Cutting-Edge Innovations in Fluorescence Microscopy Illuminate the Way Forward

Microscopy is integral to the visualization and unraveling of biological processes across space and time, but its inherent limitations and potentially complicated set-ups can hinder the experimental process. The ability to selectively label proteins and lipids with fluorescent markers alongside newer and numerous ways to optically clear tissue has fueled innovation over the last few decades, perhaps best illustrated by newer iterations of light sheet fluorescence microscopy (LSFM), super resolution microscopy, and confocal imaging. Artificial intelligence (AI) is also upgrading and automating microscopes across the board.

Here we will look at emerging technology in fluorescence microscopy, as well as considerations for choosing the best microscope for the research questions at hand.

Light sheet fluorescence microscopes (LSFMs) image large samples in 3D with minimal tissue damage

While the initial development of LSFMs dates back to 1903, their resurgence and widespread implementation in the past two decades can be attributed to advancements in fluorescent labeling, tissue clearing techniques, and enhanced computational capabilities. The fundamental design involves an orthogonal arrangement of illumination and detection paths. A narrow sheet of light emitted by one or two illumination objectives excites fluorophores confined to the focal plane, thereby averting issues such as photobleaching and toxicity to the surrounding sample. The laser light can be shaped into a Gaussian beam or other types to generate specific intensity distributions. Volumetric imaging relies on scanning the sample, increasing the depth of focus, or moving the sheet and the detection. LSFMs are particularly valuable for imaging large, optically cleared samples, such as an entire rodent brain, as well as facilitating long-term live cell observations.

“Light sheet microscopy is ubiquitous across the life sciences, especially in neuroscience, developmental biology, and organoid studies, owing to its rapid volumetric imaging and gentle illumination approach,” says Peter Favreau, Product Market Manager at ZEISS. The company offers several options for “gentle volumetric imaging of either optically cleared spheroids, brains, and fixed tissues, or whole living model specimens such as zebrafish, organoids, or Drosophila embryos.” The Lightsheet 7, for example, has dual-sided imaging and a set of holders to accommodate specimens of a variety of shapes, sizes, and refractive indices. “These advancements allow for unparalleled imaging of large optically cleared specimens and long timelapse imaging with minimal photodamage to delicate samples, opening new avenues for specimen interrogation across time.”

Lightsheet 7 image generated using cleared (Translucence panel) murine brain showing Thy-1 EGFP. Image courtesy of ZEISS.

According to Favreau, lattice light sheet microscopy (LLSM), developed by Nobel laureate Eric Betzig, is another exciting development in fluorescence microscopy. Using a modified Bessel beam light sheet (slimmer than Gaussian versions) created by a spatial light modulator and projected onto the sample, there is “next to no photodamage over time, allowing for imaging experiments of up to several days.” ZEISS's Lattice Lightsheet 7 has an inverted objective configuration for standard sample carriers and a 5-axis stage for precision in all dimensions, with automatic leveling. Favreau notes, “early iterations of lattice light sheet technology required cumbersome alignment protocols and restrictive sample carriers” limiting accessibility of LLSM for researchers. This microscope, however, offers user-friendly implementation with auto-alignment; and since standard sample preparations can be used, imaging can get underway within minutes. “There are many biological questions currently unable to be answered because we simply are unable to observe those samples over a long enough time,” Favreau points out. The Lattice Lightsheet 7 aims to change that.

Super resolution microscopes reveal cellular and molecular dynamics with unprecedented clarity

The light sheet microscopes fall under “widefield microscopy,” says Favreau. ZEISS, conversely, also offers a line of super resolution microscopes that perform structured illumination microscopy (SIM). While light sheet microscopy restricts illumination to the focal plane, SIM uses a light grid pattern combined with image processing to perform optical sectioning.

The Elyra 7, part of this classification, boasts double the resolution of conventional SIM technology, and is able to discriminate fine sub-organelle structures down to 60 nm laterally. This super resolution microscope uses high-dynamic imaging up to 255 frames per second, again without the need for any special preparation of fixed or living specimens. “The Elyra 7 provides scientists with a powerful tool for observing subcellular structures and molecular events in real-time, greatly advancing our understanding of sub-organelle cellular processes,” says Favreau.

ZEISS also launched a new family of Lattice SIM microscope solutions at the end of November, including Lattice SIM 3, Lattice SIM 5, and Elyra 7 with Lattice SIM, which will further build on the innovations described above giving scientists more options for larger samples and for capturing cellular events quickly and with the continued theme of increased resolution. This line uses a lattice grid pattern instead of a rotating grid line pattern, used in traditional SIM. The newer version of the Elyra 7 expands on previous “capabilities with higher sensitivity cameras,  quantification down to the molecular level, and the widest range of super-resolution technologies in one system,” explains Favreau.

Building further on confocal capabilities

Confocal microscopes came to market over 25 years ago and have long been considered the gold standard for optical sectioning. Whether specimens are fixed, thinly cut sections, or living cells attached to glass, excellent high-resolution images are attainable. More complex in design than LSFMs, confocal microscopes continue to dominate the research arena and most universities have at least a couple in their core facilities. However, even these stalwarts continue to evolve with next-generation models flush with novel abilities and resolutions to old problems.

One of the main limitations associated with confocal imaging is photobleaching or toxicity. However, Antonio Ortiz, Senior Biosystems Technical Manager at Nikon, explains how this is becoming less of a problem. “Nikon came out with two products this year. The Nikon Spatial Array Confocal (NSPARC) detector and the Nikon ECLIPSE Ji. The NSPARC detector is different than other products on the market for its exceptional ultra-low noise profile, improved signal-to-noise ratio, and it allows for imaging with lower excitation power, which leads to less photobleaching and phototoxicity of scientific samples.” NSPARC consists of an array of 25 detectors operating like an extremely sensitive camera. Emission light is directed to this point scanning detector by the optical lenses, facilitating use with a variety of objectives and magnifications, as well as a user-defined illumination spot on the detector array. The result is the collection of ultrafine structural information typically unobtainable in traditional detection.

Cleared mouse paw with blood vessels stained with Alexa Fluor 488 wheat germ agglutinin imaged on Nikon’s AX/AX R confocal system

The NSPARC improves on Nikon’s AX/AX R confocal system, which harnesses the power of AI and expands on the number of colors while also improving on pixel density, sensitivity, and speed. The AX/AX R has one of the largest fields of view (FOV), at 25 mm diagonally, of any confocal microscope. Its high-throughput capabilities are worth noting, as well. According to Ortiz, there is immense interest by labs in high-content and high-throughput screening as is used in drug discovery.

Utilizing AI to maximize efficiency in the lab

In September of this year, Nikon launched the ECLIPSE Ji, a digital inverted “smart” microscope that uses AI to automate image acquisition and analysis. Ortiz says the new system was “built on the DNA of our [ECLIPSE] Ti2, but has been redesigned to maximize ease-of-use even for those who are not used to performing imaging.” The Ji Assay and Ji Research configurations offer scientists a more tailored approach. “The Ji Assay is different than offerings from other companies because it’s preconfigured with optimized assay experiments in order to speed up the workflow.” The newest AI technology “minimizes human error and maximizes data output.”

Scientists want more but shouldn’t lose track of what they need

“The trend in the last several years is ‘more,’” says Ortiz. “Scientists want more information from their samples, more fluorescence colors and channels, and more sensitivity.” They also demand more of the microscope itself, he says. They want increased speed, ease-of-use, and a bigger field of view. Favreau agrees; he adds that researchers want more resolution and more versatility. “High-resolution imaging is crucial for discerning subcellular structures and molecular interactions, which is why advanced microscopes like Lattice Lightsheet 7 and Elyra 7 are popular choices.”

“This demand for more has given rise to new fields like spatial biology, and more information being collected in the fields of high-throughput screening and whole organism imaging,” notes Ortiz. “This has led to an explosion of data, and the need to find new ways to analyze them.”

Both Ortiz and Favreau say in order to avoid decision fatigue, researchers should carefully consider their specific needs and limitations. “This includes things like which objectives, light sources, imaging techniques, and cameras to configure on your microscope,” says Ortiz. “It is crucial to identify the desired resolution, magnification, and imaging modalities [for example,] widefield, confocal, light sheet, or super resolution," adds Favreau. “Furthermore, consideration to service options and technical support can ensure the optimal performance and longevity of the equipment, allowing users to maximize their investment and achieve the best possible results with their fluorescent microscope.”

Hero image at the top is a screenshot from a Lattice Lightsheet 7 movie generated using AICS-0013 (laminB1-mEGFP) from the Allen Institute. Image courtesy of ZEISS