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Revision as of 07:05, 17 October 2017
ChiTUcare
Digital Inline Holographic Microscopy - An iGEM Approach
In light of the iGEM competition, the need for analyzing the 3D structures of hydrogel and E.Coli at micrometer scales has arisen. Our project aims at constructing a low cost Digital Inline Holography Microscope (DIHM). The DIHM features on its ease-of-use, lens-less inline structure, and the state-of-art reconstruction algorithms from holograms to 3D visualization with micrometer resolution. The working principle of a DIHM starts with a point laser source, emanating a spherical wave through a pinhole, illuminating the object to be observed, and forming a magnified diffraction pattern at the image sensor, followed by reconstruction algorithms. The holograms collected by the image sensor already contains the difference of intensity and phase shifts, compared with the reference beam from the spherical wave, thus the inline structure without the need of a lens or beam splitter. Our project uses easily accessible hardware components: an xbox 360 pickup as the laser source, DIYouware PCB board for the alignment and laser intensity control (TO BE REVISED LATER), a 1 µm pinhole, a Pi-cam and the Raspberry Pi for taking pictures, and certain 3D printed parts to assemble the microscope. The open source library Holopy is then deployed to reconstruct the 3D volumes from the holograms.
Achievements
It achieves!
Get It
Electronics
Our initial plan was to use an Xbox DVD pickup as the laser source, as it provides 405 nm, 650 nm, and 780 nm laser point source, with possible laser alignment and intensity control by using a customized PCB board. However as we experimented with the pickup, we found out that the hologram was greatly destructed by the grating (more specs?) within the pickup. Thus we opted out on the pickup and PCB idea. Instead, our setup now consists solely of a simple (LED source + pinhole) / (Laser source + lenses + optics fiber), a micromanipulator and stage for alignment, and a Picam with RapsberryPi 3 for the recorded holograms.
As shown in the picture above, RaspberryPi 3 is connected to power, Pi cam, and the LAN cable for internet connection. A webcam server is then setup with local host configuration such that a live stream from the Picam can be accessed via a browser on another PC. The webcam server is also customized with a GUI to record images or videos, adjust camera settings such as ISO, resolution, and save the recorded data on the server for downloading later.
Working Principle
The working principle of digital holographic microscopy relies on the interference pattern, i.e., the hologram, which encodes 3D information in a 2D picture, and the 3D reconstruction algorithm, to extract the 3D information from the recorded holograms. The interference pattern comes from the joint wavefronts of the object beam and the reference beam. As indicated in the picture below, a coherent collimated light source is splitted into two beams, the object beam passing through the lens and the object to be observed, and the reference beam pertaining the phase and coherence of the light source. The joint wavefronts form the hologram, and is recorded by an image sensor. The 3D reconstruction algorithm then functions as a digital lens, cutting the joint wavefronts in minuscule distances near the object, rendering a stack of cross-sectional wavefronts. Based on the stack of wavefronts, or interference patterns, the image of the object can be reconstructed in different depths, thus achieving a axial resolution for 3D imaging.
Fig : Simple DHM Setup
The inline flavor of the DIHM kicks in when neither lenses nor beam splitters are needed as in the typical DHM setup. Instead a point light source is used, which can be produced by passing a collimated light source through a pinhole. The point source creates a spherical wave, illuminates the object, and reaches the image sensor containing the joint wavefronts of both the reference beam and the object beam. This is due to the fact that the peripheral of the wavefront passes by and remains unaffected by the object, hence the reference beam, and that the wavefront passes through the object, forming the object beam.
Fig : Simple DIHM Setup
In the above picture, a laser source of wavelength lambda emanates from a pin hole, forming a spherical wave. A small object is typically placed a few thousand wavelengths from the source, reaching the image sensor much further away such that magnification is achieved. Small object means that the object should only block a small fraction of the spherical cone wavefront recorded on the image sensor. Otherwise classical diffraction dominates the image, where 3D reconstruction based on holography would no longer work as lack of reference beam.
The 3D reconstruction algorithm uses the Kirchhoff-Helmholtz transform, to reconstruct the wavefront one at a time, on several planes at various distances near the object. When the stacks of reconstructions are made, 3D image with depth information can be built.
References
| [1] | Shiraki, A., Taniguchi, Y., Shimobaba, T., Masuda, N., Ito, T. (2012) Handheld and low-cost digital
holographic microscopy.
arXiv:1211.0336 |
| [2] | Cotte, Y., Toy, F., Jourdain, P., Pavillon, N., Boss, D., Magistretti, P., Marquet, P., Depeursinge
(2013) Marker-free phase nanoscopy Nature Photonics, 7 (2):113
DOI: 10.1038/nphoton.2012.329 |
| [3] | Giuliano, C. B., Zhang, R., Wilson, L. G. (2014) Digital Inline Microscopy (DIHM) of Weakly-scattering Subjects Journal of Visualized Experiments, DOI:10.3791/50488 |
| [4] | Molaei, M., Sheng, J. (2014) Imaging bacterial 3D motion using digital inline holographic microscopy and correlation-based de-noising algorithm Optics Express, DOI: 10.1364/OE.22.032119 |
| [5] | Braat, J., Dirksen, P., Janssen, A. J. E. M. (2003) Diffractive Read-Out of Optical Discs, Optical Imaging
Springer Verlag |
| [6] | DDeng, Y., Chu, D., (2017) Coherence properties of different light sources and their effect on the image sharpness and
speckle of holographic displays, Scientific Report,
DOI: 10.1038/s41598-017-06215-x |
| [7] | Jericho, M. H., Kreuzer, H.J., (2011), Point Source Digital In-Line Holographic Microscopy, Chapter 1, Coherent Light Microscopy, Springer Series in Surface Sciences 46, 46
DOI: 10.1007/978-3-642-15813-1_1 |
| [8] | Rostykus, M., Moser, C. (2017) Compact lensless off-axis transmission digital holographic microscope, Optics Express, DOI: 10.1364/OE.25.016652 |
| [9] | Reichert, C. C., Herkommer, A., Claus, D. (2016) Das Smartphone als Mikroskop, AT-Fachverlag GmbH,
www.biophotonik.de |
| [10] | Moon, I., Daneshpanah, M., Anand, A., Javidi, B. (2011) Cell Identification Computational 3-D Holographic Microscopy, Optics & Photonics, 22 (6), |
| [11] | Greenbaum, A., Luo, W., Su, T., Göröcs, Z., Xue, L., Isikman S., Coskun, A., Mudanyali, O., Ozcan, A. (2012) Imaging
without lenses: achievments and remaining challenges of wide-field on-chip microscopy, |
| [12] | beniroquai (2017) Blog, https://beniroquai.wordpress.com/2016/01/20/holoscope-linsenloses-holographisches-mikroskop/, last visited: 10/15/2017 |
| [13] | BDan (2015) micromanipulator, Thingiverse, https://www.thingiverse.com/thing:923865/#files, last visited: 10/15/2017 |
| [14] | "Do-it-yourself" project for steering HD-DVD pickup homepage: http://www.diyouware.com/ last visited: 10/15/2017 |
