Subtle Structural Translation Magically Modulates the Super-Resolution Imaging of Self-Blinking Rhodamines

The evolution of super-resolution imaging techniques is benefited from the ongoing competition for optimal rhodamine fluorophores. Yet, it seems blind to construct the desired rhodamine molecule matching the imaging need without the knowledge on imaging impact of even the minimum structural translation. Herein, we have designed a pair of self-blinking sulforhodamines (STMR and SRhB) with the bare distinction of methyl or ethyl substituents and engineered them with Halo protein ligands. Although the two possess similar spectral properties (λab, λfl, ϕ, etc.), they demonstrated unique single-molecule characteristics preferring to individual imaging applications. Experimentally, STMR with high emissive rates was qualified for imaging structures with rapid dynamics (endoplasmic reticulum, and mitochondria), and SRhB with prolonged on-times and photostability was suited for relatively “static” nuclei and microtubules. Using this new knowledge, the mitochondrial morphology during apoptosis and ferroptosis was first super-resolved by STMR. Our study highlights the significance of even the smallest structural modification to the modulation of super-resolution imaging performance and would provide insights for future fluorophore design

Surpassing the Background Barrier for Multidimensional Single-Molecule Localization Super-Resolution Imaging: A Case of Lysosome-Exclusively Turn-on Probe

The background barrier restricts the dimensionality of live-cell single-molecule localization super-resolution imaging. Ideally, a probe exclusively turned on by its target, without any nonspecific fluorescence signals from off-target molecules, constitutes a practical solution to surpass this barrier. Yet, few such fluorophores have been developed. A lysosome with a unique acidic lumen was chosen as the target for demonstrating the concept advantage. A representative lyso-tracker Lyso-R (piperazine rhodamine) with high brightness has been spirocyclized with o-phenylenediamine to form Lyso-Ropa. This probe shifted its bright-dark spirocyclization balance to a strong acidity domain (pKa = −0.18). Consequently, under no-wash conditions, Lyso-Ropa showed almost undetectable background photons (only one-sixtieth of that of Lyso-R) in a neutral cellular environment, and it formed sparsely brightened molecules at a low ratio (∼1 × 10–3%) in lysosomes. This background-free probe enabled super-resolution imaging and modeling of live-cell lysosomes in four dimensions at 2 s resolution, with quantitative determination of lysosomal volume expansion and deformation at starvation. Our molecular approach sheds new light on surpassing the background barrier for multidimensional super-resolution imaging

Surpassing the Background Barrier for Multidimensional Single-Molecule Localization Super-Resolution Imaging: A Case of Lysosome-Exclusively Turn-on Probe

The background barrier restricts the dimensionality of live-cell single-molecule localization super-resolution imaging. Ideally, a probe exclusively turned on by its target, without any nonspecific fluorescence signals from off-target molecules, constitutes a practical solution to surpass this barrier. Yet, few such fluorophores have been developed. A lysosome with a unique acidic lumen was chosen as the target for demonstrating the concept advantage. A representative lyso-tracker Lyso-R (piperazine rhodamine) with high brightness has been spirocyclized with o-phenylenediamine to form Lyso-Ropa. This probe shifted its bright-dark spirocyclization balance to a strong acidity domain (pKa = −0.18). Consequently, under no-wash conditions, Lyso-Ropa showed almost undetectable background photons (only one-sixtieth of that of Lyso-R) in a neutral cellular environment, and it formed sparsely brightened molecules at a low ratio (∼1 × 10–3%) in lysosomes. This background-free probe enabled super-resolution imaging and modeling of live-cell lysosomes in four dimensions at 2 s resolution, with quantitative determination of lysosomal volume expansion and deformation at starvation. Our molecular approach sheds new light on surpassing the background barrier for multidimensional super-resolution imaging

Surpassing the Background Barrier for Multidimensional Single-Molecule Localization Super-Resolution Imaging: A Case of Lysosome-Exclusively Turn-on Probe

The background barrier restricts the dimensionality of live-cell single-molecule localization super-resolution imaging. Ideally, a probe exclusively turned on by its target, without any nonspecific fluorescence signals from off-target molecules, constitutes a practical solution to surpass this barrier. Yet, few such fluorophores have been developed. A lysosome with a unique acidic lumen was chosen as the target for demonstrating the concept advantage. A representative lyso-tracker Lyso-R (piperazine rhodamine) with high brightness has been spirocyclized with o-phenylenediamine to form Lyso-Ropa. This probe shifted its bright-dark spirocyclization balance to a strong acidity domain (pKa = −0.18). Consequently, under no-wash conditions, Lyso-Ropa showed almost undetectable background photons (only one-sixtieth of that of Lyso-R) in a neutral cellular environment, and it formed sparsely brightened molecules at a low ratio (∼1 × 10–3%) in lysosomes. This background-free probe enabled super-resolution imaging and modeling of live-cell lysosomes in four dimensions at 2 s resolution, with quantitative determination of lysosomal volume expansion and deformation at starvation. Our molecular approach sheds new light on surpassing the background barrier for multidimensional super-resolution imaging

Surpassing the Background Barrier for Multidimensional Single-Molecule Localization Super-Resolution Imaging: A Case of Lysosome-Exclusively Turn-on Probe

The background barrier restricts the dimensionality of live-cell single-molecule localization super-resolution imaging. Ideally, a probe exclusively turned on by its target, without any nonspecific fluorescence signals from off-target molecules, constitutes a practical solution to surpass this barrier. Yet, few such fluorophores have been developed. A lysosome with a unique acidic lumen was chosen as the target for demonstrating the concept advantage. A representative lyso-tracker Lyso-R (piperazine rhodamine) with high brightness has been spirocyclized with o-phenylenediamine to form Lyso-Ropa. This probe shifted its bright-dark spirocyclization balance to a strong acidity domain (pKa = −0.18). Consequently, under no-wash conditions, Lyso-Ropa showed almost undetectable background photons (only one-sixtieth of that of Lyso-R) in a neutral cellular environment, and it formed sparsely brightened molecules at a low ratio (∼1 × 10–3%) in lysosomes. This background-free probe enabled super-resolution imaging and modeling of live-cell lysosomes in four dimensions at 2 s resolution, with quantitative determination of lysosomal volume expansion and deformation at starvation. Our molecular approach sheds new light on surpassing the background barrier for multidimensional super-resolution imaging

Surpassing the Background Barrier for Multidimensional Single-Molecule Localization Super-Resolution Imaging: A Case of Lysosome-Exclusively Turn-on Probe

The background barrier restricts the dimensionality of live-cell single-molecule localization super-resolution imaging. Ideally, a probe exclusively turned on by its target, without any nonspecific fluorescence signals from off-target molecules, constitutes a practical solution to surpass this barrier. Yet, few such fluorophores have been developed. A lysosome with a unique acidic lumen was chosen as the target for demonstrating the concept advantage. A representative lyso-tracker Lyso-R (piperazine rhodamine) with high brightness has been spirocyclized with o-phenylenediamine to form Lyso-Ropa. This probe shifted its bright-dark spirocyclization balance to a strong acidity domain (pKa = −0.18). Consequently, under no-wash conditions, Lyso-Ropa showed almost undetectable background photons (only one-sixtieth of that of Lyso-R) in a neutral cellular environment, and it formed sparsely brightened molecules at a low ratio (∼1 × 10–3%) in lysosomes. This background-free probe enabled super-resolution imaging and modeling of live-cell lysosomes in four dimensions at 2 s resolution, with quantitative determination of lysosomal volume expansion and deformation at starvation. Our molecular approach sheds new light on surpassing the background barrier for multidimensional super-resolution imaging

Surpassing the Background Barrier for Multidimensional Single-Molecule Localization Super-Resolution Imaging: A Case of Lysosome-Exclusively Turn-on Probe

The background barrier restricts the dimensionality of live-cell single-molecule localization super-resolution imaging. Ideally, a probe exclusively turned on by its target, without any nonspecific fluorescence signals from off-target molecules, constitutes a practical solution to surpass this barrier. Yet, few such fluorophores have been developed. A lysosome with a unique acidic lumen was chosen as the target for demonstrating the concept advantage. A representative lyso-tracker Lyso-R (piperazine rhodamine) with high brightness has been spirocyclized with o-phenylenediamine to form Lyso-Ropa. This probe shifted its bright-dark spirocyclization balance to a strong acidity domain (pKa = −0.18). Consequently, under no-wash conditions, Lyso-Ropa showed almost undetectable background photons (only one-sixtieth of that of Lyso-R) in a neutral cellular environment, and it formed sparsely brightened molecules at a low ratio (∼1 × 10–3%) in lysosomes. This background-free probe enabled super-resolution imaging and modeling of live-cell lysosomes in four dimensions at 2 s resolution, with quantitative determination of lysosomal volume expansion and deformation at starvation. Our molecular approach sheds new light on surpassing the background barrier for multidimensional super-resolution imaging

Surpassing the Background Barrier for Multidimensional Single-Molecule Localization Super-Resolution Imaging: A Case of Lysosome-Exclusively Turn-on Probe

The background barrier restricts the dimensionality of live-cell single-molecule localization super-resolution imaging. Ideally, a probe exclusively turned on by its target, without any nonspecific fluorescence signals from off-target molecules, constitutes a practical solution to surpass this barrier. Yet, few such fluorophores have been developed. A lysosome with a unique acidic lumen was chosen as the target for demonstrating the concept advantage. A representative lyso-tracker Lyso-R (piperazine rhodamine) with high brightness has been spirocyclized with o-phenylenediamine to form Lyso-Ropa. This probe shifted its bright-dark spirocyclization balance to a strong acidity domain (pKa = −0.18). Consequently, under no-wash conditions, Lyso-Ropa showed almost undetectable background photons (only one-sixtieth of that of Lyso-R) in a neutral cellular environment, and it formed sparsely brightened molecules at a low ratio (∼1 × 10–3%) in lysosomes. This background-free probe enabled super-resolution imaging and modeling of live-cell lysosomes in four dimensions at 2 s resolution, with quantitative determination of lysosomal volume expansion and deformation at starvation. Our molecular approach sheds new light on surpassing the background barrier for multidimensional super-resolution imaging

Recruiting Rate Determines the Blinking Propensity of Rhodamine Fluorophores for Super-Resolution Imaging

Live-cell single-molecule localization microscopy has advanced with the development of self-blinking rhodamines. A pKcycling of <6 is recognized as the criterion for self-blinking, yet a few rhodamines matching the standard fail for super-resolution reconstruction. To resolve this controversy, we constructed two classic rhodamines (pKcycling < 6) and four sulfonamide rhodamines with three exhibited exceptional larger pKcycling characteristics (6.91–7.34). A kinetic study uncovered slow equilibrium rates, and limited switch numbers resulted in the reconstruction failure of some rhodamines. From the kinetic disparity, a recruiting rate was first abstracted to reveal the natural switching frequency of spirocycling equilibrium. The new parameter independent from applying a laser satisfactorily explained the imaging failure, efficacious for determining the propensity of self-blinking from a kinetic perspective. Following the prediction from this parameter, the sulfonamide rhodamines enabled live-cell super-resolution imaging of various organelles through Halo-tag technology. It is determined that the recruiting rate would be a practical indicator of self-blinking and imaging performance

MOESM3 of A low-cost and open-source platform for automated imaging

Additional file 3. Three additional views of the HyperScanner