Multiphoton effects have been predicted by the PhD student Maria Goeppert in the twenties of the last century. However, it took more than 30 years to prove her theory. In 1961, one year after the discovery of the laser, Kaiser et al. demonstrated two-photon excited fluorescence. Shortly after the introduction of a first ultrashort laser…
Two-photon excited autofluorescence (AF) is based on the fluorescence of the endogenous intracellular fluorophores NADH and NADPH, flavins, porphyrins and keratin. Furthermore, the pigment melanin is fluorescent. Also the extracelluar matrix proteins elastin and collagen are fluorescent. However, the fluorescence of collagen is weak compared to the SHG signal. Photo: Autofluorescence of human cells recorded…
The Second Harmonic Generation (SHG) signal occurs exactly at half the laser wavelength in forward direction. SHG can be measured in vivo in tissues due to multiple scattering. The major source is collagen. Also muscle tissue exhibits SHG. Photo: SHG signal produced by collagen fibers (red) and AF signal produced by elastin fibers and cells…
Fluorescence lifetime imaging (FLIM) is a technique to visualize the lifetimes of the fluorescent samples in an image by fitting the exponential decay. Each voxel of a FLIM image represents an autofluorescence lifetime corresponding to the excited molecule located in the specified voxel. The lifetimes correlate with the metabolism of the coenzymes. Therefore, alterations of…
Raman spectroscopy provides additional chemical information on tissues such as lipids, water, and proteins. The Raman signal is extremely weak and in vivo Raman imaging not realistic due to long investigation times. However, rapid Raman tissue imaging can be realized when using Coherent Anti-Stokes Raman Scattering (CARS). CARS requires the use of two overlapping NIR…
Optical Metabolic Imaging (OMI) is a technique to gain information from the autofluorescent properties and the fluorescent lifetimes of reduced nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD). Particularly the optical redox ratio, defined by the NADH fluorescence intensity devided by the FAD fluorescence intensity, is calculated and compared with the FLIM images of…
