Second harmonic generation (SHG) is a microscopic technique applicable to a broad spectrum of biological and medical imaging due to its excellent photostability, high signal-to-noise ratio (SNR) and narrow emission profile. Current SHG microscopy techniques rely on two main contrast modalities. These are endogenous SHG generated by tissue structures, which is clinically relevant but cannot be targeted to another location, or SHG nanoprobes, inorganic nanocrystals that can be directed to proteins and cells of interest, but cannot be applied for clinical imaging due to their chemical composition. Here we analyzed SHG signal generated by large-scale peptide assemblies. Our results show the sequence of peptides play an important role on both the morphology and SHG signal of the peptide assemblies. Changing peptide sequence allows confinement of large number of peptides to smaller voxels, generating intense SHG signal. With miniaturization of these peptides and their proper functionalization strategies, such bioinspired nanoparticles would emerge as valuable tools for clinical imaging.
Fluorescence microscopy has profoundly changed how cell and molecular biology is studied in almost
every aspect. However, the need of characterizing biological targets is largely unmet due to deficiencies
associated with the use of fluorescent agents. Dye bleaching, dye signal saturation, blinking, and tissue
autofluorescence can severely limit the signal-to-noise ratio (SNR). Given the photophysical properties are
fundamentally different to the fluorescent agents currently used in biomedical research, second harmonic
generating (SHG) nanoprobes can be suitable for biomedical imaging and can eliminate most of the
drawbacks encountered in classical fluorescence systems.
In biological imaging of fluorescent molecules, multiphoton laser scanning microscopy (MPLSM) has become the favorite method of fluorescence microscopy in tissue explants and living animals. The great power of MPLSM with pulsed lasers in the infrared wavelength lies in its relatively deep optical penetration and reduced ability to cause potential nonspecific phototoxicity. These properties are of crucial importance for long time-lapse imaging. Since the excited area is intrinsically confined to the high-intensity focal volume of the illuminating beam, MPLSM can also be applied as a tool for selectively manipulating fluorophores in a known, three-dimensionally defined volume within the tissue. Here we introduce localized multiphoton photoactivation (MP-PA) as a technique suitable for analyzing the dynamics of photoactivated molecules with three-dimensional spatial resolution of a few micrometers. Short, intense laser light pulses uncage photoactivatable molecules via multiphoton excitation in a defined volume. MP-PA is demonstrated on photoactivatable paGFP in Drosophila wing imaginal discs. This technique is especially useful for extracting quantitative information about the properties of photoactivatable fusion proteins in different cellular locations in living tissue as well as to label single or small patches of cells in tissue to track their subsequent lineage.