Digital Inline Holographic Microscopy - An iGEM Approach

Introduction

Digital inline holography microscopy is a special technique to recover object properties from interference patterns. We will explain the working principle in greater detail later on, but first let us show the basic properties of our DIHM setup in a short video. The light coming from a bright LED is sent 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.

Achievements

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Micromanipulators

In order to align our DIHM setup, it is necessary to focus the brightest spot of the light source to the pinhole. Due to the tiny dimension of this setup, we need to manipulate the objects in the micrometer level. Therefore, we tested different micromanipulators. We started with a concept of BDan from Thingiverse, which lacks flexibility and proportion. Later on, we tried 3D printed joint micromanipulators, movable with a screw. They failed on accuracy and the ability to move in more than one dimension. Our last and final approach is to split the movement axes using screws for control, as demonstrated below:

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Light Sources

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, which 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 an 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. Due to the fact that the peripheral of the wavefront passes by and remains unaffected by the object, the reference beam, and that the wavefront passes through the object, forming the object beam.

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.

 

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