研究目的
To achieve the ultimate speed limit of FRET-PAINT microscopy by optimizing camera speed, dissociation rate of DNA probes, and bleed-through of donor signal to the acceptor channel, and to increase imaging speed by 8-fold for obtaining high-quality super-resolution images in tens of seconds.
研究成果
The optimized FRET-PAINT microscope achieves an 8-fold increase in imaging speed, allowing for high-quality 40-nm resolution super-resolution images of microtubules in tens of seconds. Key improvements include faster camera and DNA probe dissociation, reduced bleed-through, and higher signal-to-noise ratio. Future work should address photo-induced DNA damage to enable even faster imaging and broader applications, such as 3D imaging of neural tissues.
研究不足
Photo-induced damage to DNA probes limits further increases in imaging speed and observation time. The damage varies sample-to-sample and is not fully understood, preventing the use of even higher frame rates or probe concentrations. The study used a specific cell type (COS-7) and may not generalize to other samples.
1：Experimental Design and Method Selection:
The study optimized FRET-PAINT microscopy by selecting a high-speed sCMOS camera, shorter DNA donor strands for faster dissociation, and improved fluorophore pairs and filters to reduce background noise and bleed-through. Theoretical models included exponential decay fitting for dissociation times and Fourier ring correlation for resolution analysis.
2：Sample Selection and Data Sources:
COS-7 cells were used as a model system, with microtubules immunostained using anti-tubulin antibodies conjugated to docking DNA strands. DNA oligonucleotides were purchased and labeled with fluorophores.
3：List of Experimental Equipment and Materials:
Equipment includes an sCMOS camera (ORCA-Flash 4.0 V2, Hamamatsu), EMCCD camera (iXon Ultra DU-897 U-CS0-#BV, Andor), inverted microscope (IX71, Olympus), oil-immersion objective (100×1.4 NA, UPlansSApo, Olympus), band-pass filter (ET700/75 m, Chroma), long-pass filter (640 nm), and lasers (473 nm excitation). Materials include DNA strands, fluorophores (AF488, CF488A, Cy5, CF660R), antibodies, and imaging buffers.
4：0 V2, Hamamatsu), EMCCD camera (iXon Ultra DU-897 U-CS0-#BV, Andor), inverted microscope (IX71, Olympus), oil-immersion objective (100×4 NA, UPlansSApo, Olympus), band-pass filter (ET700/75 m, Chroma), long-pass filter (640 nm), and lasers (473 nm excitation). Materials include DNA strands, fluorophores (AF488, CF488A, Cy5, CF660R), antibodies, and imaging buffers. Experimental Procedures and Operational Workflow:
4. Experimental Procedures and Operational Workflow: Cells were cultured, fixed, and immunostained. Single-molecule imaging was performed with optimized parameters: high frame rates (100-200 Hz), short exposure times (5-10 ms), high excitation power (1.5 kW/cm2), and specific DNA probe concentrations. Data was acquired and analyzed using software like ThunderSTORM.
5：5 kW/cm2), and specific DNA probe concentrations. Data was acquired and analyzed using software like ThunderSTORM. Data Analysis Methods:
5. Data Analysis Methods: Dissociation times were fitted with exponential decay functions. Image resolution was assessed using Fourier ring correlation. Localization density and signal-to-noise ratio were calculated from Gaussian fits and statistical analysis.
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EMCCD camera
iXon Ultra DU-897 U-CS0-#BV
Andor
Used for fluorescence imaging in the previous microscope setup, with a maximum frame rate of 56 Hz.
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sCMOS camera
ORCA-Flash 4.0 V2
Hamamatsu
Used for high-speed fluorescence imaging in the new microscope setup, with a maximum frame rate of 400 Hz.
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Inverted microscope
IX71
Olympus
Base microscope for imaging setup.
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Oil-immersion objective
UPlansSApo
Olympus
Used for high-resolution imaging.
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Spectrophotometer
Nanodrop 2000
Thermo Fisher Scientific
Used to measure DNA labeling efficiency.
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Band-pass filter
ET700/75 m
Chroma
Used to filter acceptor signal and reduce bleed-through in the new setup.
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Long-pass filter
640 nm
Used in the previous setup for filtering.
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Alexa Fluor 488
A20000
Invitrogen
Fluorophore used as donor in previous experiments.
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CF488A
92120
Biotium
Fluorophore used as donor in optimized experiments.
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Cy5
PA15101
GE Healthcare
Fluorophore used as acceptor.
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CF660R
92134
Biotium
Fluorophore tested as an alternative acceptor.
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