研究目的
To develop a robust and versatile implementation of image scanning microscopy (ISM) that overcomes the limitations of current methods, particularly incompatibility with fluorescence lifetime imaging (FLIM), and enables super-resolution FLIM along with enhanced multicolor, live-cell, and deep imaging.
研究成果
The SPAD array-based ISM implementation provides a robust and versatile platform that achieves super-resolution FLIM, enhances multicolor and live-cell imaging, and allows deep tissue imaging. It overcomes key limitations of existing ISM methods by enabling adaptive reconstruction without calibration and integrating temporal resolution. This paves the way for widespread adoption of ISM in confocal microscopy.
研究不足
The method requires post-processing for image reconstruction, which may not be real-time without preloaded parameters. The SPAD array has a limited number of elements (5x5), which could affect resolution and field of view. Compatibility with very high-speed scanning (e.g., resonant scanners) may be constrained by the dynamic range and data handling capabilities.
1：Experimental Design and Method Selection:
The study involved designing and integrating a novel single-photon avalanche diode (SPAD) array module into both custom and commercial confocal laser-scanning microscopes to enable ISM. Methods included pixel-reassignment (PR) and adaptive PR (APR) for image reconstruction, fluorescence lifetime analysis, and phasor plot calculations.
2：Sample Selection and Data Sources:
Samples included gold beads (80 nm diameter), fluorescent beads (ATTO 647 N, 23 nm), fixed and live cells (e.g., HeLa, HEK, PC-3) stained with various fluorophores (e.g., Abberior STAR red, Alexa Fluor dyes), and cleared mouse brain tissue expressing E-YFP.
3：List of Experimental Equipment and Materials:
Custom and commercial microscopes (Nikon A1R), SPAD array detector (5x5 elements), pulsed diode lasers (LDH-D-C-640 and LDH-D-C-485 from Picoquant), acoustic optical modulators (MT80-A1-VIS from AA Optoelectronic), galvanometric mirrors (6215HM40B from CT Cambridge Technology), objective lenses (e.g., Nikon CFI Plan Apo VC 60x/1.4-NA oil), time-to-digital converter (Time Tagger 20 from Swabian Instruments), and various optical components (e.g., dichroic mirrors, filters from AHF Analysentechnik and Chroma).
4：4-NA oil), time-to-digital converter (Time Tagger 20 from Swabian Instruments), and various optical components (e.g., dichroic mirrors, filters from AHF Analysentechnik and Chroma). Experimental Procedures and Operational Workflow:
4. Experimental Procedures and Operational Workflow: The SPAD array was integrated into the detection arm of microscopes. For ISM, the pinhole was opened, and photons were detected by the array elements. Data acquisition was synchronized with scanning mirrors. For FLIM, time-tagged data were collected and analyzed using exponential fitting or phasor approaches. Image reconstruction was performed using PR or APR methods.
5：Data Analysis Methods:
Data were analyzed using custom software (carma and Matlab) for image reconstruction (PR, APR, deconvolution), fluorescence lifetime fitting (FLIMfit), Fourier ring correlation (FRC) for resolution measurement, and phasor plot generation.
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Pulsed diode laser
LDH-D-C-640
Picoquant
Provides excitation light at 640 nm for fluorescence imaging.
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Pulsed diode laser
LDH-D-C-485
Picoquant
Provides excitation light at 485 nm for fluorescence imaging.
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Dichroic mirror
F43-491
AHF Analysentechnik
Combines or separates light beams based on wavelength.
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Band-pass filter
685/70 nm
AHF Analysentechnik
Filters fluorescence light to specific wavelengths.
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Band-pass filter
525/50 nm
AHF Analysentechnik
Filters fluorescence light to specific wavelengths.
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Aspheric lens
300 mm focal length
Thorlabs
Focuses light in the optical path.
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SPAD array
5x5 elements
Custom designed
Detects single photons for image scanning microscopy, enabling super-resolution and fluorescence lifetime imaging.
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Acoustic optical modulator
MT80-A1-VIS
AA Optoelectronic
Controls the intensity/power of excitation lasers.
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Galvanometric mirrors
6215HM40B
CT Cambridge Technology
Deflects excitation and emission beams for scanning.
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Objective lens
CFI Plan Apo VC 60x/1.4-NA
Nikon
Focuses light onto the sample and collects fluorescence.
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Time-to-digital converter
Time Tagger 20
Swabian Instruments
Measures photon arrival times for fluorescence lifetime imaging.
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Multi-band dichroic mirror
ZT 405-488-594-640-775
Chroma
Reflects or transmits specific wavelengths for multi-color imaging.
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Data acquisition card
NI USB-7856R
National Instruments
Acquires and processes signals from the SPAD array.
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Confocal microscope
Nikon A1R
Nikon
Commercial microscope used for integrating the SPAD array for ISM.
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