Figure 1
Figure 2
Figure 3
Figure 4
100
How does OLS achieve its large field of view (FOV) without sacrificing spatial resolution?
A) By employing a stationary oblique beam that illuminates the sample homogeneously, reducing variability in excitation intensity across the imaging plane.
B) By shaping and focusing a thin optical light sheet in the back focal plane and dynamically scanning it with a galvanometric mirror, ensuring uniform illumination across the FOV.
C) By using a cylindrical lens system to generate a parallelized light sheet that uniformly excites fluorophores while minimizing out-of-focus excitation.
B) By shaping and focusing a thin optical light sheet in the back focal plane and dynamically scanning it with a galvanometric mirror, ensuring uniform illumination across the FOV.
100
The slow moving fraction of PCNA is only observed in cells in which cell-cycle phase?
S phase
100
When comparing TCF4 diffusion in the presence of monovalent vs. bivalent β-catenin, what experimental result validated OLS as a tool for studying protein complex formation?
A) A decrease in diffusion coefficient with increasing β-catenin concentration, with bivalent β-catenin producing a more pronounced effect than monovalent.
B) A sigmoidal relationship between β-catenin concentration and TCF4 diffusion, indicating cooperative binding kinetics.
C) A time-dependent fluorescence decay curve correlating with the transition of TCF4 from a monomeric to oligomeric state.
A) A decrease in diffusion coefficient with increasing β-catenin concentration, with bivalent β-catenin producing a more pronounced effect than monovalent.
100
What two fluorophores were used in Fig 4a?
JF549 and JF646
200
What was the effect of increasing the frame rate in OLS-enabled SMT experiments on KEAP1 diffusion measurements?
A) At higher frame rates, mean trajectory length and linking precision improved, with diffusion estimates stabilizing around 400 Hz, indicating an optimal sampling frequency.
B) Increasing the frame rate led to a significant drop in signal-to-noise ratio (SNR), making it difficult to accurately track fast-diffusing molecules beyond 250 Hz.
C) Higher frame rates caused a decrease in detected diffusion coefficients due to photobleaching effects, limiting the ability to observe long-term molecular trajectories.
A) At higher frame rates, mean trajectory length and linking precision improved, with diffusion estimates stabilizing around 400 Hz, indicating an optimal sampling frequency.
200
What are the three compartments the cells were split into for this analysis?
Nuclear
Perinuclear
Cytoplasmic
200
How does OLS contribute to improving drug discovery applications in protein-protein interaction (PPI) studies?
A) By enabling high-speed tracking of protein complexes in live-cell environments without the need for chemical crosslinking.
B) By allowing real-time quantification of small-molecule inhibitors’ effects on diffusion coefficients, distinguishing bound vs. unbound states.
C) By leveraging structured illumination microscopy to improve spatial resolution, reducing the need for fluorescence-based tracking.
B) By allowing real-time quantification of small-molecule inhibitors’ effects on diffusion coefficients, distinguishing bound vs. unbound states.
200
What is one of the SMLM techniques they were interested in testing with OLS?
Either:
STORM
or
FRAP
300
What key advantage does OLS offer for single-molecule tracking (SMT) compared to HILO?
A) OLS enables deeper tissue penetration by using a collimated beam that minimizes scattering effects, leading to better single-molecule detection.
B) OLS increases the effective imaging field while maintaining signal-to-noise ratio (SNR), leading to significantly more detected trajectories per field of view.
C) OLS reduces photobleaching by limiting fluorophore excitation to a sub-diffraction volume, thereby extending the lifetime of single-molecule signals.
B) OLS increases the effective imaging field while maintaining signal-to-noise ratio (SNR), leading to significantly more detected trajectories per field of view.
300
Knockdown of what effects VCP protein dynamics in ONLY the perinuclear compartment?
FAF2
300
What key feature of OLS enables precise assessment of competitive protein binding interactions?
A) Its ability to capture high-throughput single-molecule trajectories while maintaining a homogeneous signal-to-noise ratio across the full field of view.
B) Its capacity to selectively excite fluorophores based on their molecular conformation, providing real-time structural insights.
C) Its reliance on deep-learning-based tracking algorithms that compensate for photobleaching-induced signal decay.
A) Its ability to capture high-throughput single-molecule trajectories while maintaining a homogeneous signal-to-noise ratio across the full field of view.
300
Figure 4k,l shows that instead of capturing the intensity recovery after photobleaching over time, the authors measured what?
the normalized spot density after recovery