Arrhythmias (irregular heartbeats) are experienced by millions of people. Frequently brought on by age, congenital defects, or heart disease, arrhythmia has traditionally been mediated medically by implanting artificial pacemakers which impact the patient’s quality of life.
For children, these electronic devices are too large, so for this reason there is a research laboratory striving to develop a “hardware free” solution, i.e., producing functional pacemaker cells from ordinary heart muscle . This lab research requires the use of 3D cell culture and spheroids which present several challenges for high resolution observation with microscopy .
While widefield microscopy offers advantages for speed, viability, and cost, imaging large spheroids with up to four fluorescent channels using traditional light microscopy methods creates significant out-of-focus haze which limits the ability to document the growth and function of the specimens.
Spheroids were derived from neonatal rat ventricular myocytes (NRVM) which were grown on special plates. Afterwards, spheroids were covered with a monolayer cell culture . The spheroids were stained with alpha-actin and vimentin antibodies. A Z-stack of approximately 60 µm was acquired with a THUNDER Imager Live Cell which also applied the opto-digital technique Instant Computational Clearing (ICC) . For comparison, a maximum projection of raw widefield and THUNDER ICC images was produced.
The THUNDER images processed with Computational Clearing show more details of the spheroids compared to the classical widefield images. Moreover, the images shown in figure 2 below enable more of the dynamics between the monolayer cells and those in the spheroid to be explored.
- S.I. Grijalva, J.‐M. Gu, J. Li, N. Fernandez, J. Fan, J. Hoon Sung, S. Yup Lee, C. Herndon, E.M. Buckley, S.‐J. Park, F.H. Fenton, H. Cheol Cho, Engineered Cardiac Pacemaker Nodes Created by TBX18 Gene Transfer Overcome Source–Sink Mismatch, Advanced Science (2019) vol. 6, iss. 22, 1901099, DOI: 10.1002/advs.201901099.
- C. Greb, Introduction to Mammalian Cell Culture: Morphology and Cell Types & Organization, Science Lab (2017) Leica Microsystems.
- J. Schumacher, L. Bertrand, THUNDER Technology Note: THUNDER Imagers: How Do They Really Work?, Science Lab (2019) Leica Microsystems.