STED Microscope
Super-resolution imaging has tremendous implications,
and microscopes need to be multifunctional platforms that can be adapted to specific applications. Te following sections will delve into three cases that require specific solutions to realize the full potential of STED (Figure 3).
Focus Cases Enhanced multi-color imaging and resolution
Figure 2: abberior’s one-roof approach.
• Upgradeability: Considering the fast pace of the field, it makes sense to design modular microscopes that can be retrofitted with essential enhancements. Tis allows con- stant upgrades and access to new features, as opposed to frequently purchasing a new system.
Tis combination of deep knowledge in organic chemistry
and optics (Figure 2), together with daily involvement in life science issues, has allowed STED to become a true multi-talent technique.
improvement with time-resolved detection. The first example highlights the impact of time-resolved detection on daily microscopy work. Basically, in-depth structural studies often require the use of multi-color imaging to visualize sample context and for analyzing spatial relation- ships and interactions. The ability to unambiguously sort multiple dyes into different detection channels is a major factor in experimental design and usually not a simple feat, especially in connection with super- resolution microscopy. Traditionally, this is achieved by selective activation of dyes with different excitation lasers followed by selective detec- tion, where the emitted signal is separated based on spectral bands. Unfortunately, excitation and emission spectra are broad and hard to distinguish when growing in number, even with spectral unmixing. When using STED, more con- straints are added because the dyes also must be sufficiently de-excitable at the available STED wavelengths. The use of long Stokes-shift dyes is a remedy; for example, STAR 460L in combination with STAR ORANGE and STAR RED, which allows for easy three-color imaging with a single 775 nm STED laser. But to fully lift the restrictions imposed by over- lapping spectra, all of the fluorophores’ characteristics have to be used (Figure 4C). As an example, the dye combination STAR ORANGE,
STAR 580, STAR RED, and STAR 635 consists of four of the best STED-dyes, yet they cannot be combined using strict spectral separation. Effectively, it would only yield a two-color image, since the spectra of STAR ORANGE and
Figure 3: Optimization of STED microscopy. These are further discussed in the text and subsequent figures by the use of three examples showing structural analy- sis, live-cell dynamics, and deep tissue imaging.
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www.microscopy-today.com • 2022 July
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