Obtaining 3D topology from a single 2D image of more than 16,000 live cells using Standing Wave Mesoscopy

Session

New and Emerging Concepts in Microscopy

Authors

Ms Shannan Foylan (3), Dr Jana Katharine Schniete (2), Ms Lisa Sophie Kölln (3, 4), Dr John Dempster (3), Dr Carsten Gram Hansen (4, 6), Dr Michael Shaw (1, 5), Prof Gail McConnell (3)

Affiliations

1. Department of Computer Science, Faculty of Engineering Sciences, University College London
2. Strathclyde Institute of Pharmacy and Biomedical Scienc
3. Strathclyde Institute of Pharmacy and Biomedical Sciences
4. University of Edinburgh Centre for Inflammation Research, Institute for Regeneration and Repair
5. Biometrology Group, National Physical Laboratory
6. ncer Research UK Edinburgh Centre, Institute of Genetics and Cancer, University of Edinburgh

Keywords

Fluorescence, super-resolution, live-cell, mesoscopy

Abstract text

SUMMARY:

Standing Wave (SW) illumination requires the simple addition of a first surface reflector in place of a coverslip. The method generates an interference pattern to excite fluorescence from labelled cellular structures and produces high-resolution images of three-dimensional specimen in a two-dimensional image. SW microscopy is generally performed with high magnification, high numerical aperture (NA) objective lenses: this results in high resolution images but only within a small field of view (FOV) so the cell numbers are limited. Here, we present a technique for performing SW imaging at the at the mesoscale using the Mesolens. In our FOV which extends across 4.4 mm x 3.0 mm, we performed topological mapping of more than 16,000 live cells simultaneously.  

Standing Wave (SW) microscopy generates an interference pattern by utilising two beams from the same or separate illumination sources to form a sinusoidal excitation pattern along the optical axis of a microscope ¹. When this illumination pattern is used to excite fluorescence from labelled specimens, emission is only detected from fluorophores within the anti-nodal maxima of the SW, giving rise to axially separated bands of fluorescence in a 2D image. These anti-nodal fringes are separated by λ/2n, where λ is the wavelength of excitation light and n is the refractive index of the specimen and the fringe thickness is λ/4n, giving rise to planes of axial illumination smaller than the depth of field of the imaging objective lens. As such, SW microscopy is an axial super-resolution technique that provides 3D information within a 2D image. It is simple to introduce to an upright microscope system: a thin, high flatness first surface reflector in place of a coverslip. Previous work has demonstrated a multi-wavelength illumination method for filling the axial information gaps generated by the nature of a SW ².

The Mesolens is a custom giant objective lens designed with the unusual lens prescription of 4x/0.47NA ³. This gives rise to a 4.4 mm x 3.0 mm FOV in widefield acquisition mode ⁴ with sub-cellular lateral resolution throughout. The Mesolens is flat field corrected across this field and is chromatically corrected across the visible spectrum. Previous studies have shown the functionality of the Mesolens operating in laser-scanning confocal ³ and light-sheet illumination ⁵, both of which allow for optically sectioned imaging of 3D millimetre scale volumes. However, pixel-wise scanning of large specimens with point-scanning illumination can take several days to acquire. While light sheet mesoscopy was shown to reduce the imaging time by a factor of 14, 3D image acquisition is still on the order of hours and the setup is highly complex ⁵. 

Here, we demonstrate how the combination of multi-wavelength SW illumination with the large FOV of the Mesolens yields 3D topological information from a single 2D acquisition of up to more than 16,000 live cells. The single image acquisition speed is only limited by the camera detector, which in our case for the sensor shifting camera to acquire the full 4.4 mm x 3.0 mm FOV is 5 s. 

In this study, we first used a non-biological glass plano-convex test specimen to characterise the SW illumination pattern of the Mesolens LED epi-illuminators. This non-biological specimen was fluorescently coated and placed convex side down on the same high flatness mirrors used for further imaging with biological specimen. The curvature of the lens allowed for the height of the anti-nodal planes from the mirror surface to be extracted from the lateral planes of fluorescence in the acquired 2D image.

Following characterisation, we prepared and imaged fixed fibroblast specimens labelled for F-actin with SW mesoscopy. Finally, live human red blood cells were collected and labelled for their membranes and studied with SW mesoscopy under static and flow conditions, allowing for observation of both morphological and rheological changes to the > 16,000 red blood cells in 3D acquired in a single time series. We will present this work alongside a discussion of the potential future applications of SW mesoscopy., such as facilitating the diagnosis of parasitaemia, hereditary spherocytosis, sickle cell disease, hereditary stomatocytosis or elliptocytosis.

 

References

1. Bailey, B., Farkas, D. L., Lansing Taylor, D., & Lanni, F. (1993). Enhancement of axial resolution in fluorescence microscopy by standing-wave excitation. Nature, 366, 44-48.

2. Schniete, J. K., Tinning, P. W., Scrimgeour, R. C., Robb, G., Kölln, L. S., Wesencraft, K., Paul, N. R., Bushell, T. J., & McConnell, G. (2021). An evaluation of multi-excitation-wavelength standing-wave fluorescence microscopy (TartanSW) to improve sampling density in studies of the cell membrane and cytoskeleton. Scientific Reports, 11, 2903.

3. McConnell, G., Tragardh, J., Amor, R., Dempster, J., Reid, E. & Amos, W.B. (2016). A novel optical microscope for imaging large embryos and tissue volumes with sub‐cellular resolution throughout. eLife, 5, e18659.

4. Schniete, J., Franssen, A., Dempster, J., Bushell, T. J., Amos, W. B., & McConnell, G. (2018). Fast optical sectioning for widefield fluorescence mesoscopy with the Mesolens based on HiLo microscopy. Scientific Reports, 8, 16259.

5. Battistella, E., Schniete, J., Wesencraft, K., Quintana, J. F., & McConnell, G. (2022). Light-sheet mesoscopy with the Mesolens provides fast sub-cellular resolution imaging throughout large tissue volumes. iScience, 25, 104797.