Exploration of the two-photon excitation spectrum of fluorescent dyes at wavelengths below the range of the Ti:Sapphire laser

We have studied the wavelength dependence of the two-photon excitation efficiency for a number of common UV excitable fluorescent dyes; the nuclear stains DAPI, Hoechst and SYTOX Green, chitin- and cellulose-staining dye Calcofluor White and Alexa Fluor 350, in the visible and near-infrared wavelength range (540-800 nm). For several of the dyes, we observe a substantial increase in the fluorescence emission intensity for shorter excitation wavelengths than the 680 nm which is the shortest wavelength usually available for two-photon microscopy. We also find that although the rate of photo-bleaching increases at shorter wavelengths, it is still possible to acquire many images with higher fluorescence intensity. This is particularly useful for applications where the aim is to image the structure, rather than monitoring changes in emission intensity over extended periods of time. We measure the excitation spectrum when the dyes are used to stain biological specimens to get a more accurate representation of the spectrum of the dye in a cell environment as compared to solution-based measurements. Layman's abstract UV absorbing dyes and fluorophores are important for bio-imaging, encompassing dyes for imaging calcium signalling in cells, nuclear stains to image the cell nucleus, which can be used as a marker to find the cell in a microscope, dyes that only show up in cells with metabolic activity, indicating that the cell is still alive and functioning, endogenous fluorophores, such as the amino acid tryptophan, allowing label free imaging of cells and tissue, as well as the neurotransmitter serotonin. The UV radiation can, however, potentially damage cells and tissue. A more gentle way of exciting the dye is two photon excitation, where two photons of half the energy are simultaneously absorbed, their combined energy being enough to excite the dye. The lower energy of the light would cause less damage to the cell. The likeliness of this process happening, the cross section, is, however, low. In order to get a sizable signal, one uses lasers with short, 100 femtoseconds long intense pulses of light, so that many photons arrive at the same time, although the average power is low enough to not damage the sample. There are a limited number of such lasers available, and the most commonly used is the Ti:Sapphire laser. This laser only emits wavelengths longer than 680 nm, which is suitable for dyes excitable in the visible, but half of 680 nm does not get you very far out in the UV. In this paper we use an add-on to this laser called an Optical Parametric Oscillator (OPO) which can emit such short pulses in the visible wavelength range, resulting in two photon excitation deep in the UV, without harmful radiation being applied to the specimen. We investigate what the optimum two-photon excitation wavelength is for a number of biologically relevant dyes. We investigate these dyes when used as stains in biological samples, i.e. in the cell environment they are intended to be used in, to get the most representative information about the optimum excitation wavelength.