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

Summary

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.

Abstract

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.

Introduction

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 them^2,3. 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 "superlens," 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 objects^4,5,6,7.

Recently, the hyperlens has emerged as a novel approach to real-time far-field optical imaging^8,9,10,11,12. 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 symmetry^14,15,16.

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.

Protocol

1. Substrate Preparation

1. Obtain highly refined quartz wafer. For the fabrication reported here, use a wafer with a 500 µm thickness.
2. Spin-coat the quartz wafer with a positive photoresist at 2,000 rpm and bake for 60 s at 90 °C.
   NOTE: The positive photoresist layer is coated to prevent damage during the subsequent cutting step.
3. Use a dicing machine to cut the wafer with photoresist into small pieces 20 x 20 mm² in size.
4. Blow using a compressed nitrogen gun to remove particulates resulting from the cutting step.
5. Place it in an ultrasonic bath in de-ionized (DI) water for 5 min at 45 °C. Remove the photoresist layer using an ultrasonic bath in acetone for 5 min at 45 °C. Clean the substrate using two ultrasonic baths, acetone and isopropyl alcohol, each for 5 min at 45 °C.
6. Dry the substrate with a compressed nitrogen gun.

2. Etching the Mask Pattern

1. Load the clean quartz substrates into a high-vacuum electron beam evaporation system. Ensure that substrate rotation is enabled.
2. Deposit the chromium layer with a deposition rate of 2 Å/s.
   NOTE: A layer at least 100 nm-thick should be deposited for the etching mask to prevent pinholes made from deposition.
3. Press the vent button to vent the chamber and mount a sample on the focused ion beam (FIB) holder using conducting copper tape.
4. Load the FIB holder into the FIB chamber.
5. Close the chamber door and press the pump button to evacuate the chamber.
6. Select "beam on" under the beam control tab and set the ion beam current (7.7 pA) and acceleration voltage (30 kV) for FIB mode.
7. Turn on the ion beam system.
8. Select "beam on" under the beam control tab to turn on the electron beam and focus the image with low magnification using software.
9. Set the working distance (WD) at 4 mm under the navigation tab in scanning electron microscope (SEM) mode.
10. Set the tilt angle of the holder to 52° and take the SEM images at different magnifications before hole array mask pattern fabrication.
11. 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.
12. After finishing, turn off the electron beam and ion beam systems and cool down the system.
13. Press the vent button and vent the chamber with nitrogen gas. Take the holder out of the chamber.
14. Close the chamber door and evacuate the chamber by pressing the pump button.

3. Wet-etching Process and Removal of the Mask Layer

1. 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 µm-diameter hemisphere can be obtained within 5 min.
2. 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.
3. Dry the sample with compressed nitrogen gas.
4. 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 µm in diameter can be obtained.
5. Put the patterned substrate into DI water to clean it (5 min).

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.

1. Press the vent button of the electron beam evaporation system and wait until the vent is over.
2. Load the patterned substrate into a high-vacuum electron beam evaporation system after the vent.
3. 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.
4. Deposit the Ag layer with a growth rate of 1 Å/s and deposit a 15 nm-thick Ag layer.
5. After the deposition of the Ag layer, cool down the substrate for 5 min.
6. Change the pocket of the electron beam evaporation system by choosing another crucible and deposit the TiO₂ layer with a growth rate of 1 Å/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.
7. After the deposition of the TiO₂ layer, cool down the substrate for 5 min.
8. 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.
9. Change the pocket of the electron beam evaporation system and deposit the chromium layer at a thickness of 50 nm.
10. 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.
11. After the vent, open the chamber door and take the mount holder out of the chamber. Strip off the fabricated hyperlens device.
12. Close the chamber door and evacuate the chamber by pressing the pump button.
13. Mount the hyperlens deposited with chromium into the FIB milling system and pattern a nano-sized structure, per the manufacturer's instructions.

5. Setting Up the Imaging System and Imaging Procedure

1. Place a conventional transmission-type optical microscope on the optical table.
   NOTE: Here, an inverted optical microscope was used as the main body.
2. Connect a white-light source to the microscope illumination path using an adapter.
3. 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.
4. 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.
5. 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.

Results

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...

Discussion

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...

Materials

List of materials used in this article

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