The overall goal of this procedure is to achieve lens free on chip three dimensional imaging of biological specimen over a large imaging volume. This is accomplished by first placing the sample directly on the sensor chip and providing partially coherent illumination. The second step is to laterally shift the light source of different positions to record.
Subpixels shifted holographic images of the sample. Next subpixels shifted. Holographic images are recorded at multiples of illumination for tomographic imaging.
The final step is data processing where the acquired data set is digitally reconstructed to ultimately obtain three dimensional tomographic images of the sample. The main advantage of this technique over existing methods like conventional light microscopy, is that it provides high throughput imaging in a compact architecture. This makes our platform particularly useful for integration with Lebanon chip platforms.
In this report, the basic imaging setup for a benchtop implementation toward tomography of static samples is described. A monochromator with a xenon lamp was adjusted to provide an output with approximately one to 10 nanometer spectral width around a center wavelength of 450 to 650 nanometers. This partially coherent output is then coupled to a multi-mode optical fiber to deliver partially coherent light to the system.
The optical fiber is mounted on a motorized rotation stage to change the angle of illumination. The motorized stage with the light source attached is mounted on a two dimensional linear XY stage, which is used to achieve in plain shifts of the light source at a given angle for detection. A-C-M-O-S sensor array having five megapixels with a pixel size of 2.2 microns is used.
The detector is used to record the wide field of view holographic images of samples. It is critical that the sensor array is positioned at the same axial plane as the axis of rotation of the light source to ensure that we check if the sensor receives sufficient light at large angles of illumination. While lens free optical tomography can image a variety of objects such as cells and microorganisms, the basic principles are demonstrated here by performing three dimensional microscopy of a sea elgan sample using a scalpel or a spatula.
Take a small piece of AGA from the Petri dish containing the sealer organs culture. A cubic piece of several millimeters along each dimension will contain hundreds of nematodes. Place the small chunk of agar in a polypropylene vial containing one milliliter of deionized water.
Gently vortex for 30 seconds to one minute. After 10 to 15 minutes, the worm should crawl out of the agar into the deionized water. Note that the worms are too small to see here in order to temporarily immobilize the worms and one milliliter of five to 10 millimolar Lima.
So solution and wait for 10 minutes. Pipette five to 10 microliters of sample from the bottom of the vial and sandwich between two cover slips. This sample containing a large number of temporarily immobilized worms can be placed on the detector to start data acquisition.
For demonstration purposes, the image acquisition steps for a typical lens free optical tomography or LOT experiment are summarized in this section, however, the entire process has been automated by using the custom developed lab view interface. To begin manual steps, adjust the initial angle of the rotation stage to negative 50 degrees, where zero degrees corresponds to the vertical position of the light source. Adjust the initial position of the XY stage to zero zero, which is the home position.
Adjust the exposure time of the detector to best utilize its dynamic range such that the image is as bright as possible without any saturated pixels without changing the angle of the rotation stage. Capture nine images for each image. Move the XY stage to a new position in a three by three square grid, such that each image shifts by approximately one quarter of a pixel compared to the previous one.
Acquiring more images at each angle might improve resolution depending on the type of the object and signal to noise ratio. After collecting nine images at the initial angle, adjust the position of the XY stage back to the zero zero position. Then increase the angle of the rotation stage by increments of two degrees until reaching positive 50 degrees.
After each increment at each new angle, repeat the described steps. The angular increments can be finer or coarser depending on the optimization of acquisition time versus imaging resolution. Following data acquisition, a set of 459 images is acquired, which contain nine subpixels shifted images for each of the 51 different angles of illumination.
Each set of nine images is digitally processed using pixel super resolution algorithms to obtain one high resolution projection. Hologram per angle pixel super result are then digitally reconstructed to obtain 51 projection images. The data processing is shown here using a laptop.
The reconstruction would take less than one second for full field of view using graphical processing units. This set of 51 projection images is then back projected using Tom OJA plugin. For Image J, the projection images are first loaded to image J, after which the Tom OJ plugin is invoked.
A lookup table that provides the viewing angle for each projection image is loaded to to OJ.Using the weighted back projection method, three dimensional tomographic images of the specimen can be obtained. The large field of view of lens free optical tomography is demonstrated here as the sample is placed directly on the top of the detector array. Holographic images of the objects can be recorded over a field of view of 24 millimeter square, which can be further increased using emerging detector arrays with larger active areas.
Although pixel size of the detector array limits the resolution of the recorded holographic images pixel super resolution techniques mitigate this problem shown here are pixel super resolved holograms, along with the reconstructed projection images that offer sub micrometer spatial resolution for three different angles of illumination. The projection images can be combined using tomographic image reconstruction techniques to compute TOM grounds of the specimen. Three slice images in the XY plane through the anterior of the worm are shown here where the pharyngeal tube is visible only in the slice through Z equals eight microns, as expected from this roughly cylindrical structure with an approximate five micron outer diameter.
Moreover, the cross-sectional image in the XZ plane clearly shows the boundaries of the worm and the pharyngeal tube inside demonstrating successful 3D imaging of the pharynx. With its development, this technique can pave the way for researchers to perform three dimensional microscopy over tens of millimeter tubes of imaging volume. Therefore, lens free optical tomography can be a useful tool for high throughput imaging applications in Lebanon chip platforms.
