A white light confocal microscope for spectrally resolved multidimensional imaging.

Area of Science:

• Optical microscopy
• Spectroscopy
• Biophotonics

Background:

• Confocal microscopy is a powerful tool for biological imaging.
• Characterizing fluorophores requires spectrally resolved excitation and emission data.
• Existing systems may have limitations in spectral range or tunability.

Purpose of the Study:

• To develop and demonstrate a spectrofluorometric imaging confocal microscope.
• To enable spectrally resolved fluorescence excitation and detection.
• To provide comprehensive spectral information for each point in a 3D image.

Main Methods:

• Utilized a supercontinuum laser as a broad-spectrum excitation source.
• Integrated a custom-built prism spectrometer for spectrally resolved detection.
• Employed an acousto-optic tunable filter for precise excitation wavelength selection (1-nm bandwidth).

Main Results:

• Achieved spectrally resolved confocal imaging from 450 to 700 nm.
• Demonstrated simultaneous selection of eight excitation wavelengths.
• Obtained fluorescence excitation and emission spectra for each location in 3D images.
• Showcased sensitivity comparable to standard confocal systems.
• Confirmed suitability for live-cell imaging due to scan speed.

Conclusions:

• The developed microscope system provides extensive fluorophore characterization capabilities.
• It offers optimal access to a wide range of fluorophores.
• Modest chromatic aberration effects do not significantly impact spatial resolution.