The overall goal of this procedure is to demonstrate a lens less on-chip fluorescent microscopy platform for imaging fluorescent objects over an ultra wide field of view. To accomplish this, a thin absorption filter and a fiber optic face plate are gently placed on top of an opto electric sensor array, or CCDC moss, and then a microfluidic chip is positioned on top of the face plate. Next, a prism is assembled with the microchip through the use of index matching oil.
Fluorescent citation is achieved through the side facet of a rhomboid prism and the holographic illumination is done through the top facet of the same prism. Ultimately, high resolution microscopic images can be obtained from the acquired fluorescent images by decoding the raw images through digital algorithms. If needed, the decoded fluorescent objects can be automatically counted using a custom developed interface.
On chip lens. Imaging in general aims to replace bulky lens-based optical microscopes with simpler and more compact designs. This emerging technology platform has the potential to eliminate the need for bulky and the costy appeal components through the help of novel theories and the digital reconstruction algorithms.
The lens list on on-chip imaging platform comprises several optical components, including a digital sensor array, an incoherent illumination source, a prism, an absorption filter, and a fiber optic face plate, or a fiber optic taper. In this platform, different types of sensor arrays can be used for detection of the fluorescent signal from target micro objects. For the purposes of this video, a charge coupled device will be used, an incoherent light source, such as a simple light emitting diode is used for fluorescence excitation.
Two different excitation filtering methods are used in parallel to create the required dark field background as shown in this diagram. First, a glass prism or a glass hemisphere is used to create total internal reflection or TIR at the bottom surface of the microfluidic chip that hosts the samples of interest. In parallel to this, an inexpensive absorption filter is used to remove the weekly scattered excitation lights that does not obey the TIR process.
Upon successful rejection of the excitation using these two mechanisms, only the fluorescent emission of the samples is acquired at the detector plane. Despite such a large detection numerical aperture, the raw fluorescent spots at the sensor array become fairly wide to engineer and better control the spatial spreading of this fluorescent signal. A fiber optic face plate will be placed between the object and the sensor planes as an alternative to a regular face plate.
A tapered fiber optic face plate, which has a significantly larger density of fiber optic cables on its top facet compared to the bottom one, can be used for higher resolution imaging through magnification. The microfluidic chips that are used in this platform are fabricated using poly dimethyl Sloane walls placed on glass slides to create the micro channels that are needed for on-chip imaging to begin assembly of the landless on-chip imaging platform. First, remove the cover glass of the optoelectronic sensor array using a vacuum pen, gently place a thin absorption filter on the top of the detector active area.
Position a fiber optic face plate on top of this absorption filter. Then place the fabricated microfluidic chip directly on top of the fiber optic array. Next, a glass prism is assembled above the microfluidic chip use index matching oil such that the refractive index of the interface is matched with the refractive index of the glass.
Connect the light sources to fiber optic cables through the use of FC connectors. One of the most difficult aspects of this procedure is the alignment of the components. Carefully align the distances between the components, the position and angle of the illumination.
Finally, move the side illumination fiber closer to the prism and adjust its angle to ensure that total internal reflection occurs at the glass air interface corresponding to the microfluidic chips. Bottom substrate, various cells or small animal models can be imaged using this on-chip platform, but white blood cells will be imaged for the purposes of this demonstration. To label the white blood cells with a fluorescent dye first mix around 100 microliters of whole blood with around one milliliter of red blood cell rising buffer incubate for around three minutes of room temperature, then centrifuge the lies blood solution.
After removing the top layer using a micro pipette, resuspend the pellet layer in phosphate buffered saline to label the nucleic acid of the cells with fluorescent dye, add SYTO 16 to the resus suspension and then incubate the sample for around 30 minutes in the dark at room temperature. After 30 minutes, centrifuge the labeled sample. Remove the supinate to decrease the background noise due to fluorescent emission from unbound dyes.
Reese has bend the white blood cell pellet layer in around five milliliters of PBS. The sample is now ready to be loaded into the microfluidic chip for lens free on chip fluorescent imaging. Prior to imaging the white blood cells, the imaging platform must be calibrated using fluorescent microbeads.
Prepare diluted microbead sample using deionized water and raw bead solution. Using a sharp needle syringe. Inject this initial sample into the microfluidic chip from the side of the PDMS walls.
After assembling the components to achieve on-chip fluorescent imaging, acquire raw images of fluorescent beads using the custom developed lab view interface through a computer. Make sure to check that there are at least 20 isolated fluorescent bead images across the entire field of view. Save the calibration data, which will be used later to calibrate and extract the point spread function of the lens less fluorescent microscopy platform.
Remove the microfluidic chip containing the fluorescent bead sample and detach the prism from this chip. Inject the previously prepared white blood cell sample into another unused microfluidic chip from the side of the PDMS walls. A assemble the components once again with the same experimental conditions, especially with regard to the distances and excitation power.
Acquire the raw images of white blood cells using the custom made lab view with the same software parameters used for the fluorescent microbeads. The calibration data and the raw white blood cell images are then used to create higher resolution images in MATLAB through digital processing. Representative results of wide field lens fluorescent on-chip imaging of a mixture containing four micron and 10 micron green fluorescent particles are shown here for comparison purposes, 10 x microscope.
Objective images are also provided, which agree well with the lens less fluorescent images shown. Next, a results of wide field lens, less fluorescent imaging of a mixture containing 10 micron green and 10 micron red fluorescent particles. The lens less fluorescent images provide a decent match to 10 x microscope objective images of the same samples.
This figure provides a performance comparison of the Lucy Richardson Deconvolution and compressive sampling based on decoding algorithms for imaging of various 10 micron bead pairs. The top row illustrates the lens less raw fluorescent images in which inset images show the microscope comparisons of the same particles that are required using a 10 x objective lens. The middle row demonstrates the compressive decoding results while the bottom row illustrates the Lucy Richardson Deconvolution results DDCS and DLR refer to the center to center distances and microscope images, CS decoded lens images, and LR de convoluted lens less images respectively.
Next, digital processing of lens, less fluorescent raw images is illustrated. A compressive sampling based algorithm is used to achieve less than four microns spatial resolution by resolving closely packed two micron diameter bead pairs. The insets show 40 x microscope objective comparisons, which agree very well with the decoded fluorescent images.
Here D refers to the center to center distances in microscope images. While DCS refers to the center to center distances in the CS decoded lens, less fluorescent images. This figure shows the results of lens less imaging of fluorescently labeled white blood cells.
The raw lens free images are rapidly decoded using a CS based decoder, which agrees very well with a conventional microscope image of the same sample that is acquired with a 10 x objective lens. In addition to imaging if needed, the decoded fluorescent objects can also be automatically counted using a custom developed interface for on-chip cytometry applications Illustrated here is a custom designed, automated fluorescent object counting interface in matlab. We have just demonstrated the procedure for landless fluorescent microscopy of labeled cells and micro beats over a field of view of eight centimeter square.
Such a compared ship imaging platform with the rapid compressive decoder behind. It'll be quite variable for high throughput cytometry, micro rare analysis, and real cell research applications.
