Varied Length Stokes Shift BODIPY-Based Fluorophores for Multicolor Microscopy

Abstract

Multicolor microscopy tools necessary to localize and visualize the complexity of subcellular systems are limited by current fluorophore technology. While commercial fluorophores cover spectral space from the ultraviolet to the near infrared region and are optimized for conventional bandpass based fluorescence microscopy, they are not ideal for highly multiplexed fluorescence microscopy as they tend to have short Stokes shifts, restricting the number of fluorophores that can be detected in a single sample to four to five. Herein, we synthesized a library of 95 novel boron-dipyrromethene (BODIPY)-based fluorophores and screened their photophysical, optical and spectral properties for their utility in multicolor microscopy. A subset of our BODIPY-based fluorophores yielded varied length Stokes shifts probes, which were used to create a five-color image using a single excitation with confocal laser scanning microscopy for the first time. Combining these novel fluorophores with conventional fluorophores could facilitate imaging in up to nine to ten colors using linear unmixing based microscopy approaches.

Superresolution microscopy with novel BODIPY-based fluorophores

“… The maximum absorbance wavelength vs. maximum emission wavelength for conventional fluorophores excited using a standard Cy3 imaging channel/561 nm laser line ( x ) and Cy5 imaging channel/647 nm laser line ( + ) result in a gap in spectral emission space from 615–650 nm. This spectral space can be filled using novel BAA fluorophores with long Stokes shifts (○) [ 13 ]. Four additional BAA fluorophores with short Stokes shifts (●) were also evaluated for their SMLM imaging utility.…”

“…There are currently limited commercially available fluorophores with optimal spectral and photoswitching properties for multicolor SMLM imaging, as most commercially available photoswitchable fluorophores all have relatively short Stokes shifts (<30 nm), with an average of ~20 nm [ 11 , 12 ]. Using fluorophores with short Stokes shifts in the conventional Cy3 and Cy5 imaging channels results in a lack of fluorophores that emit between 615–650 nm [ 2 , 4 , 10 , 11 , 13 – 17 ]. Filling this gap in spectral emission space with novel photoswitchable fluorophores would enhance multicolor SMLM imaging.…”

“…Understanding of optimal photoswitching buffers for BODIPY-based fluorophores could substantially improve the SMLM color palette as BODIPY-based fluorophores have the potential to provide a variety of short and long Stokes shift probes. Commercially available BODIPY fluorophores emit at various wavelengths throughout the visible range with short Stokes shifts [ 25 ], and recently a BODIPY-based fluorophore library was synthesized containing fluorophores with Stokes shifts of varying length that are optimally excited using a 561-nm laser [ 13 ], providing the opportunity to fill the spectral void in the currently available photoswitchable probes and expand the number of fluorophore options available for multicolor SMLM.…”

“…SMLM imaging of immunolabeled microtubules confirmed the optimal imaging buffer conditions for BODIPY FL, which also resulted in high quality SMLM immunolabeled microtubule images using AF647. Six novel BODIPY-based fluorophores with long Stokes shifts (>30 nm) and four novel BODIPY-based fluorophores with short Stokes shifts (<30 nm) [ 13 ] were selected for further evaluation of photoswitching and SMLM image quality. Photoswitching properties of our novel BODIPY fluorophores were compared to Alexa Fluor ™ 568 (AF568), a widely used photoswitchable fluorophore imaged in the standard Cy3 channel [ 10 , 16 ].…”

Light-responsive nanomedicine for biophotonic imaging and targeted therapy

“…Fluorophores are representative light-responsive materials, which absorb light energy and emit photons with particular wavelengths [3,39]. By simple loading or conjugating dyes to the targeting domain, such as small molecule ligands, peptides, proteins, antibodies, and polymers, many imaging nanoprobes have been developed and applied in bioimaging and therapy [7,9,40–46].…”