In this video, proof of concept is shown for a lens list on-chip imaging termed Lucas. Lucas stands for lens list ultra wide field of view cell monitoring array platform based on shadow imaging. Unlike conventional imaging setups, the Lucas platform does not require any microscope objectives or other bulky optical components.
For Lucas, the specimen of interest is positioned on a microscope slide and is gently sandwiched by an identical cover glass. Using a vacuum pen, the sample is loaded onto the active region of the optoelectronic sensor array. The imaging platform then records the shadows or lens list digital holograms of each cell of interest within its field of view.
Automated digital processing of these cell shadows can determine the type, the count, and the relative positions of cells within the solution. The field of view can span as large as 18 centimeter squared with the depth of field of about five millimeters. The acquired diffraction images are then digitally processed by a custom developed decision algorithm to enable both characterization and identification of target cells or particles.
Lucas offers a miniaturized platform that reduces the cost and significantly increases the throughput for imaging based experiments, making it compatible with advanced state-of-the-art technologies in microfluidics and biological research. Hi, I am ROM Bio and Nano Laboratory in the Electrical engine Department of University of California, Los Angeles, And I'm Anthony Erlinger, also from the Nano and Biophotonics lab. Today we will show you a procedure for high throughput and lens for imaging of a heterogeneous SA solution such as whole blood sample.
This procedure in our laboratory utilizes a novel imaging technique, which we call lens list ultra wide field cell array monitoring platform based on shadow imaging, which we call Lucas for short. So let's get started. To begin the process, obtain three milliliters of whole blood and polystyrene microbeads of various diameters such as five, 10, and 20 micrometers, and bring them to room temperature for 30 minutes.
Using a serological pipette, add two milliliters of RPMI 1640 classic liquid media as a diluent into a sterile five milliliter polypropylene tube. After a 30 minute sedimentation period, withdraw 10 microliters of erythrocyte specimen from the bottom of the whole blood collection tube and transfer it into the RPMI solution. Add a total volume of 20 microliters of polystyrene microbeads into the erythrocyte RPMI dilution and agitate the cell solution by gently pipetting Transfer five to 15 microliters of the heterogeneous solution onto a cover slip using forceps.
Place a second identical cover slip over the solution. The sample should be evenly sandwiched between the two cover slips. Then using a vacuum pen, load the sample onto the active region of the sensor array for imaging.
Then prepare aqueous staining reagent by combining 0.1%buffered as SNY and diluted new methylene blue or NMB solution At a volume ratio of two to one, please refer to the accompanying written protocol for details on preparing as SNY and pum methylene blue solutions. Then in a polypropylene beaker, combine ACOA staining reagent with the whole blood specimen at a volume ratio of one to one, using a magnetic stir mix for two minutes. Then incubate an additional 10 minutes at room temperature after the solution is incubated at 1.99 milliliters of RPMI 1640 classic liquid solution into a five milliliter polypropylene tube.
Next, transfer 10 microliters of stained blood solution into the RPMI to acquire a volume ratio of one to 200. Then vortex the solution for 30 seconds, pipette 10 to 100 microliters of the stained cell solution between cover slips or within a microfluidic reservoir. Then using a vacuum pen, place the specimen onto the active region of sensor array.
The Lucas platform utilizes an optoelectronic sensor array to digitally record landless cell holograms. For this purpose charged, couple devices or CCDs are utilized in order to enable tunable wavelength illumination, a monochromator with a xenon lamp along with a standard grade fused silica fiber, which consists of a bundle of fibers and pinhole of about 100 micrometer diameter located at about five to 10 centimeters above the sensor surface can be used tunable wavelength. Illumination provides a flexible platform where the holographic signatures of the cells can be fine tuned and hybrid digital signatures can be synthesized by merging more than one wavelength to improve the signal to noise ratio and the contrast of the Lucas image.
For better characterization, accuracy samples sandwiched between two cover slips are loaded onto the active region of the sensor array, uniformly illuminated by the light source. The diffraction holograms of each cell or micro object within a heterogeneous solution fall onto the sensor array forming a Lucas image. The acquired Lucas Diffraction images are processed by a custom developed decision algorithm, which uses a statistical diffraction image library for automated characterization and identification of target cells or micro objects.
This figure shows a conventional 40 x objective image of a heterogeneous solution of five, 10, and 20 micrometer, polystyrene, microbeads, and red blood cells. 3D morphology, refractive index, and intercomp. Complexity of these particles are all different from one another.
Some of these features can be distinguished under a conventional microscope, but over a very limited field of view of less than 0.5 millimeters squared in Lucas platform. All these microparticles exhibit uniquely different holographic patterns like a fingerprint. Therefore, they can be characterized without any ambiguity based on their 2D textures.
Lucas can monitor an ultra wide field of view that can be made as large as 10 to 20 centimeters squared. Orders of magnitude larger than standard lens-based microscopes. This is a 40 x objective image of a stained whole blood sample using the Lucas platform.
Inner features of stained white blood cells vary efficiently by modifying the refractive index and scattering properties of cell nuclei. Distinctive diffraction patterns of each individual cell are recorded by the sensor array. This exhibits another advantage of the Lucas platform as a powerful tool for point-of-care cytometry and diagnostic purposes.
As a fully automated platform for rapid characterization of cells of interest, Lucas diffraction images are digitally processed using a custom developed decision software. In this figure, a heterogeneous mixture of red blood cells and microbeads is processed by the interface. The algorithm is robust in detecting high density regions as well as particles with low signal to noise ratio, such as the three micrometer beads.
A processed image in the Lucas custom interface is shown here. The software allows input for various specifications such as the sensor pixel size or the wavelength of illumination. Additionally, selections of the image can be made and cell patterns can be defined.
The sample is counted based on this data and the marked image is displayed to the user. We have just shown you how to image and characterize cells using high throughput platform of Lucas. In doing this procedure, it's important to remember that the demonstrated experiments can be applied to a wide variety of cells and microparticles.
It also enables ultra wide field and high throughput cell characterization of a heterogeneous solution. So that's it. Thanks for watching and good luck with your experiments.
