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.
Digital inline holography microscopy is a special technique to recover object properties from interference patterns. We explain the working principle in greater detail, but first let us show the basic properties of our DIHM setup in short video. A the light from a bright LED is send through a pinhole. The light waves are spherical diffracted. At small samples e.g. E. coli, the wavefront is scattered. Small fringes are created. The unscattered waves serves as reference template. Everything is captured on a camera screen.
The resulting hologram needs to be computational analyzed. We present an 'easy-to-use' interface for a commonly used analyzation package in our software section. First, we invited to check-out what we could achieve and then how to get our own DIHM.
In the above figure, the green stack functions as a frame to fixate the screws, with one end attached with springs, allowing more bounce and flexibility. The yellow and the red stacks enable the alignment control in the x and y direction, correspondingly. The camera is then placed on the red stack to record holograms once the light source, the object to be observed, and the camera is properly aligned.
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.
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.
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.
In the above picture, a laser source of wavelength 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.
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|||BDan (2015) micromanipulator, Thingiverse, https://www.thingiverse.com/thing:923865/#files, last visited: 10/15/2017|
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