Fluorescence Microscopy: Solving Common Documentation Challenges

Fluorescence has proven to be a useful technique for identifying, tracking, and studying molecules of interest in biological specimens. In fact, fluorescence is among the fastest growing microscopy techniques for analyzing both fixed and living specimens. With the recent development of new research methodologies and an ever-increasing number of fluorescence probes optimized for tagging structures of interest, scientists have used fluorescence to study intra- and intercellular behavior; tissue structure and function; ion fluctuations (i.e., Ca2+, pH, etc.); and more, extending their study to plant, animal, and even human samples.

Researchers have exploited and adapted rapid advances in the technology to expand the depth of their scientific investigations. But documenting research findings for presentation or publication can be a challenge. Aside from the nuances associated with sample preparation and experimental design, the process of documentation itself poses many complex variables. Five fluorescence documentation challenges are outlined here, with thoughts on how laboratories can overcome these hurdles.

The fluorescence phenomenon is difficult to see. Generally speaking, fluorophores absorb excitation light at one wavelength, and emit light at a longer wavelength. Many samples fluoresce very dimly, briefly, intermittently, or all three. Scientific cameras are very sensitive and capture any photons entering the optical path to the detector (i.e., charge-coupled device [CCD]), regardless of whether the photons are from the specimen or room light. To aid in minimizing the impact of ambient light and to prevent photobleaching, many researchers place their fluorescence instrumentation in a darkened room. Isolating the imaging system from ambient light reduces noise from extraneous light sources, making dimly fluorescing cells easier to view. The goal is to collect only enough light to illuminate the process or structure the scientist wants to view, while avoiding additional light that can lead to photodamage or photobleaching.

However, darkrooms cannot compensate for the issues of fluorescent “cross-talk,” the detection of signal in one channel that originates from an unrelated fluorophore. Due to some spectral overlap of fluorophores, shorter wavelengths (moving toward ultraviolet wavelengths) may excite or photobleach fluorophores typically excited with longer wavelengths (green or red), thus contributing to this cross-talk. One technique to minimize cross-talk is to collect the longer (closest to the red or infrared) wavelengths first, working gradually down to the shortest wavelengths. Where cross-talk still occurs, several microscope manufacturers and image analysis software providers offer spectral unmixing algorithms to remove the contaminating signal.

The dimness of observed phenomena can also be addressed by imaging over longer periods of time. The ability to image over long time spans allows more flexibility in collecting photons, but brings the researcher to a second key challenge. Vibration contributes to poor images, and the high magnifications often used with microscopes to view phenomena on a cellular level only exacerbate the issue. Vibration sources can be quite varied, and often include air handling machinery in the facility and other laboratory equipment (i.e., centrifuges, freezers, etc.). However, even external sources may be implicated (i.e., trains, traffic, construction, etc.). To reduce or eliminate vibrations, scientists often use vibration-dampening techniques. The most common vibration reduction methods and materials include rubber cushions, marble slabs, and air tables. Faster cameras can capture images at up to thousands of frames per second, and can diminish the effect of vibration by “freezing” the biological phenomenon in time. Although these high-speed video instruments have great utility, resolution can be sacrificed for speed. In addition, most such video cameras capture so much information that, due to data storage limitations, they are not well suited to recording changing or repeating events over long periods, like hours, days, or weeks. However, they are ideal for collecting short bursts of fast-changing image data.

Keeping live cells happy presents a third challenge to fluorescence documentation. Many cell cultures are sufficiently resilient to tolerate a quick image on the microscope, but longer term imaging experiments can possibly lead to cell death in response to a prolonged change in environment. Several types of microscope incubators have been developed to provide improved environmental control during experiments that extend beyond a few minutes. One style of incubator encloses the entire top of the microscope, including the stage. The entire system is appropriately heated and can be charged with the optimal gas mixture for the cells. Stage-top incubators offer similar control, but can only accommodate small dishes or chambers. However, thermal drift—changes in focus due to the expansion and contraction of the microscope and its own optics in response to fluctuations in ambient temperature—still poses a challenge to consistent imaging. Recently, microscopes have been integrated into traditional tissue culture incubators to provide the best of both worlds: high-resolution imaging without drifting out of focus, in a cell-friendly environment. This type of incubator–microscope system makes it possible to image multiple samples simultaneously, without ever removing the cells from their life-sustaining incubator environment.

Yet another challenge to fluorescence documentation is the complexity of the equipment. Often, a user’s microscope, software, and/or camera may not be properly or ideally configured for fluorescence documentation. Today’s instrumentation has extraordinary capabilities not even dreamed of a decade ago, and with these capabilities come intricacies that can be difficult to navigate. In some cases, the microscope may have originally been purchased for another purpose and later retrofitted to accommodate the demands of fluorescence documentation. In other cases, the system may be a compromise of function, quality, and convenience, or the last user may have failed to restore the microscope to a neutral starting configuration. Of course, there are many researchers who master the operation of their high-end instrumentation and software very effectively. Some laboratories benefit from a resident microscope expert who guides colleagues through the process of generating quality digital images. In other laboratories, staff turnover is a concern since it creates the need to train new personnel again and again, which can distract senior researchers from productive work.

Knowledgeable microscopy specialists are also found in core imaging facilities, where they routinely assist researchers in getting the best results on highly specialized microscopy equipment. Access to these experts and the highly advanced microscope imaging systems in their facilities is a boon to many researchers, even if the capability of the equipment far exceeds the need of some experimental protocols. Core imaging facilities often provide highly advanced microscopy stations where, in many cases, imaging tasks range from documentation of basic fluorescent protein expression to highly advanced techniques. For some core facility directors, users with less demanding imaging requirements provide welcome traffic through the facility, and often lead to the use of more advanced microscope techniques by their clients. Yet other directors fret about the potential for damage to sensitive microscope systems, and the additional maintenance and training workload to support these systems. With a finite number of high-end research instruments on board and only limited staff time, it can be difficult to provide adequate support to meet the needs of researchers at all levels of experience throughout the facility.

Training personnel to get the most from their fluorescence microscopes can be handled in various ways. First, manufacturers’ sales representatives generally provide basic training upon microscope installation and, in many cases, will offer refresher training upon request. Second, microscope manufacturers may offer training courses on particular microscope methodologies; these are generally oriented toward advanced techniques. Finally, many institutions offer courses and workshops that cover topics from optical theory to advanced fluorescence techniques such as Förster resonance energy transfer (FRET), fluorescence lifetime imaging microscopy (FLIM), and total internal reflection fluorescence (TIRF) microscopy. Well-known microscopy training courses for selected researchers are sponsored at such prominent locations as the Marine Biological Laboratory at Woods Hole, MA; Cold Spring Harbor, NY; Mount Desert Island, ME; and Yale University, New Haven, CT. The popularity of these high-level courses demonstrates how much demand there is to learn advanced research imaging methodologies for fluorescence and other microscopy imaging. For all but the most advanced microscope users, researchers using fluorescence are focused on their investigations and do not see the need to spend time maintaining or learning to operate complex equipment.

Figure 1 - Cross-section of mouse colon stained with Alexa Fluor* 350-wheat germ agglutinin (blue), Alexa Fluor 568-phalloidin (red), and SYTOX Green nucleic acid stain (green). Scale bar indicates 200 µm. All figures were captured using the FSX100 fluorescence microscope; figures were provided courtesy of Olympus America Inc.
*Alexa Fluor®, MitoTracker®, and SYTOX® are trademarks or registered trademarks of Molecular Probes, Inc. (Eugene, OR). Olympus and FSX are trademarks or registered trademarks of Olympus Corp. (Tokyo, Japan), Olympus America Inc. (Center Valley, PA), and/or their affiliates.

Figure 2 - Mouse stomach fundus stained with Alexa Fluor 350-wheat germ agglutinin (blue), Alexa Fluor 568-phalloidin (red), and SYTOX Green nucleic acid stain (green). Scale bar indicates 100 µm.

It is understandable, however, that the majority of fluorescence-based documentation is predictable and regular. Recent developments in fluorescence probes, staining techniques for fixed specimens, expression vectors for fluorescent proteins, and equipment design have contributed to making fluorescence documentation easier and more readily integrated into research projects at every level and every point in the process. Accessibility of equipment, both in terms of location and available time, can become an issue when researchers with narrow time frames for imaging specific phenomena, or with the need to image for long periods of uninterrupted time, must adjust their research protocols to the availability of instrumentation in core facilities. In addition, many researchers say they would prefer to locate their fluorescence microscope closer to where they prepare their specimens (i.e., slides, cell culture, etc.) to avoid transporting specimens down the hall to the darkroom or across campus to the core facility. However, laboratories are crowded places, with space at a premium and limited ability to operate in darkness for minutes or hours at a time to facilitate fluorescence imaging. The cost of bringing in major fluorescence microscopy systems is another consideration.

Figure 4 - OK (opossum kidney) epithelial cells stained with Hoechst 33342 (blue), Alexa Fluor 488-phalloidin (green), and MitoTracker Red CMXRos (red). Scale bar indicates 30 µm.

Figure 3 - NIH/3T3 mouse embryo fibroblasts stained with DAPI (blue) and MitoTracker Red CMXRos (red). Scale bar indicates 25 µm.

The FSX100TM  (Olympus America Inc., Center Valley, PA) is a compact, all-in-one fluorescence and brightfield microscope and imaging system that solves many of the challenges associated with routine fluorescence image documentation (Figures 1–6). It combines the same high-quality components found on traditional microscopes with an intuitive, software-based work flow that reduces the need for training. Additionally, the microscope creates its own darkroom; thus it can be installed practically anywhere a 3’× 3’ tabletop space is available, even in a fully lit laboratory. 

Figure 5 - Rat brain (horizontal section, 8 µm thick) stained with Hoechst 33342 (blue), Alexa Fluor 488-NFiH (green), and Alexa Fluor 568-GFAP (red).

Figure 6 - OK (opossum kidney) epithelial cells stained with Hoechst 33342 (blue), Alexa Fluor 488-phalloidin (green), and MitoTracker Red CMXRos (red). Scale bar indicates 20 µm.

Core imaging facilities such as that at the University of Wyoming (Laramie) use the FSX100 to address routine documentation and liberate the more advanced systems for even more sophisticated imaging techniques such as laser scanning confocal microscopy. In another application, a discovery research group at a major pharmaceutical company has adopted the FSX100 as its primary documentation system for both live and fixed cells. The group develops cell-based toxicological models, and uses the imaging system to document cell culture densities, protein expression levels, and time point and endpoint analyses.

Summary

Advancements in fluorescence microscopy have been a boon to researchers applying this rapidly growing technique for documentation of both living and fixed cells. Hardware and software developments continue to offer researchers new paradigms for answering scientific questions. But fluorescence imaging has numerous challenges that can sometimes turn the imaging and documentation step into a project itself, rather than a means to an end. Improving work flows and developing instruments that help conquer some of the key challenges of fluorescence imaging provides the promise of even brighter days to come.

Mr. Clymer is Product Manager, Olympus America Inc., 3500 Corporate Pkwy., P.O. Box 610, Center Valley, PA 18034, U.S.A.; tel.: 484-896-5000; e-mail: [email protected].

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