High quality microscopy is increasingly used by scientists in new areas of research.
In the life sciences, light microscopy has long been a pillar of research methodology. However, microscopy today is rarely used to the exclusion of other research techniques. Instead, light microscopy has become a means to a larger end, and more scientists than ever are expected to be able to provide optical images of phenomena being studied via other mechanisms. Sometimes, researchers whose primary field of endeavor is molecular biology, sequencing, proteomics, mass spectrometry, flow cytometry, the study of animal models, and even human clinical trials now use microscope analysis and images in publications to support their work. As a result, there is an expanding need to make high-quality microscopy available to scientists whose expertise lies in other fields.
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Figure 1. Z-stack projection showing differentiated human neural stem cells after 14 days in culture. ß-tubulin II (green) indicates immature neurons, glial fibrillary acidic protein (GFAP, red) shows glial cells, and Hoechst (blue) reveals cell nuclei. Image captured using the Olympus FluoView FV10i confocal microscope system at 25×. (Source: Daniel Haus PhD and Katja Pillti PhD, the Cummings Lab at the University of California, Irvine) |
Although fluorescence and optical sectioning methods have traditionally been among the most complex aspects of light microscopy to master, they are among the areas of microscopy that have expanded the most throughout the last decade. The ability to introduce fluorescent reporter molecules into live cells has transformed fluorescence microscopy into a direct measure of cellular processes and molecular interactions. Monolayer tissue culture provides only limited models of biologic processes, but in vivo imaging poses troublesome issues such as specimen movement and inaccessibility. Three-dimensional cultures are increasing in popularity due to the development of matrices and supports that can mimic tissue architecture and more faithfully reproduce cell interactions.
Optical Sectioning
Optical sectioning methods, combined with carefully selected objective lenses and growth substrates along with control of such environmental factors as temperature and pH, can enhance the validity of time lapse live cell imaging. Optical sectioning may be achieved by several means:
• Deconvolution in which the effect of the microscope optics on a point of light are calculated and mathematically removed from each image
• Structured illumination in which multiple images are acquired using slightly different illumination schemes and unwanted light is removed by comparing images
• Excluding out-of-focus light by using a confocal laser scanning or spinning disk aperture
• Multiphoton excitation, where excitation of fluorophores occurs solely at the focal plane.
The methods above are listed in order according to their ability to image specimens at increasing depths. Each method performs well in a certain range of specimen thickness and acquisition speed. Optical sectioning using confocal laser scanning microscopy (CLSM) provides excellent depth resolution for many tissue culture models and model organisms in the range of 100 microns in thickness. These include cultures grown on three-dimensional matrices, migration, invasion assays, and microfluidic cell culture devices. In addition, confocal laser scanning has been a mainstay for imaging self-assembling multicellular structures such as tumor spheroids, neurospheres, embryoid bodies, endothelial cell tubes, and polarized epithelia. Tissue explants such as brain slices and pancreatic islets, along with model organisms such as Xenopus oocytes, zebrafish, and Drosophila embryos, often are chosen partly because their dimensions fit well with the optical sectioning capabilities of CLSM.
Increasing the Reach of Advanced Microscopy
The laboratory of Brian Cummings, PhD, of the University of California, Irvine, focuses on stem cell research, investigating the mechanisms of recovery mediated by human stem cell transplantation after spinal cord injury. The laboratory also looks at factors affecting the survival, engraftment, migration and differentiation of stem cells both in animal models and in cell culture models (Figure 1). For many such researchers, the compelling feature of fluorescence and confocal imaging is the ability to label different proteins or cellular features with probes of different colors and record multiparametric data. The cellular morphology also can be recorded by means of a label-free modality using transmitted light to give a grayscale image. CLSM technology collects all of these images simultaneously and in perfect registration. The ability of confocal microscopy to make crisp images of three-dimensional objects without the blur from out-of-focus light has propelled it to a “must-have” component of corroborative data. The rejection of unwanted light makes the fluorescent intensities a more accurate representation of molecular distribution than the corresponding “widefield” microscope image.
Making advanced microscopy available to a broader range of research scientists is far from trivial. While the need for light microscopy has grown, its complexity also has increased exponentially, making it more and more difficult for scientists who lack extensive experience to access the additional data necessary to support their work and publications. In addition, as university education has changed and required more specialization, graduate students now often arrive in laboratories without the training in optical microscopy that they need to support their principal investigator’s research protocols adequately.
Today’s most advanced confocal laser scanning microscopes are typically found in core facilities. This is because they are expensive, often need dedicated set-ups with darkened rooms and anti-vibration tables, and are complex enough so experts must operate them. The microscopists who operate them make use of laser scanning technologies for biophotonics applications that allow them to observe and image dynamic events going on within cells, such as FRAP, FRET, photoconverting optical highlighters, uncaging bioactive molecules, stimulating optogenetic probes, and others. Yet, despite the capabilities of the most advanced confocal microscopes, the majority of publications using confocal microscopy do not require these advanced biophotonics techniques. Instead, they include multicolor confocal images, some with multiple planes in the Z (depth) dimension for three-dimensional rendering or time-lapse imaging of live cell processes.
For many scientists who want access to the power of confocal microscopy to supplement their work, the power and complexity of top-level confocal instruments can be daunting. While core facilities have staff specifically for this purpose, there are challenges to live cell imaging that can make the process quite complex. For instance, many live cells in culture are exquisitely sensitive to environmental changes in such parameters as temperature along with the level of key gases including oxygen and carbon dioxide. To be successful, researchers need to carry out imaging tasks as close as possible to the incubator and with as little perturbation of cultures as is feasible. Transporting cells down the hall or across campus to another facility may make such imaging complex, if not impossible. The need for a darkened room may be another factor in limiting the type of work that can be done right in the lab. Finally, it can be difficult at times to coordinate the availability of key instruments and personnel with the experimental requirements for when imaging is to begin and how long it is to last. Though some researchers have brought in relatively inexpensive personal confocal instruments, in many cases these instruments may not deliver the performance required.
Within the core facility itself there are other issues at stake regarding management of resources. With limited numbers of top-level instruments, core facility managers have to decide who gets access to the most sophisticated two-photon and confocal instruments, and what staff must be dedicated to their operation. There is a need to deliver the vast majority of research images easily and reliably, without the necessity of extensive staff involvement or the most costly instruments.
Finally, there are experimental challenges. In order to draw meaningful conclusions from cellular imaging, a statistically significant number of cells should be imaged. This means scheduling image acquisition from multiple fields of view, and in time-lapse studies, revisiting the same field over time. For thicker samples such as three-dimensional cultures or polarized epithelia, multiple planes in the Z-axis may be acquired to accurately represent cellular detail. The increasing number of images required thus demands documentation software that facilitates image annotation and data storage.
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The Olympus FV10i is a self-contained Confocal Laser Scanning Microscope. (Source: Olympus America Inc.) |
Addressing the Challenges
Recently, Olympus has addressed the challenges of bringing CLSM to the lab bench with the introduction of the FluoView FV10i system, a totally self-contained confocal laser scanning microscope that is streamlined to satisfy the requirements of the majority of users while providing the highest quality confocal image data. Available with an oil immersion (60× NA 1.35) objective, it is designed to provide the highest quality confocal imaging to meet the needs of researchers. It provides a range of magnifications from 10× to 60× via its optical zoom.
Self-contained instruments are a useful addition to core facilities, freeing up expert operator time as well as increasing fee-for-use income by making confocal imaging more popular as it becomes more cost-effective. The optical path follows an inverted microscope configuration for maximum versatility and the stage insert accepts several different substrates including slides, chambered cover glass and culture dishes.
Instruments like the FV10i are compact and enclosed, so they can sit on the bench in any laboratory with no need to build a darkroom. Laboratory remodeling no longer means expensive re-installation service calls. The instrument can even be moved to a different lab when retooling for the next biotechnology or pharmaceutical discovery campaign. The long lifetime of diode lasers and simplified construction can result in cost savings in terms of reduced maintenance, wear and tear and replacement parts such as mercury burners.
Individual client laboratories that regularly pay for confocal usage may opt to purchase their own instruments because publication-quality images are easier to capture at relatively low cost with the new all-in-one systems. Conversely, confocal laser scanning experts will have more time to develop protocols for newly emerging biophotonics applications that will bring in more sophisticated users.