Multiphoton excitation spectra in biological samples

Area of Science:

• Biophotonics and Imaging
• Cell Biology

Background:

• Multiphoton microscopy offers advantages for live and fixed cell imaging due to nonlinear excitation at the focal plane.
• Ultrafast near-infrared lasers enhance depth penetration and excitation efficiency in multiphoton microscopy.
• Tunable lasers allow for the acquisition of multiphoton excitation spectra.

Purpose of the Study:

• To demonstrate a method for acquiring and analyzing multiphoton excitation spectra from biological samples.
• To utilize excitation fingerprinting for improved fluorochrome separation and autofluorescence elimination.
• To optimize multiphoton microscopy for single and multilabel experiments and study environmental influences.

Main Methods:

• Acquisition of excitation lambda stacks using a software-tunable laser (Coherent Chameleon) and a Zeiss LSM 510 META NLO microscope.
• Analysis of excitation spectra by plotting mean pixel intensity against excitation wavelength.
• Application of linear unmixing algorithms to separate signals from different dyes and elimination of autofluorescence.

Main Results:

• Excitation fingerprinting successfully separated fluorochromes with overlapping emission spectra, outperforming traditional emission fingerprinting.
• The technique effectively eliminated autofluorescence, producing crosstalk-free images.
• Excitation fingerprinting performed with nondescanned detectors (NDDs) provided flexibility for deep-tissue imaging.

Conclusions:

• Excitation fingerprinting is a valuable technique that complements existing multiphoton microscopy capabilities.
• This method enables the optimization of excitation wavelengths for complex multicolor imaging experiments.
• The approach allows for the investigation of how the cellular environment affects nonlinear excitation processes.