Read the Application Note to find out how STELLARIS can take you beyond traditional limitations:
- Image longer without photodamage, and with enhanced sensitivity for the fluorophores you use most.
- Study dynamic events at exceptionally high frame rates—capturing up to 428 frames per second without compromising spatial resolution.
- Multiplex beyond spectral options, resolving overlapping fluorescence spectra, and adding the fluorescence lifetime dimension to your experiments.
In recent years, the field of live-cell imaging has evolved rapidly due to groundbreaking advances in confocal technology together with an ever expanding toolbox of fluorescent dyes and genetically encoded tags, as well as better biological models-such as stem cell-derived cultures, spheroids, and organoids that more closely mimic the complex physiology and architecture of in vivo systems. While great progress has been made, live cell confocal imaging still presents significant challenges when it comes to practical applications in the lab. Chief among them is how to avoid the classic trade-offs between spatial resolution, signal-to-noise ratio (SNR), and acquisition speed-all the while ensuring that cells remain healthy for the duration of the experiment.
Cell health, phototoxicity and photobleaching
Maintaining sample health is paramount to ensuring the validity of any live imaging experiment. Most tissues and cell types in multicellular organisms are shielded from light during their natural life span in vivo. Consequently, uncontrolled doses of light can perturb their behavior, activate stress responses, impair metabolic function, and alter vital structures. Phototoxicity has been reported for many popular fluorescent dyes, including Hoechst, Draq5, Fluo4-AM, Mitotracker, and Rhodamine B. When fluorophores are photobleached, the risk of phototoxicity increases, due to the generation of reactive intermediates that persist for longer, giving them more time to inflict damage .
Because any exposure to light has the potential to perturb living systems, there is always a limit to the length of time a live specimen can be imaged without causing damage. Great care must be taken to avoid oversampling, minimize the amount of light exposure, and control for phototoxic effects whenever possible. This fact has implications not only for experimental set-up and execution, but also when it comes to the selection of the microscopy system itself.
Despite the growing diversity and spectral range of existing fluorophores, finding the right probes for a live-cell experiment can still be a big stumbling block. In multicolor imaging experiments, the different fluorophores in the sample need to be distinguishable from each other and bright enough to enable adequate contrast at the light dose levels needed to preserve cell health. They also need to be photostable enough to withstand repeated
imaging without photobleaching and phototoxic effects.
While fluorescent proteins (FP) are generally less photolabile than chemical dyes due to caging of the fluorophore within a protective polypeptide structure, photobleaching and phototoxicity may still be an issue depending on the experimental conditions, sample type, and FP species. Achieving sufficient brightness can be particularly problematic when working at endogenous expression levels or with weak red-emitting FPs. Spectral overlap between fluorophores can also lead to bleed-through artifacts. Endogenous fluorescence is a further complicating factor that can contribute to poor image quality and limit multiplexing capacity. Many naturally occurring fluorescent molecules-for example, NAD, FAD, lipofuscin and chlorophyll- emit in the green and yellow regions of the spectrum, where they compete with signal from commonly used fluorophores like Calcein-AM and EGFP.
The pyramid of frustration
Three essential elements to consider when imaging live biological samples are signal-to-noise ratio (SNR), spatial resolution, and temporal resolution. Because these factors are interdependent, finding the optimal imaging conditions for a given sample usually involves compromise. For instance, to study a rapid dynamic event like calcium signaling, contrast and spatial resolution may need to be sacrificed to boost the speed of image acquisition. Such trade-offs are an everyday reality for most live cell experiments. Add to these factors the overriding priority of ensuring that the chosen settings do not damage specimen health, and users experience what has come to be known as “the pyramid of frustration” (Figure 1)-the time-consuming and extremely difficult task of balancing all four of these interdependent parameters.
Addressing the challenges described above with traditional microscopy systems can be time-consuming and frustrating, with no guarantee of success. To overcome these limitations and break the “pyramid of frustration”, Leica Microsystems has designed its new STELLARIS confocal platform with the specific challenges of live-cell imaging in mind. All components have been carefully selected and harmonized to boost efficiency of photon detection, while enabling high spatiotemporal resolution with minimal sample damage. By addressing all four vertices of the pyramid in its novel design, STELLARIS makes it easier and quicker than ever to find the optimal imaging conditions for any application.
Frustration-free imaging starts with high-performance detectors
The detector is a crucial consideration for optimal imaging, since it determines the level of sensitivity to fluorescent labels in the sample, what structures can be resolved, and the degree of temporal resolution possible when capturing dynamic events. The STELLARIS Power HyD family of detectors offers users more flexibility to achieve ultra-high sensitivity across the spectrum and for varied applications. The new photon-counting capability-Power Counting-improves image contrast, for more quantitative results and a higher dynamic range .
The Power HyD S detectors at the core of STELLARIS provide enhanced sensitivity in the blue-green spectral range-where it matters most for many live cell experiments. With photon detection efficiencies (PDE) up to 56%, Power HyD S detectors outperform Gallium arsenide phosphide (