Combined intracellular three-dimensional imaging and selective nanosurgery by a nonlinear microscope

Area of Science:

• Cell Biology
• Biophysics
• Microscopy

Background:

• Studying the mechanics of cell division requires precise manipulation of cellular structures.
• Green fluorescent protein (GFP) is a widely used fluorescent marker in cell biology.
• Multiphoton excitation offers advantages for deep tissue imaging and targeted molecular manipulation.

Purpose of the Study:

• To develop and validate a combined microscopy and nanosurgery technique using near-IR femtosecond laser pulses.
• To demonstrate the precise dissection of cellular structures labeled with GFP in living cells.
• To assess the viability and precision of this technique in fission yeast.

Main Methods:

• Utilized near-infrared femtosecond laser pulses for both 3D microscopy and nanosurgery.
• Employed green fluorescent protein (GFP) to label microtubule structures in fission yeast.
• Performed cell cycle analysis and precise microtubule dissection using varying laser powers.

Main Results:

• Achieved 3D reconstructions of GFP-labeled microtubules during different cell cycle phases.
• Successfully dissected microtubules with submicrometer precision using higher laser power.
• Confirmed minimal collateral damage to the fission yeast cells post-nanosurgery.
• Demonstrated that GFP primarily absorbs laser energy, protecting other cellular components.

Conclusions:

• Combined laser microscopy and nanosurgery with GFP is a viable technique for precise cellular manipulation.
• This method enables targeted disruption of specific cellular structures in living cells.
• The technique holds potential as a valuable tool for cell biology research, particularly in studying cell division mechanics.