The so-called “natural killer” (NK) cells, also known as large granular lymphocytes (LGL), play an important role in the immune system [1,2]. They can rapidly destroy virus-infected, malignant or stressed cells, ensuring a quick and specific defense response. The study of NK cells as a therapeutic means to relieve ailments, like immunodeficiencies, impaired cell maturation, and chronic pain, among others, have led to investigations of the contact-dependent mechanism of NK cell differentiation and migration . These studies often involve multidimensional imaging techniques which include widefield fluorescence, confocal, and super-resolution microscopy.
NK cells can be derived from induced pluripotent stem cells (iPSC). These iPSCs, derived from skin or blood cells, can be re-programmed into an embryonic-like pluripotent state which can be exploited to provide any type of cell needed for therapeutic purposes . Recently, there has been an increase in the number of studies which use iPSC-derived NK cells due to their important advantages, such as easy generation from accessible sources, retained pluripotency during expansion, and long-term storage capability [3,5]. When iPSCs are cultured, they quite often form thick layers or “domes”. Subsequently, fluorescent labels can be used to identify specific proteins within the cells, as well as to identify and track the cells.
To study thick cell layers, it is practical to have a microscope which allows fast screening over a large area of the specimen, but also enables higher magnification imaging with very good contrast at points deep inside it. Camera-based fluorescence microscopy offers ease of use, speed, and detection sensitivity. However, there are several important challenges for the study of multilayers of live cells in culture. Imaging of thick specimens often leads to a typical “haze” appearing in the images which significantly reduces contrast. This haze is produced by detected fluorescence signals which are emitted from out-of-focus planes in the specimen. With widefield microscopy there can also be the difficulty to focus clearly on places deep inside thick specimens. Additionally, the phenomenon of refractive index mismatch for specimens immersed in aqueous media can occur.
A THUNDER Imager 3D Cell Culture equipped with a long-working-distance achromatic water objective (FL PLAN, 25x, 0.95 NA) was used to image deep inside thick cell layers. The use of a water immersion objective helps to correct for mismatches between the refractive indices of the cells and aqueous media in which they were cultured. Additionally, the long working distance enables sharp focusing deep inside the cell layers. To clear the images, especially of out-of-focus information, Large Volume Computational Clearing (LVCC), an opto-digital method developed by Leica Microsystems , was applied during acquisition. Additionally, background substraction and adaptive deconvolution were applied to z stacks used for 3D image reconstruction so that subcellular structures could be observed with higher contrast and in sharper detail .
Images of thick iPSC stem cell layers recorded with a THUNDER Imager 3D Cell Culture are shown below in figure 1.
The results showed that images free of out-of-focus blur or haze and scattered light could be acquired of relatively thick induced pluripotent stem cell (iPSC) layers using a THUNDER Imager 3D Cell Culture and Large Volume Computational Clearing (LVCC) . The application of background subtraction and adaptive deconvolution  allowed the iPSC cells, showing fluorescently labelled adhesion and transmembrane proteins, as well as chromatin in the nuclei, to be observed with higher contrast and in sharper detail throughout the z stack used for 3D image reconstruction.
- A.G. Freud, B.L. Mundy-Bosse, J. Yu, M.A. Caligiuri, The Broad Spectrum of Human Natural Killer Cell Diversity, Immunity (2017) vol. 47, iss. 5, pp. 820-833, DOI: 10.1016/j.immuni.2017.10.008.
- A. Mandal, C. Viswanathan, Natural killer cells: In health and disease, Hematology/Oncology and Stem Cell Therapy (2015) vol. 8, iss. 2, pp. 47-55, DOI: 10.1016/j.hemonc.2014.11.006.
- B.J. Lee, E.M. Mace, From stem cell to immune effector: how adhesion, migration, and polarity shape T-cell and natural killer cell lymphocyte development in vitro and in vivo, Molecular Biology of the Cell (2020) vol. 31, no. 10, DOI: 10.1091/mbc.E19-08-0424.
- T. Hamazaki, N. El Rouby, N.C. Fredette, K.E. Santostefano, N. Terada, Concise Review: Induced Pluripotent Stem Cell Research in the Era of Precision Medicine, Stem Cells (2017) vol. 35, iss. 3, pp. 545-550, DOI: 10.1002/stem.2570.
- J. Zeng, S.Y. Tang, L.L. Toh, S. Wang, Generation of “Off-the-Shelf” Natural Killer Cells from Peripheral Blood Cell-Derived Induced Pluripotent Stem Cells, Stem Cell Reports (2017) vol. 9, iss. 6, pp. 1796-1812, DOI: 10.1016/j.stemcr.2017.10.020.
- J. Schumacher, L. Bertrand, THUNDER Technology Note: THUNDER Imagers: How Do They Really Work? Science Lab (2019) Leica Microsystems.