Multichannel Fluorescence Microscopy: Advantages of Going beyond a Single Emission

Abstract

Fluorescent microscopy has enabled the study of intracellular processes and revealed the most intricate details of the subcellular structure. This has benefitted not only the basic biological science, but also has had an impact in numerous biomedical applications. Basic fluorescent sensing techniques use the change in the absolute emission of a fluorescent sensor. This entails some disadvantages as the signal might be influenced by factors not directly related to the process under study (e.g., fluctuations in the excitation source). To overcome these drawbacks, one can use multiple emissions of a single or various fluorophores. There are numerous examples of multichannel fluorescence microscopy techniques that have given rise to numerous ratiometric methods and multiplexing assays. Herein, how the use of multiple emission channels has impacted fluorescence microscopy in terms of speed, sensitivity, and resolution is reviewed. Using recent examples, how the easy implementation of multichannel detection can overcome current limitations of the main used fluorescence techniques and promote the development of novel microscopy methods is shown.

Single‐Benzene Dual‐Emitters Harness Excited‐State Antiaromaticity for White Light Generation and Fluorescence Imaging

“…Successful imaging of the PFC droplets in live cells prompted us to test whether 7 might be suitable for multicolor imaging with other commercial dyes to track the intracellular processes of PFC droplets [70] . With a short excitation wavelength (350–410 nm) and broad emission with large Stokes shifts (Figure 8a), 7 is spectrally well separated from other commercial dyes that require excitation at visible wavelengths (488–635 nm) [71] .…”

Electrochemical Diselenation of BODIPY Fluorophores for Bioimaging Applications and Sensitization of 1O2

“…Finally, the selenium derivatives exhibited significantly larger Stokes shifts (~2000 cm −1 ) compared to the starting BODIPYs (~500 cm −1 ), previously reported compounds (~400 cm −1 ), [1f,3d,g] and even BODIPY lipid probes, that usually show a strong overlap between the fluorescence and absorption spectra, [20] resulting in a substantial reduction of the inner filter effect. The selenium‐containing BODIPYs showed broad emission compared to reported BODIPYs, which could be used to enable imaging [21a] . In this case, the observed broad emission is likely associated with intrinsic vibronic coupling and structural relaxation of the S 1 state, a phenomenon commonly observed in π‐conjugated organic fluorophores [21b] .…”

Cy5 Dye Cassettes Exhibit Through-Bond Energy Transfer and Enable Ratiometric Fluorescence Sensing

“…As shown in Figure , the mixture of 1B , 2-hydroxyisopropyl β-cyclodextrin, and pyranine acted as a ratiometric fluorescent pH sensor with a near-linear change in output signal over the pH range of 6–8. Looking to future applications, it should be feasible to incorporate the binary mixture of 1B and pyranine within suitable nanoparticles and create robust nanoscale sensing probes for ratiometric diagnostics and multiplex microscopy using a single excitation wavelength. ,,, …”

“…Looking to future applications, it should be feasible to incorporate the binary mixture of 1B and pyranine within suitable nanoparticles and create robust nanoscale sensing probes for ratiometric diagnostics and multiplex microscopy using a single excitation wavelength. 13,14,45,46 ■ CONCLUSIONS Five new Cy5-cassettes were created by directly attaching a pyrene, TPE, or biphenyl(ethynylene) component, as an ultraviolet-absorbing energy donor, to the pentamethine chain of a deep-red Cy5 acceptor. The fluorescent Cy5-cassettes exhibited large pseudo-Stokes shifts and high TBET efficiencies upon excitation with ultraviolet light.…”