Slanted channel microfluidic chip for 3D fluorescence imaging of cells in flow.

Veerendra Kalyan Jagannadh,Ajoy K Kar,Parama Pal,Mark D Mackenzie,Sai Siva Gorthi

doi:10.1364/oe.24.022144

Veerendra Kalyan Jagannadh, Ajoy K Kar + Show 3 more

Open Access

https://doi.org/10.1364/oe.24.022144

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Abstract

Three-dimensional cellular imaging techniques have become indispensable tools in biological research and medical diagnostics. Conventional 3D imaging approaches employ focal stack collection to image different planes of the cell. In this work, we present the design and fabrication of a slanted channel microfluidic chip for 3D fluorescence imaging of cells in flow. The approach employs slanted microfluidic channels fabricated in glass using ultrafast laser inscription. The slanted nature of the microfluidic channels ensures that samples come into and go out of focus, as they pass through the microscope imaging field of view. This novel approach enables the collection of focal stacks in a straight-forward and automated manner, even with off-the-shelf microscopes that are not equipped with any motorized translation/rotation sample stages. The presented approach not only simplifies conventional focal stack collection, but also enhances the capabilities of a regular widefield fluorescence microscope to match the features of a sophisticated confocal microscope. We demonstrate the retrieval of sectioned slices of microspheres and cells, with the use of computational algorithms to enhance the signal-to-noise ratio (SNR) in the collected raw images. The retrieved sectioned images have been used to visualize fluorescent microspheres and bovine sperm cell nucleus in 3D while using a regular widefield fluorescence microscope. We have been able to achieve sectioning of approximately 200 slices per cell, which corresponds to a spatial translation of ∼ 15 nm per slice along the optical axis of the microscope.

Highlights

Cells, the fundamental building blocks of life constitute all living organisms from simple single celled organisms such as bacteria to vastly complex multicellular organisms like humans and whales [1]
We present the design and fabrication of a slanted channel microfluidic chip for 3D fluorescence imaging of cells in flow
We demonstrate the retrieval of sectioned slices of microspheres and cells, with the use of computational algorithms to enhance the signal-to-noise ratio (SNR) in the collected raw images

Summary

Introduction

The fundamental building blocks of life constitute all living organisms from simple single celled organisms such as bacteria to vastly complex multicellular organisms like humans and whales [1]. Focal stack collection involves acquiring images of the sample, while different parts of the specimen/cell are in the focal plane/depth of field (DOF) of the microscope objective. In other words, it is the process of scanning the sample along the optical axis of the microscope. The cells traverse through the focal plane of the objective, enabling acquisition of cell images at different depths/focus in successive frames This approach of slanted channel acquisition enables automated focal stack collection, without the need for high precision translation stages and very high-speed cameras. The solution proposed here forms a key element in furthering the development of flow based 3D imaging modalities which rely on focal stack acquisition

Device design

Device fabrication

Characterization of focal stack acquisition with fluorescent microspheres

Conclusion