Cells used in pre-clinical research are traditionally cultured on a 2-dimensional surface, typically on the bottom of a plastic container. Even for a non-cell biologist, like me, it is easy to understand that this is very far from a natural cell environment. In our bodies, cells live in a protein gel similar to the gel created by using ordinary cooking gelatin. Cell biologists call this gel the extracellular matrix.
3D cell culture
To make experiments in pre-clinical research more realistic and to reduce animal testing, researchers are increasingly culturing cells embedded in a 3-dimensional layer of collagen – the main structural protein of the extracellular matrix. Conventional microscopy has a very limited focal depth of just a few micrometers. This makes it difficult to create focused images of the cells, as they move out of focus when they move about in the 3-dimensional gel.
In contract to conventional microscopy, our HoloMonitor® instrument digitally focuses the image after it is recorded. This enables HoloMonitor to create sharp images of the cells wherever they are located in the gel.
The hologram recorded by HoloMonitor is an interference pattern, created by joining the sample and reference laser beam.
An autofocused HoloMonitor time-lapse video of cells cultured in a 3D gel.
Fine focusing is done entirely in software, after recording. The digitally recorded hologram is computationally processed to create holographic images over a range of focal distances. The image in focus (green frame) is selected as the final image.
Poster image: As the complexity of the formulations increases, there is a decrease in the high optical thickness and a corresponding increase in the low optical thickness consistent with increasing cell death.
Using HoloMonitor and a public domain image processing software called Image J, the researchers from Northeastern University have developed a method for cell experiments based on cells cultured in a 3-dimensional collagen gel. They show that this new method is clearly superior to conventional methods, based on cells cultured on a 2-dimensional surface, and conclude:
“We developed a novel holographic imaging method using HoloMonitor and Image J. The new method is an important step in developing methods that better emulate biological processes and offer the possibility of evaluating effects of drugs at lower cost and experimental complexity than methods based on animals.”