Researchers develop an experimental dual-modality 3D imaging system

Researchers at the Indiana University School of Optometry (Bloomington, IN, USA; https://optometry.iu.edu/) have developed a multimodal imaging system that combines structural and functional imaging to enable 3D visual assessment of cellular-level activities involved in developmental conditions or eye diseases.

Specifically, the researchers used high-resolution optical coherence microscopy (HR-OCM), a 3D structural technique, and confocal scanning dual-channel fluorescence microscopy (DC-SCFM), a functional technique, to simultaneously record the imaging information. The researchers say the combined system could lead to imaging processes that allow biologists to assess changes in cell activity in the eyes of small animals over time. For example, scientists could label and then track a specific protein in a cell using the functional system and the area around the cell, such as neighboring cells, with the structural system.

“We want to be able to capture not only the functional aspect of cell activities, but also the structure around it, so that we can fully understand how cells behave in their natural environment”, explains Patrice Tankam, assistant. professor at the Indiana University School of Optometry.

It is also important to follow the same animals. If scientists follow different animals or tissue samples, they don’t know if the differences they see are the result of biological changes or inherent variations in the subject, Tankam adds. Overall, he says, “our goal is: how can we track these biological processes in the same animals over time? »

This type of investigative work not only improves scientists’ understanding of eye biology, but could also become a first step in the development of new drugs for humans. “It will help scientists find a cure for certain diseases,” says Reddikumar Maddipatla, research associate at Indiana University School of Optometry.

The researchers chose to co-register functional and structural imaging information because it would reduce the potential for misinformation more effectively than a sequential approach where you “capture the structural information, then move the mice to a different system and collect functional information,” explains Tankam. .

To create the dual-modality imaging system, the researchers overcame technical challenges so that the HR-OCM and DC-SCFM systems recorded visual information from the same location at the same time. They wanted to maximize the resolution of side and depth images.

Since the two systems operate in different parts of the light spectrum – OCM uses near infrared light and fluorescence uses visible light – they tend to focus on two different planes, which is called the mismatch. of the focal plane, explains Maddipatla.

In addition to the focal plane mismatch issues, the two systems also register depth information differently, Maddipatla says.

The researchers addressed these issues in the design of the dual-modality system, which includes an HR-OCM reference arm and both a DC-SCFM fluorescence detection arm and an excitation arm. They defocused the light beam from the HR-OCM system to align the systems along the sample plane.

To combine the light signals of the two systems, Tankam and Maddipatla used those of Semrock (Rochester, NY, USA; www.semrock.com) dichroic mirror. From there the signal went to a dual-axis galvanometer scanner from Cambridge Technology, a brand owned by Novanta Photonics, (Stockport, UK; www.novantaphotonics.com), which then relayed the light onto the sample.

In the HR-OCM system, the light, which is transmitted through the mirror, is then detected by a spectrometer from Wasatch Photonics (Morrisville, NC, USA; https://wasatchphotonics.com). The spectrometer is equipped with a Teledyne e2v OctoPlus (Milpitas, CA, USA; imagery.teledyne-e2v.com) high-speed linescan camera operating at 250 kHz.

The electrical output signal of the spectrometer is then recorded by an Axion CameraLink acquisition card from Bitflow (Woburn, MA, USA; www.bitflow.com.)

In the second imaging system, the fluorescence emission signal from the fluorophores travels from the sample to the mirror where it is reflected back to the fluorescence system through two channels – red and green. The light is then focused by a Thorlabs aspherical lens and transmitted through Thorlabs (Newton, NJ, USA; www.thorlabs.com) 10 µm Pinhole Aperture – P1, a product that only passes rays from one plane into the specimen while blocking light rays from other planes or depths. Then another lens, also from Thorlabs, adjusts the size of the beam before channeling it through a photomultiplier tube from Hamamatsu (Hamamatsu, Japan; www.hamamatsu.com). The output of the tube is converted into voltage using FEMTO amplifiers (Berlin, Germany; www.femto.de). A four-channel digitizer from Alazar Technologies (Pointe-Claire, Canada; www.alazartech.com) is used to record the voltage of amplifiers.

For the final step, the researchers recorded and synchronized the HR-OCM and DC-SCFM image data using custom software they developed using National Instruments (Austin, TX, USA ; www.ni.com) LabVIEW 2017. They used both LabVIEW and MATLAB from MathWorks (Natick, MA, USA; www.mathworks.com) for further processing of the image data.

To test the system, they used custom-made phantoms representing mice, which were constructed using imaging fluorescence microspheres embedded in multi-layer tape and silicone.

The combined system achieved a speed of 250 kHz and a lateral resolution of 2 µm, and an axial resolution of 2.4 µm was captured over a field of view of 1.1 mm × 1.1 mm.

For the next phase, Tankam and Maddipatla plan to test the system on tissue samples before tackling their ultimate goal of tracking cell activity in the eyes of small animals.

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