研究目的
To develop and test a method based on photoswitching properties of donor molecules to measure FRET in cells, offering advantages over existing techniques like FLIM and pbFRET.
研究成果
psFRET is a viable alternative for FRET imaging that mimics advantages of FLIM but is more accessible. It allows repeated measurements and can be used with nonfluorescent acceptors. The method was validated with various constructs and applications, though corrections for cycle-dependent kinetics may be needed for long-term studies.
研究不足
The method requires fitting decay curves, which can be complex. Photoswitching kinetics depend on illumination power, and heterogeneity in illumination or sample environment can affect results. The use of photoswitchable donors limits fluorophore choices, and cycle-dependent changes in Dronpa photoswitching kinetics can introduce artifacts in time-lapse experiments.
1：Experimental Design and Method Selection:
The study designed experiments to test psFRET using photoswitchable fluorescent proteins (e.g., Dronpa as donor) and acceptors (e.g., mCherry or Ultramarine). The method involves monitoring photoswitching kinetics of the donor in the presence and absence of an acceptor to calculate FRET efficiency.
2：Sample Selection and Data Sources:
COS 7 cells were transfected with various tandem dimer constructs of fluorescent proteins (e.g., Dronpa-mCherry with different linkers) and imaged. Control samples included donor alone and acceptor alone.
3：List of Experimental Equipment and Materials:
A homebuilt microscope with specific lasers, objectives, cameras, and image splitters was used. Fluorescent proteins (Dronpa, mCherry, Ultramarine) and cell culture materials (e.g., DMEM-HG medium, FBS) were employed.
4：Experimental Procedures and Operational Workflow:
Cells were imaged under 488-nm excitation to monitor photoswitching off of the donor, with images collected at intervals. Acceptor photobleaching was performed for comparison. Data were analyzed using ImageJ plugins for curve fitting and FRET efficiency calculation.
5：Data Analysis Methods:
Photoswitching decays were fitted to exponential equations to determine rate constants. FRET efficiencies were calculated using derived equations. Sensitized emission analyses and pixel-by-pixel fitting were also conducted.
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Sapphire laser
488 nm
Coherent, Inc.
Excitation source for donor fluorescence and photoswitching.
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Sapphire laser
568 nm
Coherent, Inc.
Excitation source for acceptor fluorescence and photobleaching.
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Dichroic mirror
Di03-R405/488/561/635
Semrock
Direct excitation light to the objective.
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EMCCD camera
897
Andor Technology Corp.
Detection of fluorescence signals.
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Camera
PCO Edge 4.2 LT
PCO AG
Detection of fluorescence signals.
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Power sensor
S170C
Thorlabs, Inc.
Measurement of laser power levels.
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LaserBoxx
405-nm
Oxxius
Excitation source for photoswitching and imaging.
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AOTF
Gooch & Housego PLC
Control of laser lines for excitation.
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Objective lens
100× 1.4 N.A. Plan-Apo
Nikon
Microscopy imaging with high magnification and numerical aperture.
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Objective lens
40× 1.0 N.A. Oil Plan-Apo
Nikon
Microscopy imaging with moderate magnification.
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Microscope
TE2000
Nikon
Platform for fluorescence imaging experiments.
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Dual-View image splitter
Photometrics
Simultaneous collection of green and red emission signals.
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Optical fiber
Andor Technology Corp.
Direction of laser beams to the microscope.
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Cell culture dish
Delta-T
Bioptechs
Cultivation of COS 7 cells for experiments.
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DMEM-HG medium
Invitrogen, Life Technologies
Cell culture medium.
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Glutamax
Invitrogen
Supplement for cell culture medium.
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Sodium pyruvate
Invitrogen
Supplement for cell culture medium.
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FBS
Invitrogen
Serum for cell culture medium.
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Transfection reagent
XtremeGene HP
Roche
Transfection of plasmids into COS 7 cells.
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PCR enzyme
Phusion
New England Biolabs
PCR for plasmid construction.
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