Extended Dual-Focus Microscopy for Ratiometric-Based 3D Movement Tracking

Seohyun Lee,Hideo Higuchi,Hyuno Kim

doi:10.3390/app10186243

Seohyun Lee, Hideo Higuchi + Show 1 more

Open Access

https://doi.org/10.3390/app10186243

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Abstract

Imaging the three-dimensional movement of small organelles in living cells can provide key information for the dynamics of drug delivery and virus transmission in biomedical disciplines. To stably monitor such intracellular motion using microscope, long depth of field along optical axis and accurate three-dimensional tracking are simultaneously required. In the present work, we suggest an extended dual-focus optics microscopy system by combining a bifocal plane imaging scheme and objective lens oscillation, which enables accurate localization for a long axial range. The proposed system exploits high-resolution functionality by concatenating partial calibration result acquired each axial imaging level, maintaining the practical advantages of ratiometric method.

Highlights

Intracellular imaging is one of the most important techniques in biomedical optics, and offers immediate visual information on the specific positions and movements of subcellular organelles [1,2].In a practical imaging microscopy system for a living cell, a fully stable, single focal plane or simultaneous imaging at multiple focal planes are frequently required, depending on the movement range of the intracellular organelle of interest
To track a target organelle traveling a long axial range, such as in the case of endocytosis where a vesicle navigates from the cell membrane to the nucleus periphery, multiple images taken at different focal planes are demanded for acquiring the accurate geometric information, in order not to miss or misconceive the actual dynamics of the target organelle
The images were captured by the dual-focus optics [10], with the focal spacing parameter e, by adjusting the focal length of either optical path before collected by an electric multiplying charge coupled device (EMCCD) camera image sensor (Andor iXon 885, 1004×1002, 14-bit, Belfast, Northern Ireland)

Summary

Introduction

Intracellular imaging is one of the most important techniques in biomedical optics, and offers immediate visual information on the specific positions and movements of subcellular organelles [1,2].In a practical imaging microscopy system for a living cell, a fully stable, single focal plane or simultaneous imaging at multiple focal planes are frequently required, depending on the movement range of the intracellular organelle of interest. To track a target organelle traveling a long axial range, such as in the case of endocytosis where a vesicle navigates from the cell membrane to the nucleus periphery, multiple images taken at different focal planes are demanded for acquiring the accurate geometric information, in order not to miss or misconceive the actual dynamics of the target organelle This is because an organelle in the cytoplasmic area can be transported by cytoskeletal networks in three dimensions [4,5], which refers that the conventional imaging system with a single focal plane cannot chase the fast-changing axial position of the organelle of interest, once it leaves the focal plane.

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