Auto-fluorescent proteins have become an indispensible tool for cell biology research over the past decade. Originating from sea creatures such as jellyfish and sea anemone, these fluorescent proteins come in a rainbow of colors and are used to monitor cellular processes like protein localization, receptor signaling, and protein trafficking.
A typical cell biologist spends much of his or her time planning experiments and culturing cells in a biosafety-controlled laboratory environment. Before the next test can be arranged, it is necessary to evaluate results from previous experiments. Given the increased role of “fluorescent tools” available to cell biologists, the need for fluorescence microscopy and experimental evaluation in the cell culture laboratory has increased. By design, however, the traditional fluorescence microscope functions optimally in the dark, leading to separate microscopy facilities. These facilities tend to house many complex microscope systems that require training and consistent use to maintain the skills needed to produce results.
Location, features, and the need for training are the main barriers to the accessibility of fluorescence microscopes. First, the location of the instrumentation is paramount: As cell culture takes place in ambient room light, so too must the evaluating of results. The device itself must be able to meet the needs of the cell biology researcher who, for example, typically uses many different types of sample vessels and often shares and documents his or her work. The required training needed to learn fluorescence microscopy is also a barrier that initially exists for all researchers, and can come at the cost of taking time away from making scientific advancements. Finally, instrumentation expense can hold back individual labs or smaller colleges, costing $50k-$250k.
In this article, we will look at how fluorescence microscopy has been made accessible through a paradigm shift in the design and engineering of benchtop imaging devices.
Setting the Stage: Traditional Microscopes
Traditional fluorescence microscopes are built for maximal flexibility and may best be likened to flying a jet aircraft. There are knobs, buttons, and levers to control nearly every aspect of the device. With the flexibility, however, comes several drawbacks. Designed for the dark, traditional fluorescence microscopes are kept in rooms devoid of light, much like a film-processing dark room. Complexity results from each element including mirror alignment, focus, light intensity, and filter configuration having the ability to be adjusted, and perhaps incorrectly adjusted, by a novice or untrained user. Physical size is bulky due to each component (microscope base unit, camera, light source, power supply, computer, and monitor) being a separate part. Ergonomically speaking, the traditional microscope appears to be designed based on functionality first and user experience second. Lastly, the user interface is typically cluttered and contains many adjustable parameters, making it only available to experienced users.
Fluorescence Microscopy in Room Light
Industrial design is often considered important for aesthetics and utility, but it can also become an enabling feature. By placing a light shield over where the sample goes, tools like the FLoid™ cell imaging station from Life Technologies Corp. (see image above) capture fluorescence images under normal, ambient room lighting. The light shield effectively blocks room light from entering the optical light path at the objective lens. This industrial design element places the device in a cell culture lab, and effectively brings fluorescence imaging to the cell biologist. The light shield also hinges upward to allow samples to still be precisely positioned on the stage. The light shield design element makes fluorescence microscopy accessible by overcoming the barrier of location.
Technology Advancement: A Simplified Optical Path
Typically each color acquired by a fluorescence microscope involves a separate set of optical filters which must be moved each time a new color channel is captured. The movement of filters can be manual or automated, but either way this movement takes energy and can break down over time. Oblique illumination has been used to increase contrast in brightfield imaging of nearly transparent samples. Its application to fluorescence imaging, however, is new.
Oblique illumination combined with a multi-bandpass optical filter provides a robust method to leave optical filters stationary while still able to collect blue, green, and red fluorescent images. Oblique illumination, a technology enabled in the FLoid station, features a light-emitting diode and excitation filter in position to directly illuminate the specimen, without traversing the objective lens. The multi-bandpass emission filter appropriately corresponds to the emission wavelengths expected based on the excitation filters. By locking this method, a fixed optical system is created, thereby eliminating the need for users to verify that instrument components have not been changed without their knowledge.
Tools like the EVOS® fl fluorescence microscope from Advanced Microscopy Group (Bothell, WA) use integrated filter cubes with LEDs. These LED light cubes are interchangeable to offer more wavelength options than the oblique illumination system. Either method decreases the specialized knowledge required to operate the system and makes the technique of microscopy more accessible.
User Interface Design
User interface design is critical to the adoption of most consumer electronics and therefore must be given adequate attention as most science equipment is judged on a similar scale. Figure 2 illustrates how common icons, few words, and image thumbnails can be used to design an intuitive interface. Key items to consider are the use of recognizable features such as a camera icon for snapping a picture and common elements like the “X” used to delete a thumbnail image.
The overall theme is to simplify the user interface and make it more intuitive so that researchers can access the technology without having to become experts in the use of a device. The LED light output, camera gain, and exposure time, for example, can all be controlled by the “Light” scale as a simple slider bar. The alternative to the “Light” scale could be three separate control elements that allow users to define exact parameters for LED intensity, camera gain, and exposure time. With the increased flexibility, however, comes increased complexity.
The “Light” scale is an attempt to allow all three parameters to be optimized by pre-determining the optimal configuration for each of the parameters under the conditions of a weak to a strong fluorescent sample. By pre-setting the parameters, a simple slider can be used to adjust sample brightness and therefore decrease the feeling of confusion when trying to image.
Trying to find focus is the hardest part of day-to-day microscope operations. A digital encoder, a device that converts motion into a sequence of digital pulses, can be used to track the position of the focus mechanism and output an integer value. This value can be related by end users to a particular sample vessel and greatly aid them in finding the focal plane of a sample. The integer value is displayed on the user interface as the “Focus Assist” scale (Figure 3), helping remove the feeling of being “lost” when trying to focus the sample.
An Integrated Technology
Ultimately if the goal is to make a cell imaging device for an already crowded cell culture facility, the footprint must be minimized. Standard fluorescence microscopes include the base unit, plus light source and power supply, camera and power supply, computer, and monitor. Newer devices integrate the computer, microscope optics, camera, and LCD screen into a single device.
The LCD screen also serves as an ergonomic advancement. The EVOS fl fluorescence microscope, for example, mounts a screen on the device so that researchers can stand and use the device on a lab bench. Previously microscopes were too low, and users had to lean in order to use them. Traditional microscopes use eye pieces that allow only one user at a time to view a sample. The LCD instead provides a convenient way to instantly share results with fellow researchers.
Besides the reduced footprint, an integrated design is less intimidating and can provide better ergonomics, which makes fluorescence microscopy accessible to new user groups and novice users. Benchtop devices such as the FLoid cell imaging station and the EVOS fl fluorescence microscope have the power to accelerate scientific discovery by reducing the obstacles often required to operate scientific instrumentation.
This article was written by George Hanson, PhD, senior staff scientist at Life Technologies Corp. (Eugene, OR). For more information, Click Here .