the overall goal of this protocol is to prepare and use an asymmetric detection time stretch optical microscopy system for label-free high contrast single-cell imaging in ultra-fast microfluidic flow. The main advantage of ATOM is that it's allows ultra-fast image capture with line scan rates of up to tens of megahertz while enhancing the image contrast on the unlabeled cells. ATOM enables complex morphological analysis of cellular and subcellular structures with an uncentered cell-based assays at up to 100, 000 cells per second, which is not possible with standard flow cytometry.
to begin setting up the system use a fiber collimator to couple a broadband femto or pico second near IR pulsed laser to a single mode dispersive optical fiber connected to an optical amplifier. Illuminate a transmission diffraction grading with the laser to produce a spectral shower. Orient the grading close to the littrow configuration to maximize the diffraction efficiency.
Configure a telescopic relay lens assembly in a 4f imaging system to direct the spectral shower onto the rear focal plane of an objective lens below a sample platform. Ensure that the spectral shower fills the rear aperture of the objective lens. Place another objective lens with a plane mirror at its rear aperture above the sample platform.
Adjust the objective lenses until their focal planes overlap. Verify that the spectral shower double passes the image plane and follows the same path back to the grading. Place a microfluidic chip on the sample platform and ensure that the spectral shower falls across the microfluidic channel.
Redirect the returned light with a beam splitter after it passes through the grading. Separate the returned light into two beams with another beam splitter. Use fiber collimators to couple the beams to single mode fibers of different lengths to introduce a time delay between beams.
Connect the fibers to a real-time oscilloscope through a fiber coupler and a photodetector. Tune the optical amplifier gain until the signal-to-noise ratio of the optical signal is greater than 10 decibels. Then block approximately half of each returned beam with knife-edge beam blocks.
Adjust the beam blocks until the measured optical power in each fiber is about half of the power before the block. Verify that the oscilloscope bandwidth and sampling rate are both high enough not to influence the image resolution. To begin the experiment dilute cell samples to between 10 to the 5th and 10 to the 6th cells per milliliter with phosphate buffered saline or spring water.
Then load the cell solution into a 10 milliliter syringe. Connect the syringe to the microfluidic channel inlet. Direct the microfluidic channel outlet to a centrifuge tube.
Secure the syringe in a syringe pump and set the flow rate appropriately for the channel width. Choose the number of data points to be saved for the experiment. Then acquire and save the optical signal data.
Digitize and export the data from the oscilloscope to a computer. Process the data into 2D images and extract a library of parameters from the images. Import the images in parameter library into a visualization program.
Assign a parameter of interest to each axis and select the data set to be displayed as a scatter plot. Check the cell images associated with each group in the plot to identify trends. Use manual gating or display additional datasets for further analysis as needed.
ATOM was used to acquire high contrast single-cell images of human cells and phytoplankton. Characteristic cell structures such as vacuoles, pyrenoids, and flagella, are visible in the phytoplankton images. Parameters such as cell size and circularity were determined from the ATOM images to classify cells for cytometric analysis.
The classification results can be displayed as 2D scatter plots. Here volume versus circularity was plotted for an MCF7 shown in yellow and PBMC shown in red. Two clusters were observed for MCF7.
By identifying the high contrast images corresponding to individual data points, the lower cluster was found to correspond to cell fragments and debris. Once mastered the imaging system can be set up in two to three hours if it is performed properly. The imaging procedures can be done within an hour.
Don't forget to pay special attention to eye safety while working with the laser source and laser beam alignment. Precautions such as wearing safety goggles should always be taken while performing these procedures. While attempting this procedure remember to monitor the optical beam alignment throughout the whole imaging system setup process to ensure optimal imaging quality.
While this technique can lend insight into the detection and quantification of rare or apparent cells during disease process, it can also be applied to cell type classification, within large and heterogeneous cell populations. This technique is particularly advantageous when looking to overcome the throughput limitation of imaging cytometry, while preserving the image quality. After watching this video, you should have good understanding of how to implement ATOM from the system setup to the imaging procedures.
