An evaluation of two-photon excitation versus confocal and digital deconvolution fluorescence microscopy imaging in xenopus morphogenesis

From jellyfish to biosensors: the use of fluorescent proteins in plants

“…These features make multiphoton microscopy the technique of choice for experiments where repetitive or prolonged laser exposure are required, such as time lapse and live imaging. Another advantage of multiphoton microscopy is the ability of longer wavelength photons to penetrate deeper into the tissue, beneficial when working with thicker samples (Denk et al, 1990;Potter et al, 1996;Centoze and White, 1998;Periasamy et al, 1999;Rubart, 2004).…”

“…Increasing pinhole size to compensate for low excitation and emission results in lower Z-axis resolution. To obtain 3D pictures, the specimen can be imaged in many different layers of the Z-axis (Z-stack), but as a consequence a longer time is required for image acquisition which can result in photobleaching and photodamage of the subject (Pawley, 1995;Periasamy et al, 1999;Rubart, 2004). Spinning disc confocal microscopy overcomes some of the limitations of confocal microscopy by scanning the entire image simultaneously and collecting fluorescence through numerous pinholes.…”

Determination of Spatial Distributions of Zinc and Active Biomass in Microbial Biofilms by Two-Photon Laser Scanning Microscopy

“…Previous work and this research confirm many advantages of 2P-LSM compared to single-photon CLSM in studies of interfacial microbial ecology, including reduced photobleaching and the ability to analyze thick films (26,45). In single-photon CLSM, quinoline-based fluorescence probes will require UV excitation at 350 to 380 nm, and this wavelength is below the range commonly available in single-photon instruments.…”

“…In addition, since two-photon absorption depends quadratically on laser intensity, absorption occurs only at the focus of the laser, so that the technique provides better spatial resolution for imaging of samples (1,17,45). Thus, in comparison to single-photon CLSM, two-photon excitation allows reduced phototoxicity and photobleaching, improved spatial localization, and deeper sectioning of samples (26,41,45). While applications of 2P-LSM to microbial biofilms are beginning to appear (22,23), to our knowledge, the advantages of 2P-LSM have yet to be applied to analysis of metal distribution in microbial biofilms.…”

Poor man's two photon imaging: Scanning Laser Upconversion Microscopy

“…Any obtained image can be described as the convolution of the imaged object with the PSF of the system. Therefore, it is useful to obtain the PSF of a system as a basis for comparison 17 . To estimate the PSF of the SLUM at different saturation conditions , we have focused an stationary beam on a thin layer of UCNPs, forming an emitting object with negligible size in z-direction and near-Gaussian profile in x and y directions.…”