td><td>KVE-E4000</td><td></td></tr><tr><td>Scanning electron microscopy</td><td>Hitachi</td><td>SU6600</td><td></td></tr><tr><td>Inverted microscopy</td><td>Zeiss</td><td>Axiovert 200</td><td></td></tr><tr><td>Light source</td><td>EXCELITAS Technologies</td><td>X-Cite 110 LED</td><td></td></tr><tr><td>Band pass filter</td><td>Chroma</td><td>ET405/30M</td><td></td></tr><tr><td>Objective lens</td><td>Zeiss</td><td>Plan-Apochromat</td><td>NA=1.3, 100X</td></tr><tr><td>CCD camera</td><td>Andor</td><td>Zyla 4.2</td><td></td></tr><tr><td>Quartz wafer</td><td>CORNING</td><td>Fused Silica Corning 7980</td><td></td></tr><tr><td>Buffered oxide etchant</td><td>J.T Baker TM</td><td>J.T.Baker 5175</td><td></td></tr><tr><td>Photoresist</td><td>AZ electronic materials</td><td>GXR-601 PR</td><td></td></tr><tr><td>Chromium etchant</td><td>SIGMA-ALDRICH</td><td>651826</td><td></td></tr><tr><td>Aceton</td><td>J.T Baker TM</td><td>UN1090</td><td></td></tr><tr><td>Isopropyl alcohol</td><td>J.T Baker TM</td><td>UN1219</td><td></td></tr><tr><td>FEM simulation tool</td><td>COMSOL 5.1 Multiphysics</td><td></td><td></td></tr></tbody></table>

demonstration of a hyperlens-integrated microscope and super-resolution imaging

The use of super-resolution imaging to overcome the diffraction limit of conventional microscopy has attracted the interest of researchers in biology and nanotechnology. Although near-field scanning microscopy and superlenses have improved the resolution in the near-field region, far-field imaging in real-time remains a significant challenge. Recently, the hyperlens, which magnifies and converts evanescent waves into propagating waves, has emerged as a novel approach to far-field imaging. Here, we report the fabrication of a spherical hyperlens composed of alternating silver (Ag) and titanium oxide (TiO2) thin layers. Unlike a conventional cylindrical hyperlens, the spherical hyperlens allows for two-dimensional magnification. Thus, incorporation into conventional microscopy is straightforward. A new optical system integrated with the hyperlens is proposed, allowing for a sub-wavelength image to be obtained in the far-field region in real time. In this study, the fabrication and imaging setup methods are explained in detail. This work also describes the accessibility and possibility of the hyperlens, as well as practical applications of real-time imaging in living cells, which can lead to a revolution in biology and nanotechnology.

The ability of the hyperlens device to resolve sub-diffraction features relies on its uniformity and on a high-quality fabrication. Here, a hyperlens is composed of a multilayer of Ag and TiO2 deposited alternately. Figure 2a shows the SEM image of a well-made hyperlens17. The cross-sectional image shows that the multilayer of Ag and Ti3O5 thin film is deposited with uniform thickness on the hemispheric...

Watch this Scientific Journal Video about Demonstration of a Hyperlens-integrated Microscope and Super-resolution Imaging at JoVE.com

Demonstration of a Hyperlens-integrated Microscope and Super-resolution Imaging

The use of a hyperlens has been regarded as a novel super-resolution imaging technique due to its advantages in real-time imaging and its simple implementation with conventional optics. Here, we present a protocol describing the fabrication and imaging applications of a spherical hyperlens.

The use of super-resolution imaging to overcome the diffraction limit of conventional microscopy has attracted the interest of researchers in biology and ...

demonstration-hyperlens-integrated-microscope-super-resolution

Universidad San Sebastian

Research

JoVE Journal

Engineering

7.7K Views.  Pohang University of Science and Technology (POSTECH). The use of a hyperlens has been regarded as a novel super-resolution imaging technique due to its advantages in real-time imaging and its simple implementation with conventional optics. Here, we present a protocol describing the fabrication and imaging applications of a spherical hyperlens.

Video: Demonstration of a Hyperlens-integrated Microscope and Super-resolution Imaging

Lensless Fluorescent Microscopy on a Chip

On-chip lensless imaging in general aims to replace bulky lens-based optical microscopes with simpler and more compact designs, especially for high-throughput screening applications. This emerging technology platform has the potential to eliminate the need for bulky and/or costly optical components through the help of novel theories and digital reconstruction algorithms. Along the same lines, here we demonstrate an on-chip fluorescent microscopy modality that can achieve e.g., &lt;4&mu;m spatial resolution over an ultra-wide field-of-view (FOV) of &gt;0.6-8 cm2 without the use of any lenses, mechanical-scanning or thin-film based interference filters. In this technique, fluorescent excitation is achieved through a prism or hemispherical-glass interface illuminated by an incoherent source. After interacting with the entire object volume, this excitation light is rejected by total-internal-reflection (TIR) process that is occurring at the bottom of the sample micro-fluidic chip. The fluorescent emission from the excited objects is then collected by a fiber-optic faceplate or a taper and is delivered to an optoelectronic sensor array such as a charge-coupled-device (CCD). By using a compressive-sampling based decoding algorithm, the acquired lensfree raw fluorescent images of the sample can be rapidly processed to yield e.g., &lt;4&mu;m resolution over an FOV of &gt;0.6-8 cm2. Moreover, vertically stacked micro-channels that are separated by e.g., 50-100 &mu;m can also be successfully imaged using the same lensfree on-chip microscopy platform, which further increases the overall throughput of this modality. This compact on-chip fluorescent imaging platform, with a rapid compressive decoder behind it, could be rather valuable for high-throughput cytometry, rare-cell research and microarray-analysis.

A lensless on-chip fluorescent microscopy platform is demonstrated that can image fluorescent objects over an ultra-wide field-of-view of e.g., &#62;0.6-8 cm2 with &#60;4&#956;m resolution using a compressive sampling based decoding algorithm. Such a compact and wide-field fluorescent on-chip imaging modality could be valuable for high-throughput cytometry, rare-cell research and microarray-analysis.

On-chip lensless imaging in general aims to replace bulky lens-based optical microscopes with simpler and more compact designs, especially for ...

Bioengineering

Lensfree On-chip Tomographic Microscopy Employing Multi-angle Illumination and Pixel Super-resolution

Tomographic imaging has been a widely used tool in medicine as it can provide three-dimensional (3D) structural information regarding objects of different size scales. In micrometer and millimeter scales, optical microscopy modalities find increasing use owing to the non-ionizing nature of visible light, and the availability of a rich set of illumination sources (such as lasers and light-emitting-diodes) and detection elements (such as large format CCD and CMOS detector-arrays). Among the recently developed optical tomographic microscopy modalities, one can include optical coherence tomography, optical diffraction tomography, optical projection tomography and light-sheet microscopy. 1-6 These platforms provide sectional imaging of cells, microorganisms and model animals such as C. elegans, zebrafish and mouse embryos.
Existing 3D optical imagers generally have relatively bulky and complex architectures, limiting the availability of these equipments to advanced laboratories, and impeding their integration with lab-on-a-chip platforms and microfluidic chips. To provide an alternative tomographic microscope, we recently developed lensfree optical tomography (LOT) as a high-throughput, compact and cost-effective optical tomography modality. 7 LOT discards the use of lenses and bulky optical components, and instead relies on multi-angle illumination and digital computation to achieve depth-resolved imaging of micro-objects over a large imaging volume. LOT can image biological specimen at a spatial resolution of &lt;1 &mu;m x &lt;1 &mu;m x &lt;3 &mu;m in the x, y and z dimensions, respectively, over a large imaging volume of 15-100 mm3, and can be particularly useful for lab-on-a-chip platforms.

Lensfree optical tomography is a three-dimensional microscopy technique that offers a spatial resolution of &lt;1 &mu;m &times; &lt;1 &mu;m &times; &lt;3 &mu;m in x, y and z dimensions, respectively, over a large imaging-volume of 15-100 mm3, which can be particularly useful for integration with lab-on-a-chip platforms.

Tomographic imaging has been a widely used tool in medicine as it can provide three-dimensional (3D) structural information regarding objects of different ...

Super-resolution Imaging of the Natural Killer Cell Immunological Synapse on a Glass-supported Planar Lipid Bilayer

The glass-supported planar lipid bilayer system has been utilized in a variety of disciplines. One of the most useful applications of this technique has been in the study of immunological synapse formation, due to the ability of the glass-supported planar lipid bilayers to mimic the surface of a target cell while forming a horizontal interface. The recent advances in super-resolution imaging have further allowed scientists to better view the fine details of synapse structure. In this study, one of these advanced techniques, stimulated emission depletion (STED), is utilized to study the structure of natural killer (NK) cell synapses on the supported lipid bilayer. Provided herein is an easy-to-follow protocol detailing: how to prepare raw synthetic phospholipids for use in synthesizing glass-supported bilayers; how to determine how densely protein of a given concentration occupies the bilayer's attachment sites; how to construct a supported lipid bilayer containing antibodies against NK cell activating receptor CD16; and finally, how to image human NK cells on this bilayer using STED super-resolution microscopy, with a focus on distribution of perforin positive lytic granules and filamentous actin at NK synapses. Thus, combining the glass-supported planar lipid bilayer system with STED technique, we demonstrate the feasibility and application of this combined technique, as well as intracellular structures at NK immunological synapse with super-resolution.

We describe here a combination of the glass-supported lipid bilayer technique of forming immunological synapses with the super-resolution imaging technique of stimulated emission depletion (STED) microscopy. The goal of this protocol is to provide users with the instructions necessary to successfully carry out these two techniques.

The glass-supported planar lipid bilayer system has been utilized in a variety of disciplines. One of the most useful applications of this technique has ...

Immunology and Infection

Confocal and Super-Resolution Imaging of Polarized Intracellular Trafficking and Secretion of Basement Membrane Proteins During Drosophila Oogenesis

The basement membrane (BM) - a specialized sheet of extracellular matrix present at the basal side of epithelial cells - is critical for the establishment and maintenance of epithelial tissue morphology and organ morphogenesis. Moreover, the BM is essential for tissue modeling, serving as a signaling platform, and providing external forces to shape tissues and organs. Despite the many important roles that the BM plays during normal development and pathological conditions, the biological pathways controlling the intracellular trafficking of BM-containing vesicles and how basal secretion leads to the polarized deposition of BM proteins are poorly understood. The follicular epithelium of the Drosophila ovary is an excellent model system to study the basal deposition of BM membrane proteins, as it produces and secretes all major components of the BM. Confocal and super-resolution imaging combined with image processing in fixed tissues allows for the identification and characterization of cellular factors specifically involved in the intracellular trafficking and deposition of BM proteins. This article presents a detailed protocol for staining and imaging BM-containing vesicles and deposited BM using endogenously tagged proteins in the follicular epithelium of the Drosophila ovary. This protocol can be applied to address both qualitative and quantitative questions and it was developed to accommodate high-throughput screening, allowing for the rapid and efficient identification of factors involved in the polarized intracellular trafficking and secretion of vesicles during epithelial tissue development.

Confocal and Super-Resolution Imaging of Polarized Intracellular Trafficking and Secretion of Basement Membrane Proteins During Drosophila Oogenesis

The basement membrane is essential for tissue and organ morphogenesis during development. To better understand the mechanisms leading to proper placement of this structure, the protocol presented describes methods to visualize and characterize the intracellular trafficking and secretion of basement membrane proteins in epithelial cells using confocal and super-resolution microscopy.

The basement membrane (BM) - a specialized sheet of extracellular matrix present at the basal side of epithelial cells - is critical for the establishment ...

Developmental Biology

Photobleaching Enables Super-resolution Imaging of the FtsZ Ring in the Cyanobacterium Prochlorococcus

Super-resolution microscopy has been widely used to study protein interactions and subcellular structures in many organisms. In photosynthetic organisms, however, the lateral resolution of super-resolution imaging is only ~100 nm. The low resolution is mainly due to the high autofluorescence background of photosynthetic cells caused by high-intensity lasers that are required for super-resolution imaging, such as stochastic optical reconstruction microscopy (STORM). Here, we describe a photobleaching-assisted STORM method which was developed recently for imaging the marine picocyanobacterium Prochlorococcus. After photobleaching, the autofluorescence of Prochlorococcus is effectively reduced so that STORM can be performed with a lateral resolution of ~10 nm. Using this method, we acquire the in vivo three-dimensional (3-D) organization of the FtsZ protein and characterize four different FtsZ ring morphologies during the cell cycle of Prochlorococcus. The method we describe here might be adopted for the super-resolution imaging of other photosynthetic organisms.

Photobleaching Enables Super-resolution Imaging of the FtsZ Ring in the Cyanobacterium Prochlorococcus

Here we describe a photobleaching method to reduce the autofluorescence of cyanobacteria. After photobleaching, stochastic optical reconstruction microscopy is used to obtain three-dimensional super-resolution images of the cyanobacterial FtsZ ring.

Super-resolution microscopy has been widely used to study protein interactions and subcellular structures in many organisms. In photosynthetic organisms, ...

Super-resolution Imaging of the Bacterial Division Machinery

Bacterial cell division requires the coordinated assembly of more than ten essential proteins at midcell1,2. Central to this process is the formation of a ring-like suprastructure (Z-ring) by the FtsZ protein at the division plan3,4. The Z-ring consists of multiple single-stranded FtsZ protofilaments, and understanding the arrangement of the protofilaments inside the Z-ring will provide insight into the mechanism of Z-ring assembly and its function as a force generator5,6. This information has remained elusive due to current limitations in conventional fluorescence microscopy and electron microscopy. Conventional fluorescence microscopy is unable to provide a high-resolution image of the Z-ring due to the diffraction limit of light (~200 nm). Electron cryotomographic imaging has detected scattered FtsZ protofilaments in small C. crescentus cells7, but is difficult to apply to larger cells such as E. coli or B. subtilis. Here we describe the application of a super-resolution fluorescence microscopy method, Photoactivated Localization Microscopy (PALM), to quantitatively characterize the structural organization of the E. coli Z-ring8.
	PALM imaging offers both high spatial resolution (~35 nm) and specific labeling to enable unambiguous identification of target proteins. We labeled FtsZ with the photoactivatable fluorescent protein mEos2, which switches from green fluorescence (excitation = 488 nm) to red fluorescence (excitation = 561 nm) upon activation at 405 nm9. During a PALM experiment, single FtsZ-mEos2 molecules are stochastically activated and the corresponding centroid positions of the single molecules are determined with &lt;20 nm precision. A super-resolution image of the Z-ring is then reconstructed by superimposing the centroid positions of all detected FtsZ-mEos2 molecules.
	Using this method, we found that the Z-ring has a fixed width of ~100 nm and is composed of a loose bundle of FtsZ protofilaments that overlap with each other in three dimensions. These data provide a springboard for further investigations of the cell cycle dependent changes of the Z-ring10 and can be applied to other proteins of interest.

We describe a super-resolution imaging method to probe the structural organization of the bacterial FtsZ-ring, an essential apparatus for cell division. This method is based on quantitative analyses of photoactivated localization microscopy (PALM) images and can be applied to other bacterial cytoskeletal proteins.

Bacterial cell division requires the coordinated  assembly of more than ten essential proteins at midcell1,2. Central  to this process is the formation of ...

Biology

Simultaneous Multicolor Imaging of Biological Structures with Fluorescence Photoactivation Localization Microscopy

Localization-based super resolution microscopy can be applied to obtain a spatial map (image) of the distribution of individual fluorescently labeled single molecules within a sample with a spatial resolution of tens of nanometers. Using either photoactivatable (PAFP) or&#160;photoswitchable (PSFP) fluorescent proteins fused to proteins of interest, or organic dyes conjugated to antibodies or other molecules of interest, fluorescence photoactivation localization microscopy (FPALM) can simultaneously image multiple species of molecules within single cells. By using the following approach, populations of large numbers (thousands to hundreds of thousands) of individual molecules are imaged in single cells and localized with a precision of ~10-30 nm. Data obtained can be applied to understanding the nanoscale spatial distributions of multiple protein types within a cell. One primary advantage of this technique is the dramatic increase in spatial resolution: while diffraction limits resolution to ~200-250 nm in conventional light microscopy, FPALM can image length scales more than an order of magnitude smaller. As many biological hypotheses concern the spatial relationships among different biomolecules, the improved resolution of FPALM can provide insight into questions of cellular organization which have previously been inaccessible to conventional fluorescence microscopy. In addition to detailing the methods for sample preparation and data acquisition, we here describe the optical setup for FPALM. One additional consideration for researchers wishing to do super-resolution microscopy is cost: in-house setups are significantly cheaper than most commercially available imaging machines. Limitations of this technique include the need for optimizing the labeling of molecules of interest within cell samples, and the need for post-processing software to visualize results. We here describe the use of PAFP and PSFP expression to image two protein species in fixed cells. Extension of the technique to living cells is also described.

We demonstrate the use of fluorescence photo activation localization microscopy (FPALM) to simultaneously image multiple types of fluorescently labeled molecules&#160;within cells. The techniques described yield the localization of thousands to hundreds of thousands of individual fluorescent labeled proteins, with a precision of tens of nanometers&#160;within single cells.

Localization-based super resolution microscopy can be applied to obtain a spatial map (image) of the distribution of individual fluorescently labeled ...

Super-resolution Imaging of the Cytokinetic Z Ring in Live Bacteria Using Fast 3D-Structured Illumination Microscopy (f3D-SIM)

Imaging of biological samples using fluorescence microscopy has advanced substantially with new technologies to overcome the resolution barrier of the diffraction of light allowing super-resolution of live samples. There are currently three main types of super-resolution techniques &#8211; stimulated emission depletion (STED), single-molecule localization microscopy (including techniques such as PALM, STORM, and GDSIM), and structured illumination microscopy (SIM). While STED and single-molecule localization techniques show the largest increases in resolution, they have been slower to offer increased speeds of image acquisition. Three-dimensional SIM (3D-SIM) is a wide-field fluorescence microscopy technique that offers a number of advantages over both single-molecule localization and STED. Resolution is improved, with typical lateral and axial resolutions of 110 and 280 nm, respectively and depth of sampling of up to 30 &#181;m from the coverslip, allowing for imaging of whole cells. Recent advancements (fast 3D-SIM) in the technology increasing the capture rate of raw images allows for fast capture of biological processes occurring in seconds, while significantly reducing photo-toxicity and photobleaching. Here we describe the use of one such method to image bacterial cells harboring the fluorescently-labelled cytokinetic FtsZ protein to show how cells are analyzed and the type of unique information that this technique can provide.

Super-resolution Imaging of the Cytokinetic Z Ring in Live Bacteria Using Fast 3D-Structured Illumination Microscopy f3D-SIM

Spatiotemporal information about dynamic proteins inside live cells is crucial for understanding biology. A type of super-resolution microscopy called fast 3D-structured illumination microscopy (f3D-SIM) reveals unique information about the cytokinetic Z ring in bacteria: both its bead-like appearance and the rapid dynamics of FtsZ within the ring.

Imaging of biological samples using fluorescence microscopy has advanced substantially with new technologies to overcome the resolution barrier of the ...

Super-Resolution Live Cell Imaging of Subcellular Structures

There has long been a crucial tradeoff between spatial and temporal resolution in imaging. Imaging beyond the diffraction limit of light has traditionally been restricted to be used only on fixed samples or live cells outside of tissue labeled with strong fluorescent signal. Current super-resolution live cell imaging techniques require the use of special fluorescence probes, high illumination, multiple image acquisitions with post-acquisition processing, or often a combination of these processes. These prerequisites significantly limit the biological samples and contexts that this technique can be applied to.
Here we describe a method to perform super-resolution (~140 nm XY-resolution) time-lapse fluorescence live cell imaging in situ. This technique is also compatible with low fluorescent intensity, for example, EGFP or mCherry endogenously tagged at lowly expressed genes. As a proof-of-principle, we have used this method to visualize multiple subcellular structures in the Drosophila testis. During tissue preparation, both the cellular structure and tissue morphology are maintained within the dissected testis. Here, we use this technique to image microtubule dynamics, the interactions between microtubules and the nuclear membrane, as well as the attachment of microtubules to centromeres.
This technique requires special procedures in sample preparation, sample mounting and immobilizing of specimens. Additionally, the specimens must be maintained for several hours after dissection without compromising cellular function and activity. While we have optimized the conditions for live super-resolution imaging specifically in Drosophila male germline stem cells (GSCs) and progenitor germ cells in dissected testis tissue, this technique is broadly applicable to a variety of different cell types. The ability to observe cells under their physiological conditions without sacrificing either spatial or temporal resolution will serve as an invaluable tool to researchers seeking to address crucial questions in cell biology.

Presented here is a protocol for super-resolution live-cell imaging in intact tissue. We have standardized the conditions for imaging a highly sensitive adult stem cell population in its native tissue environment. This technique involves balancing temporal and spatial resolution to allow for the direct observation of biological phenomena in live tissue.

There has long been a crucial tradeoff between spatial and temporal resolution in imaging. Imaging beyond the diffraction limit of light has traditionally ...

Super-Resolution Microscopy of the Synaptonemal Complex Within the Caenorhabditis elegans Germline

During meiosis, homologous chromosomes must recognize and adhere to one another to allow for their correct segregation. One of the key events that secures the interaction of homologous chromosomes is the assembly of the synaptonemal complex (SC) in meiotic prophase I. Even though there is little sequence homology between protein components within the SC among different species, the general structure of the SC has been highly conserved during evolution. In electron micrographs, the SC appears as a tripartite, ladder-like structure composed of lateral elements or axes, transverse filaments, and a central element. 
However, precisely identifying the localization of individual components within the complex by electron microscopy to determine the molecular structure of the SC remains challenging. By contrast, fluorescence microscopy allows for the identification of individual protein components within the complex. However, since the SC is only ~100 nm wide, its substructure cannot be resolved by diffraction-limited conventional fluorescence microscopy. Thus, determining the molecular architecture of the SC requires super-resolution light microscopy techniques such as structured illumination microscopy (SIM), stimulated-emission depletion (STED) microscopy, or single-molecule localization microscopy (SMLM). 
To maintain the structure and interactions of individual components within the SC, it is important to observe the complex in an environment that is close to its native environment in the germ cells. Therefore, we demonstrate an immunohistochemistry and imaging protocol that enables the study of the substructure of the SC in intact, extruded Caenorhabditis elegans germline tissue with SMLM and STED microscopy. Directly fixing the tissue to the coverslip reduces the movement of the samples during imaging and minimizes aberrations in the sample to achieve the high resolution necessary to visualize the substructure of the SC in its biological context.

Super-Resolution Microscopy of the Synaptonemal Complex Within the Caenorhabditis elegans Germline

Super-resolution microscopy can provide a detailed insight into the organization of components within the synaptonemal complex in meiosis. Here, we demonstrate a protocol to resolve individual proteins of the Caenorhabditis elegans synaptonemal complex.

During meiosis, homologous chromosomes must recognize and adhere to one another to allow for their correct segregation. One of the key events that secures ...

Demonstration of a Hyperlens-integrated Microscope and Super-resolution Imaging

Summary

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Confocal and Super-Resolution Imaging of Polarized Intracellular Trafficking and Secretion of Basement Membrane Proteins During Drosophila Oogenesis

Photobleaching Enables Super-resolution Imaging of the FtsZ Ring in the Cyanobacterium Prochlorococcus

Super-resolution Imaging of the Bacterial Division Machinery

Simultaneous Multicolor Imaging of Biological Structures with Fluorescence Photoactivation Localization Microscopy

Super-resolution Imaging of the Cytokinetic Z Ring in Live Bacteria Using Fast 3D-Structured Illumination Microscopy (f3D-SIM)

Super-Resolution Live Cell Imaging of Subcellular Structures

Super-Resolution Microscopy of the Synaptonemal Complex Within the Caenorhabditis elegans Germline