研究目的
Investigating the application of multi-angle interference microscopy (MAIM) for multi-color live-cell super-resolution volume imaging to study subcellular dynamics of mitochondria and microtubule architectures during cell migration.
研究成果
MAIM is a powerful super-resolution technique that provides a combination of volume imaging, live-cell imaging, multi-color imaging, a long time series, and low photodamage, suitable for investigating dynamic molecular behaviors proximal to the cell membrane.
研究不足
The technique's imaging speed is dependent on the camera image acquisition time, and practical application requires consideration of dye concentration, quantum efficiency, photostability, and system detection efficiency.
1：Experimental Design and Method Selection:
The study combines total internal reflection fluorescence structured illumination microscopy with multi-angle evanescent light illumination to achieve super-resolution imaging.
2：Sample Selection and Data Sources:
Fixed and live U2OS and U373 cells were used, labeled with various fluorescent dyes for mitochondria and microtubules.
3：List of Experimental Equipment and Materials:
Includes high-NA oil-immersion objective, EMCCD camera, lasers for fluorescence excitation, and various optical elements.
4：Experimental Procedures and Operational Workflow:
Involves acquiring multi-angle and multi-phase raw images, reconstructing lateral super-resolution images, and segmenting interesting sample information for 3D super-resolution reconstruction.
5：Data Analysis Methods:
Utilized custom MATLAB codes for curve fitting, gradient descent, and alternating direction method of multiplier algorithms, alongside ImageJ plugins for data pre-processing and post-processing.
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optical breadboard
B7575L
Thorlabs
Minimize external mechanical vibrations
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laser
Sapphire 488-300 CW CDRH
Coherent
Fluorescence excitation
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laser
Sapphire 561-300 CW CDRH
Coherent
Fluorescence excitation
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laser
Genesis MX 639–1000
Coherent
Fluorescence excitation
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beam collimator
ZC618FC
Thorlabs
Collimate beams
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polarization beam splitter
CCM1-PBS251/M
Thorlabs
Separate parallel beams into two paths with orthogonal polarization directions
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half-wave plate
AQWP05M-600
Thorlabs
Control the intensity ratio of two paths
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scanning lens
SL50-CLS2
Thorlabs
Rectify and focus the light
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piezoelectric stage
753.1
PI
Shift the interference pattern phase
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beam splitter
CCM1-BS013
Thorlabs
Combine two focusing beams
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EMCCD camera
iXon Ultra 888
Andor
Detect the fluorescence emitted by the sample
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AOTF
AOTFnC-400.650-TN
AA Quanta Tech
Control the illumination wavelength and the intensity of the lasers
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single mode polarization maintaining fiber
Oz Optics
Couple beams into a single mode polarization maintaining fiber
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galvanometer set
CTI 8310k
Cambridge Technology
Change the incident angle and rotate the orientation of the interference pattern
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high-NA oil-immersion objective
100 × /1.49 TIRF
Nikon
Focus the light onto the sample
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