Name: compact lens-less digital holographic microscope for mems inspection and characterization
Uploaded: 2016-07-05T15:00:00.000+00:00
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Description: Watch this Scientific Journal Video about Compact Lens-less Digital Holographic Microscope for MEMS Inspection and Characterization at JoVE.com

The authors have no acknowledgements.

1. Preliminary Preparation of the Measurement
Note: The sample used for the experiment is a MEMS electrode. The gold electrodes are fabricated on a silicon wafer using lift off process. The sample is an 18 mm x 18 mm wafer with periodic structures (electrodes) with 1 mm period
<ol>
	<li>Sign into the logbook before using the system.</li>
	<li>Turn on the computer, LASER and translation stage power.</li>
	<li>Place the MEMS electrode/micro-diaphragm sample.
	<ol>
		<li>Place the MEMS sample in the middle of the sample holder using a tweezer.</li>
		<li>Adjust the sample holder to position the electrodes in the beam path. The maximum measurement field of view is defined by the camera sensor size. It is a rectangle of 2.3 mm x 1.8 mm.</li>
	</ol>
	</li>
	<li>Using the vertical direction motorized stage, move the system approximatively 1.5 cm away from the sample.</li>
</ol>
2. Software Settings Adjustment
<ol>
	<li>Open the 3DView software. 3DView is our in-house program developed in C++.</li>
	<li>Click imaging source button to select the proper camera for the experiment. Choose the monochrome CCD camera. Avoid a color camera in this setup since a monochromatic diode laser is used. Additionally, for the same number of pixels, resolution would be lower when using color cameras.
	<ol>
		<li>In the device settings tab, select Y800 (1,280 x 960) video format and 15 frames per second video rate.</li>
	</ol>
	</li>
	<li>Click yellow play button to start the camera. An image of the object with imprinted fringe patterns (Hologram) should appear.
	<ol>
		<li>Adjust optimum gain and exposure parameters to avoid image saturation if needed.</li>
	</ol>
	</li>
	<li>Using the live video window camera view, adjust the sample position to select the exact area to investigate on the sample.</li>
	<li>Open settings tab.
	<ol>
		<li>In the configuration tab, select the type of surface (reflective or transparent), wavelength of laser, and pixel size of the camera. The laser is a diode laser operating at 633 nm. The pixel size of the camera is 4,650 nm. The sample is a specular MEMS electrode device so reflective mode should be selected. 
		Note: The CDHM configuration allows only reflective surfaces to be measured. However, the software can also be used to measure transparent samples when a different digital holography system is used13. A change in this setting changes the height calculation formula from the phase. Indeed, the optical path difference calculation is slightly different for transparent samples as it includes the object refractive index.</li>
		<li>Choose the Convolution reconstruction algorithm and set the reconstruction distance to zero. Choose a reconstruction step of 1 or 2. 
		Note: The reconstruction distance parameter can be defined later by considering the intensity image obtained from the hologram and using the auto-focus. The reconstruction step defines the number of steps used to implement the Fresnel integral and simulate the beam propagation. The first method evaluate the integral once as a single Fourier Transform. A step of 2 will evaluate the integral twice. This adds more flexibility in the grid spacing but is computationally less efficient20.</li>
		<li>In the post processing tab, select the unwrapping algorithm necessary to get the final unwrapped image. Select quality mapped algorithm. 
		Note: In the software, the choice between Goldstein and Quality Mapped algorithm can be made. The later has shown robust and rapid spatial phase unwrapping. The quality mapped algorithm is based on guided phase unwrapping as described in 21.</li>
	</ol>
	</li>
</ol>
3. Data Acquisition
<ol>
	<li>Press the Fourier transform icon to open the Fourier spectrum window. One 0 order and two +1, -1 orders should appear. If this is not the case, check that the sample is in the right position, and adjust gain and exposure time again.</li>
	<li>Stop the live measurement mode. Select one of the diffracted orders (positive or negative frequency) by using the filter tool. The selected area should be large enough so that all the frequencies needed for the phase retrieval are present. Switch on the live mode again. 
	Note: The choice of the negative order will just affect the sign of the phase in the final result, i.e., the final 3D image will be inverted.</li>
	<li>Open the phase window. Check that the unwrapped mode is not enabled. Gray phase image of the object imprinted with wrapped fringes should appear.</li>
	<li>Utilize the motorized vertical stage to reduce the number of fringes in the phase image. When only 1 or 2 fringes are left on the image, stop the motorized stage. 
	Note: The system is based on interferometry. Thus it is sensitive to vibrations. After moving the z direction motorized stage, the user should wait 1 or 2 sec before the wrapped phase image appears again. It is also important to avoid vibrations during the measurement to get a stable phase image.</li>
	<li>Click the auto-focus22 button to find the best reconstruction distance. One may need to use autofocus several times to approach the optimum reconstruction distance until the intensity image appears sharp and clear. The autofocus is based on an efficient and time effective angular spectrum method as described in 22. 
	Note: The focus slider bar can be used for fine adjustment. Then, click on center focus button to record the current reconstruction distance. It appears sometimes that best focus is not found with autofocus option. In this case, manually input reconstruction distance to find the best focus.</li>
	<li>Enable the unwrapped mode to see the unwrapped phase image by clicking on the unwrapping button.</li>
</ol>
4. Data Visualization and Analysis for Static Measurement
<ol>
	<li>Open the 3D image window to see the final 3D image of the sample. Use available options to observe the final result (rotate, color map, scale display&#8230;).</li>
	<li>Click on the tile windows button to arrange the windows as non-overlapping and display all the measurements windows.</li>
	<li>Use the line ruler to draw a line on an area of interest on the unwrapped phase image. In the line plot window, a cross sectional profile plot of the area of interest can be observed. Use the two green line markers to extract an approximate height of the object (Figure 5). 
	Surface roughness can also be obtained on the flat top part of the sample.</li>
	<li>Save the final phase image in .JPEG format to import it to other software if needed.</li>
</ol>
5. Preparation of Sample and Data Analysis for Dynamic Measurement
<ol>
	<li>Place the micro diaphragm on a heating station plate. The sample will not be removed from the plate until the experiment ends.</li>
	<li>Record a hologram of the micro diaphragm at ambient temperature by following the procedure described above in section 2 and 3. It will be used as a reference for the deformation analysis.</li>
	<li>Save the phase data on the computer.</li>
	<li>Turn on the laboratory heating plate.</li>
	<li>Using the temperature knob, vary the temperature in steps of 50 &#176;C from 50 &#176;C to 300 &#176;C. For each temperature step, save the phase map image in .JPEG format.</li>
	<li>Subtract the initial ambient temperature phase map from the other phase map recorded to obtain the deformation data. 
	Note: This post processing step can be realized with simple MATLAB code. The different phases obtained are loaded into MATLAB and simple matrix subtraction is performed. Then cross sectional plots of the different deformation stages can be obtained.</li>
</ol>

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The authors have nothing to disclose.

In this review, we provide a protocol to accurately recover the quantitative morphology of different MEMS devices by using a compact system relying on digital holography. MEMS characterization in both static and dynamic mode is demonstrated. Quantitative 3D data of a micro channel MEMS is obtained. In order to validate the accuracy of the system, results have been compared between the CDHM and the AFM. Good agreement is found meaning that digital holography can be a reliable technique for 3D imaging. Results indicate tha...

Metrology of micro and nano objects is of great importance for both industry and researchers. Indeed, miniaturization of objects represents a new challenge for optical metrology. Micro electro mechanical systems (MEMS) are generally defined has miniaturized electromechanical systems and usually comprises components such as micro sensors, micro actuators, microelectronics and microstructures. It has found many applications in diverse field such as biotechnology, medicine, communication and sensing1. Recently, the increasing complexity as well as the progressive miniaturization of test object features call for the development of suitable characterization techniques for MEMS. High throughput manufacturing of these complex microsystems requires the implementation of advanced inline measurement techniques, to quantify characteristic parameters and related defects caused by the process conditions2. For instance, the deviation of geometrical parameters in a MEMS device affects the system properties and has to be characterized. In addition, industry requires high resolution measurement performance, such as full three dimension (3D) metrology, large &#64257;eld of view, high imaging resolution, and real time analysis. Thus, it is essential to ensure a reliable quality control and inspection process. Moreover, it requires the measuring system to be easily implementable on a production line and thus relatively compact to be installed on existing infrastructures.
Holography, which was first introduced by Gabor3, is a technique that allows the recovery of the full quantitative information of an object by recording the interference between a reference and an object wave into a photosensitive medium. During this process known as recording, the amplitude, phase and polarization of a field are stored in the medium. Then the object wave field can be recovered by sending the reference beam onto the medium, a process known as optical reading of the hologram. Since a conventional detector only records the intensity of the wave, holography has been a subject of great interest in the past fifty years since it gives access to additional information on the electric field. However, several aspects of conventional holography make it unpractical for industry applications. Indeed, photosensitive materials are expensive and the recording process generally requires a high degree of stability. Advances in high resolution camera sensors such as charged coupled devices (CCD) have opened a new approach for digital metrology. One of those techniques is known as digital holography4. In Digital Holography (DH), the hologram is recorded on a camera (recording medium) and numerical processes are used to reconstruct the phase and intensity information. As with conventional holography, the result can be obtained after two main procedures: the recording and reconstruction as shown in Figure 1. However, if the recording is similar to conventional holography, the reconstruction is only numerical5. The numerical reconstruction process is shown in Figure 2. Two procedures are involved in the reconstruction process. Firstly, the object wave field is retrieved from the hologram. The hologram is multiplied with a numerical reference wave to get the object wavefront at the hologram plane. Secondly, the complex object wavefront is numerically propagated to the image plane. In our system, this step is performed using the convolution method6. The reconstructed field obtained is a complex function and thus phase and intensity can be extracted providing quantitative height information on the object of interest. The capability of whole &#64257;eld information storage in holography method and the use of computer technology for fast data processing offer more flexibility in experimental configuration and significantly increase the speed of the experimental process, opening up new possibilities to develop DH as a dynamic metrological tool for MEMS and micro-systems7,8.
Use of digital holography in phase contrast imaging is now well established and was first presented more than ten years ago9. Indeed, investigation of microscopic devices by combining digital holography and microscopy has been performed in many studies10, 11, 12, 13. Several systems based on high coherence14 and low coherence15 sources as well as different types of geometry13, 16, 17 (in line, off axis, common path&#8230;) have been presented. In addition, in line digital holography has been used previously in characterization of MEMS device18, 19. However, those systems are generally difficult to implement and bulky, making them unsuitable for industrial applications. In this study, we propose a compact, simple and lens free system based on off axis digital holography capable for real time MEMS inspection and characterization. The Compact Digital Holographic microscope (CDHM) is a lens less digital holographic system developed and patented to obtain the 3D morphology of micro-size specular objects. In our system, a 10 mW, highly stable, temperature controlled diode laser operating at 638 nm is coupled into a mono-mode fiber. As shown in Figure 3, the diverging beam emanating from the fiber is split into a reference and an object beam by a beam splitter. The reference beam path comprises a tilted mirror to realize the off axis geometry. The object beam is scattered and reflected by the sample. The two beams interfere on the CCD giving the hologram. The interference pattern imprinted onto the image is called a spatial carrier and permits the recovery of the quantitative phase information with only one image. The numerical reconstruction is performed using a common Fourier transform and convolution algorithm as stated previously. The lens-less configuration has several advantages making it attractive. As no lenses are used, the input beam is a diverging wave providing a natural geometrical magnification and thus improving the system resolution. Moreover, it is free of aberrations encountered in typical optical systems. As can be seen in Figure 3B, the system can be made compact (55x75x125 mm3), lightweight (400 g), and thus can be easily integrated into industrial production lines.

<table><tbody><tr><td>2 MP Camera</td><td>Imaging Source</td><td>DMX 41BU02</td><td>used to record the hologram. 4.65&amp;nbsp;microns pixel size</td></tr><tr><td>Motorized X,Y,Z Translation Stage</td><td>Zaber Technology&amp;nbsp;</td><td>TLS28-M</td><td>Holder for the system&amp;nbsp;</td></tr><tr><td>Beam splitter</td><td>Edmund optics</td><td>49-003</td><td>Cube Beam splitter. Separate and recombine the object and reference beam</td></tr><tr><td>Laser&amp;nbsp;</td><td>Micro Laser Systems, Inc.</td><td>SRT-F635S-20/OSYS</td><td>Diode laser</td></tr><tr><td>Mirror</td><td>Edmund Optics</td><td>#43-412-566</td><td>1&quot; Dia. Protected Gold, &amp;lambda;/20 Flat Zerodur</td></tr><tr><td>monomode Fiber</td><td>Thorlabs</td><td>S405-XP</td><td>Single Mode Optical Fiber, 400 - 680 nm, &amp;Oslash; 125 &amp;micro;m Cladding</td></tr><tr><td>Sample holder</td><td>Edmund Optics</td><td>#39-930</td><td>Ideal Positioning Platform, &amp;plusmn;35&amp;nbsp;mm Travel in Both X and Y</td></tr><tr><td>Hotplate</td><td>Thermolyne Mirak hotplate</td><td>Barnstead International HP72935-60</td><td>temperature range 40-370 &amp;deg;C</td></tr><tr><td>Holoscope Software</td><td>d&#039;Optron Pte Ltd</td><td></td><td>software developed by the NTU researchers&amp;nbsp;</td></tr></tbody></table>

compact lens-less digital holographic microscope for mems inspection and characterization

A micro-electro-mechanical-system (MEMS) is a widely used component in many industries, including energy, biotechnology, medical, communications, and automotive. However, effective inspection and characterization metrology systems are needed to ensure the functional reliability of MEMS. This study presents a system based on digital holography as a tool for MEMS metrology. Digital holography has gained increasing attention in the past 20 years. With the fast development and decreasing cost of sensor arrays, resolution of such systems has increased broadening potential applications. Thus, it has attracted attention from both research and industry sides as a potential reliable tool for industrial metrology. Indeed, by recording the interference pattern between an object beam (which contains sample height information) and a reference beam on a CCD camera, one can retrieve the quantitative phase information of an object. However, most of digital holographic systems are bulky and thus not easy to implement on industry production lines. The novelty of the system presented is that it is lens-less and thus very compact. In this study, it is shown that the Compact Digital Holographic Microscope (CDHM) can be used to evaluate several characteristics typically consider as criteria in MEMS inspections. The surface profiles of MEMS in both static and dynamic conditions are presented. Comparison with AFM is investigated to validate the accuracy of the CDHM.

The protocol described above was designed to inspect and characterize MEMS and Micro devices using CDHM system. In our system, a mono-mode fiber is coupled to a diode laser operating at a 633 nm wavelength. Due to the diverging beam configuration, it is important to match the object beam and reference beam path in order to obtain a hologram that can be reconstructed. This is achieved through careful vertical positioning of the sample with respect to the system. In the calculated wrapped p...

Watch this Scientific Journal Video about Compact Lens-less Digital Holographic Microscope for MEMS Inspection and Characterization at JoVE.com

Compact Lens-less Digital Holographic Microscope for MEMS Inspection and Characterization

We present a compact reflection digital holographic system (CDHM) for inspection and characterization of MEMS devices. A lens-less design using a diverging input wave providing natural geometrical magnification is demonstrated. Both static and dynamic studies are presented.

A micro-electro-mechanical-system (MEMS) is a widely used component in many industries, including energy, biotechnology, medical, communications, and ...

compact-lens-less-digital-holographic-microscope-for-mems-inspection

Universidad San Sebastian

Research

JoVE Journal

Engineering

10.2K Views.  Nanyang Technological University. We present a compact reflection digital holographic system (CDHM) for inspection and characterization of MEMS devices. A lens-less design using a diverging input wave providing natural geometrical magnification is demonstrated. Both static and dynamic studies are presented.

Video: Compact Lens-less Digital Holographic Microscope for MEMS Inspection and Characterization

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 ...

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 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 ...

Spectral and Angle-Resolved Magneto-Optical Characterization of Photonic Nanostructures

Photonic crystals are periodic nanostructures that can support a variety of confined electromagnetic modes. Such confined modes are usually accompanied by local enhancement of electric field intensity that strengthens light-matter interactions, enabling applications such as surface-enhanced Raman scattering (SERS) and surface plasmon enhanced sensing. In the presence of magneto-optically active materials, the local field enhancement gives rise to anomalous magneto-optical activity. Typically, the confined modes of a given photonic crystal depend strongly on the wavelength and incidence angle of the incident electromagnetic radiation. Thus, spectral and angular-resolved measurements are needed to fully identify them as well as to establish their relationship with the magneto-optical activity of the crystal. In this article, we describe how to use a Fourier-plane (back focal plane) microscope to characterize magneto-optically active samples. As a model system, here we use a plasmonic grating built out of magneto-optically active Au/Co/Au multilayer. In the experiments, we apply a magnetic field on the grating in situ and measure its reciprocal space response, obtaining the magneto-optical response of the grating over a range of wavelengths and incident angles. This information enables us to build a complete map of the plasmonic band structure of the grating and the angle and wavelength dependent magneto-optical activity. These two images allow us to pinpoint the effect that the plasmon resonances have on the magneto-optical response of the grating. The relatively small magnitude of magneto-optical effects requires a careful treatment of the acquired optical signals. To this end, an image processing protocol for obtaining magneto-optical response from the acquired raw data is laid out.

Photonic band structure enables understanding how confined electromagnetic modes propagate within a photonic crystal. In photonic crystals that incorporate magnetic elements, such confined and resonant optical modes are accompanied by enhanced and modified magneto-optical activity. We describe a measurement procedure to extract the magneto-optical band structure by Fourier space microscopy.

Photonic crystals are periodic nanostructures that can support a variety of confined electromagnetic modes. Such confined modes are usually accompanied by ...

Lensless On-chip Imaging of Cells Provides a New Tool for High-throughput Cell-Biology and Medical Diagnostics

Conventional optical microscopes image cells by use of objective lenses that work together with other lenses and optical components. While quite effective, this classical approach has certain limitations for miniaturization of the imaging platform to make it compatible with the advanced state of the art in microfluidics. In this report, we introduce experimental details of a lensless on-chip imaging concept termed LUCAS (Lensless Ultra-wide field-of-view Cell monitoring Array platform based on Shadow imaging) that does not require any microscope objectives or other bulky optical components to image a heterogeneous cell solution over an ultra-wide field of view that can span as large as ~18 cm2. Moreover, unlike conventional microscopes, LUCAS can image a heterogeneous cell solution of interest over a depth-of-field of ~5 mm without the need for refocusing which corresponds to up to ~9 mL sample volume. This imaging platform records the shadows (i.e., lensless digital holograms) of each cell of interest within its field of view, and automated digital processing of these cell shadows can determine the type, the count and the relative positions of cells within the solution. Because it does not require any bulky optical components or mechanical scanning stages it offers a significantly miniaturized platform that at the same time reduces the cost, which is quite important for especially point of care diagnostic tools. Furthermore, the imaging throughput of this platform is orders of magnitude better than conventional optical microscopes, which could be exceedingly valuable for high-throughput cell-biology experiments.

Lensfree on-chip imaging and characterization of cells is illustrated. This on-chip cell imaging approach provides a compact and cost-effective tool for medical diagnostics and high-throughput cell biology applications, making it especially suitable for resource poor settings.

Conventional optical microscopes image cells by use of objective lenses that work together with other lenses and optical components. While quite ...

Biology

Uncovering Hidden Dynamics of Natural Photonic Structures Using Holographic Imaging

In this method, the potential of optics and holography to uncover hidden details of a natural system's dynamical response at the nanoscale is exploited. In the first part, the optical and holographic studies of natural photonic structures are presented as well as conditions for the appearance of the photophoretic effect, namely, the displacement or deformation of a nanostructure due to a light-induced thermal gradient, at the nanoscale. This effect is revealed by real-time digital holographic interferometry monitoring the deformation of scales covering the wings of insects induced by temperature. The link between geometry and nanocorrugation that leads to the emergence of the photophoretic effect is experimentally demonstrated and confirmed. In the second part, it is shown how holography can be potentially used to uncover hidden details in the chemical system with nonlinear dynamics, such as the phase transition phenomenon that occurs in complex oscillatory Briggs-Rauscher (BR) reaction. The presented potential of holography at the nanoscale could open enormous possibilities for controlling and molding the photophoretic effect and pattern formation for various applications such as particle trapping and levitation, including the movement of unburnt hydrocarbons in the atmosphere and separation of different aerosols, decomposition of microplastics and fractionation of particles in general, and assessment of temperature and thermal conductivity of micron-size fuel particles.

The paper is primarily focused on the combined power of optical (linear and nonlinear) and holographic methods used to reveal phenomena at the nanoscale. The results obtained from the biophotonic and oscillatory chemical reactions' studies are given as representative examples, highlighting holography's ability to reveal dynamics at a nanoscale.

In this method, the potential of optics and holography to uncover hidden details of a natural system's dynamical response at the nanoscale is exploited. ...

Chemistry

Quantifying Microorganisms at Low Concentrations Using Digital Holographic Microscopy (DHM)

Accurately detecting and counting sparse bacterial samples has many applications in the food, beverage, and pharmaceutical processing industries, in medical diagnostics, and for life detection by robotic missions to other planets and moons of the solar system. Currently, sparse bacterial samples are counted by culture plating or epifluorescence microscopy. Culture plates require long incubation times (days to weeks), and epifluorescence microscopy requires extensive staining and concentration of the sample. Here, we demonstrate how to use off-axis digital holographic microscopy (DHM) to enumerate bacteria in very dilute cultures (100-104 cells/mL). First, the construction of the custom DHM is discussed, along with detailed instructions on building a low-cost instrument. The principles of holography are discussed, and a statistical model is used to estimate how long videos should be to detect cells, based on the optical performance characteristics of the instrument and the concentration of the bacterial solution (Table 2). Video detection of cells at 105, 104, 103, and 100 cells/mL is demonstrated in real time using un-reconstructed holograms. Reconstruction of amplitude and phase images is demonstrated using an open-source software package.

Quantifying Microorganisms at Low Concentrations Using Digital Holographic Microscopy DHM

Digital holographic microscopy (DHM) is a volumetric technique that allows imaging samples 50-100X thicker than brightfield microscopy at comparable resolution, with focusing performed post-processing. Here DHM is used for identifying, counting, and tracking microorganisms at very low densities and compared with optical density measurements, plate count, and direct count.

Accurately detecting and counting sparse bacterial samples has many applications in the food, beverage, and pharmaceutical processing industries, in ...

Digital Inline Holographic Microscopy (DIHM) of Weakly-scattering Subjects

Weakly-scattering objects, such as small colloidal particles and most biological cells, are frequently encountered in microscopy. Indeed, a range of techniques have been developed to better visualize these phase objects; phase contrast and DIC are among the most popular methods for enhancing contrast. However, recording position and shape in the out-of-imaging-plane direction remains challenging. This report introduces a simple experimental method to accurately determine the location and geometry of objects in three dimensions, using digital inline holographic microscopy (DIHM). Broadly speaking, the accessible sample volume is defined by the camera sensor size in the lateral direction, and the illumination coherence in the axial direction. Typical sample volumes range from 200 &#181;m&#160;x 200 &#181;m&#160;x 200 &#181;m using LED illumination, to 5 mm&#160;x 5 mm&#160;x 5 mm or larger using laser illumination. This illumination light is configured so that plane waves are incident on the sample. Objects in the sample volume then scatter light, which interferes with the unscattered light to form interference patterns perpendicular to the illumination direction. This image (the hologram) contains the depth information required for three-dimensional reconstruction, and can be captured on a standard imaging device such as a CMOS or CCD camera. The Rayleigh-Sommerfeld back propagation method is employed to numerically refocus microscope images, and a simple imaging heuristic based on the Gouy phase anomaly is used to identify scattering objects within the reconstructed volume. This simple but robust method results in an unambiguous, model-free measurement of the location and shape of objects in microscopic samples.

Digital Inline Holographic Microscopy DIHM of Weakly-scattering Subjects

The three-dimensional locations of weakly-scattering objects can be uniquely identified using digital inline holographic microscopy (DIHM), which involves a minor modification to a standard microscope. Our software uses a simple imaging heuristic coupled with Rayleigh-Sommerfeld back-propagation to yield the three-dimensional position and geometry of a microscopic phase object.

Weakly-scattering objects, such as small colloidal particles and most biological cells, are frequently encountered in microscopy. Indeed, a range of ...

Evaluation and Manipulation of Neural Activity Using Two-Photon Holographic Microscopy

Recent advances in optical bioimaging and optogenetics have enabled the visualization and manipulation of biological phenomena, including cellular activities, in living animals. In the field of neuroscience, detailed neural activity related to brain functions, such as learning and memory, has now been revealed, and it has become feasible to artificially manipulate this activity to express brain functions. However, the conventional evaluation of neural activity by two-photon Ca2+ imaging has the problem of low temporal resolution. In addition, manipulation of neural activity by conventional optogenetics through the optic fiber can only simultaneously regulate the activity of neurons with the same genetic background, making it difficult to control the activity of individual neurons. To solve this issue, we recently developed a microscope with a high spatiotemporal resolution for biological applications by combining optogenetics with digital holographic technology that can modify femtosecond infrared laser beams. Here, we describe protocols for the visualization, evaluation, and manipulation of neural activity, including the preparation of samples and operation of a two-photon holographic microscope (Figure 1). These protocols provide accurate spatiotemporal information on neural activity, which may be useful for elucidating the pathogenesis of neuropsychiatric disorders that lead to abnormalities in neural activity.

We developed a two-photon holographic microscope that can visualize, assess, and manipulate neural activity using high spatiotemporal resolution, with the aim of elucidating the pathogenesis of neuropsychiatric disorders that are associated with abnormal neural activity.

Recent advances in optical bioimaging and optogenetics have enabled the visualization and manipulation of biological phenomena, including cellular ...

Neuroscience

Lens-free Video Microscopy for the Dynamic and Quantitative Analysis of Adherent Cell Culture

Here, we demonstrate that lens-free video microscopy enables us to simultaneously capture the kinetics of thousands of cells directly inside the incubator and that it is possible to monitor and quantify single cells along several cell cycles. We describe the full protocol used to monitor and quantify a HeLa cell culture for 2.7 days. First, cell culture acquisition is performed with a lens-free video microscope, and then the data is analyzed following a four-step process: multi-wavelength holographic reconstruction, cell-tracking, cell segmentation and cell division detection algorithms. As a result, we show that it is possible to gather a dataset featuring more than 10,000 cell cycle tracks and more than 2 x 106 cell morphological measurements.

Lens-free video microscopy enables us to monitor cell cultures directly inside the incubator. Here we describe the full protocol used to acquire and analyze a 2.7 day long acquisition of cultured HeLa cells, leading to a dataset of 2.2 x 106 measurements of individual cell morphology and 10584 cell cycle tracks.

Here, we demonstrate that lens-free video microscopy enables us to simultaneously capture the kinetics of thousands of cells directly inside the incubator ...