Extended depth of focus multiphoton microscopy via incoherent pulse splitting

Area of Science:

• Microscopy and Imaging Technologies
• Biophotonics
• Neuroscience Tools

Background:

• Multiphoton microscopy offers optical sectioning capabilities but is limited by a shallow depth of focus.
• Extending the depth of focus is crucial for imaging thicker biological specimens and reducing imaging time.
• Existing methods for depth of focus extension often involve complex setups or compromise image quality.

Purpose of the Study:

• To introduce a novel beam splitter mask for multiphoton microscopes.
• To achieve a significant five-fold extension of the depth of focus.
• To demonstrate the method's compatibility with ultrafast laser sources and multicolor imaging.

Main Methods:

• A beam splitter mask was designed and integrated into a multiphoton raster scanning microscope.
• The point spread function was characterized to evaluate the optical performance.
• Fluorescence imaging was performed on fixed brain samples labeled with Green Fluorescent Protein (GFP).

Main Results:

• The beam splitter mask successfully extended the depth of focus by five times.
• A minor reduction in lateral resolution was observed, deemed acceptable for many applications.
• The method demonstrated high light-throughput and multicolor imaging capabilities.
• Effective fluorescence imaging of GFP-labeled neurons was achieved with the extended depth of focus.

Conclusions:

• The beam splitter mask provides a low-complexity, high-throughput solution for extending the depth of focus in multiphoton microscopy.
• This technique significantly improves the ability to image thick biological samples, such as neural tissues.
• The method is versatile, supporting ultrafast lasers and multicolor imaging, making it broadly applicable in biological research.