Simple Cell Imaging for Complex Research

Figure 1 – ZOE Fluorescent Cell Imager.

The average rat brain is about the size of a pea and is divided into more than a dozen functional structures.1 As part of their research, neuroscientists, with utmost precision, inject viruses into the brain targeting the tiniest functional domains. The only way to truly know that the correct part of the brain was injected is to use a fluorescent reporter and image slices of the brain.

Jody Martin, Ph.D., lab director of the Cell & Molecular Physiology Department at Loyola University Medical Center (Maywood, Ill.), had to confirm that his viral injection experiments in rat brains targeted the right domain, and that the virus successfully infected the desired neurons. He also needed to share these results with a collaborator to continue with the next step in the experiment, immunostaining the sections.

To capture and share the validation data he was looking for, Martin used the ZOE Fluorescent Cell Imager (Bio-Rad Laboratories, Hercules, Calif.) (Figure 1). With a few taps on the imager’s LCD touchscreen, Martin was able to capture the images he needed to show precisely which neurons were infected, and easily send them to his collaborator via e-mail (Figure 2).

Importance of overlaying images in tissue culture

Figure 2 – a) Section of rat brain middle cerebral artery occlusion (MCAO) showing approximately where fluorescent staining was observed. b) Overlay of ZsGreen1 fluorescent reporter expressed in cells infected with adeno-associated virus, four weeks postinfection, and brightfield images taken with ZOE Fluorescent Cell Imager showing infected cells are mostly in the corpus callosum.

For fluorescent cell imaging to be useful, it is critical to know where the fluorescent reporter is located in relation to the whole cell or tissue. Researchers can use DNA stains to determine the location of the nucleus and brightfield imaging to show the whole cell and tissue morphology.

Martin laid the image of the fluorescent reporter over the separate brightfield image to see precisely where the injected virus infected the cells (Figure 2), an act that was readily accomplished using ZOE’s merge feature. To do this with a fluorescence microscope in the past meant separately exporting each fluorescent and brightfield image and overlaying the images using editing software. ZOE streamlines the process by allowing touchscreen image selection and tapping the merge option to quickly obtain an overlaid image.

Linda Berg Luecke, lab manager at the Blood Research Institute (Milwaukee, Wisc.), uses ZOE’s merge feature to check for transfection efficiency during lentivirus production, which requires transfection of a very high percentage of cells. A merged brightfield and fluorescence image shows the number of cells that were transfected compared with all cells to immediately determine whether to proceed with lentivirus harvesting.

Minimizing disruption of settings and preventing damage to the microscope

Figure 3 – A student uses the ZOE Fluorescent Cell Imager at the Cell & Molecular Physiology Department at Loyola University Medical Center. Shown in the background is an old microscope to illustrate its complex platform.

Due to the high number of experiments using fluorescent reporters, fluorescence microscopes are often used daily and are shared among numerous researchers from multiple labs. This can potentially disrupt important settings or damage the instrument.

A typical fluorescence microscope has multiple knobs and buttons with an external camera and requires significant training before a new user is comfortable with it. Even after training, users can easily disturb important settings because knobs are not labeled and the system can be very complicated.

Most of the new graduate and undergraduate students in Martin’s lab have no experience with cellular research and fluorescence microscopes. Martin has found that those who don’t know how to use the microscope often fiddle with all the knobs. When he replaced the old fluorescence microscope in his lab, he chose the ZOE system, partly because there were no knobs. Instead, users can perform all the steps needed to view and image cells through the imager’s touchscreen and Android-based platform (Figure 3).

Imaging channels can be changed with a few taps on the screen, and the zoom can be adjusted by pinching in or out of the live image. Only a few taps are needed to snap pictures and overlay images.

Trading in the eyepiece for a live collaborative screen

Complex research requires training—both to use an instrument and to perform an experiment. An interface like the ZOE system’s view screen that enables teachers and students to look at the same microscope slide at the same time can be a useful instructional tool. This is not possible with a traditional fluorescence microscope, where only one person at a time can look through the eyepiece. Use of ZOE’s live display makes it possible for everyone to look at the same feature in an instant.

Bringing microscopy out of the darkroom

Figure 4 – Exosomes (green) endocytosed by human marrow stromal cells (HMSCs) imaged on a ZOE Fluorescent Cell Imager to verify staining before confocal imaging.

A great nuisance of the traditional fluorescence microscope is where it lives—the small darkroom is a difficult place to work and prone to overcrowding. A darkroom is usually needed in fluorescence microscopy because ambient light makes fluorescence difficult to see. ZOE has an integrated light shield that allows imaging in any light condition; thus it can be used in the cell culture room or on a lab bench with the lights on. Time-sensitive experiments are easier and faster in cases in which the darkroom is housed in another building.

The fluorescence microscope previously used by Sriram Ravindran, principal investigator in the Department of Oral Biology at the University of Illinois at Chicago, required a darkroom, used a halogen lamp light source that had to warm up for five minutes and had an expensive bulb that needed to be replaced often. Now, Ravindran’s lab has a ZOE, which needs no warm-up time and uses an LED light source that provides thousands of hours of illumination.

The system helps Ravindran’s lab evaluate if stained cells are of sufficient quality to be sent for confocal imaging, which saves time and money (Figure 4).

Conclusion

As demonstrated by the above examples, the ZOE Fluorescent Cell Imager can save time and money, provides high-level imaging functions and is designed for use by multiple researchers in a collaborative and fast-paced environment.

Reference

  1. Korbo, L.; Pakkenberg, B. et. al. An efficient method for estimating the total number of neurons in rat brain cortex. J. Neurosci. Meth. 1990, 31, 93–100.

Veronika Kortisova Descamps is product manager, Cell Biology, Bio-Rad Laboratories, 200 Alfred Nobel Dr., Hercules, Calif. 94547, U.S.A.; tel.: 510-741-4070; fax: 510-741-5811; e-mail: [email protected]  http://www.bio-rad.com/en-us/product/zoe-fluorescent-cell-imager

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