Multi‐photon excitation microscopy in intact animals

Abstract

SummaryTwo-photon excitation fluorescence microscopy and backscattered-second harmonic generation microscopy permit the investigation of the subcellular events within living animals but numerous aspects of these experiments need to be optimized to overcome the traditional microscope geometry, motion and optical coupling to the subject. This report describes a stable system for supporting a living instrumented mouse or rabbit during endogenous reduced nicotinamide adenine dinucleotide and exogenous dye two-photon excitation fluorescence microscopy measurements, and backscattered-second harmonic generation microscopy measurements. The system was a modified inverted LSM510 microscope (Carl Zeiss, Inc., Thornwood, NY, U.S.A.) with a rotating periscope that converted the inverted scope to an upright format, with the objective located approximately, 15 cm from the centre of the microscope base, allowing easy placement of an instrumented animal. An Olympus 20 × water immersion objective was optically coupled to the tissue, without a cover glass, via a saline bath or custom hydrated transparent gel. The instrumented animals were held on a specially designed holder that poised the animal under the objective as well as permitted different ventilation schemes to minimize motion. Using this approach, quality images were routinely collected in living animals from both the peripheral and body cavity organs. The remaining most significant issue for physiological studies using this approach is motion on the micrometre scale. Several strategies for motion compensation are described and discussed.

Expression of plasmid DNA in the salivary gland epithelium: novel approaches to study dynamic cellular processes in live animals

“…For live imaging, a carbomer-940-based gel (Snowdrift Farm, Tucson, AZ) was used to couple the sample to the objective, as described previously (32). The temperature of the exposed glands was controlled using an objective heater (Bioptechs, Butler, PA), and the body temperature of the animal was maintained by heated pads (Kent Scientific, Torrington, CT), as described previously (23).…”

“…IN THE LAST FIVE YEARS, the development of intravital microscopy has enabled imaging of various cellular and subcellular processes in living animals, allowing, for the first time, dynamic studies in physiological conditions (7,19,23,32,34,43). A breakthrough to this approach that would open the door to new and exciting discoveries would be the ability to transfer or ablate genes in a specific tissue.…”

Contribution of Macromolecular Structure to the Retention of Low-Density Lipoprotein at Arterial Branch Points

“…24 Emission spectra ( Figure I of the online Data Supplement) reveals that at Ͻ840-nm excitation the near-ultraviolet second-harmonic-generation (SHG) signal was suppressed, likely because of the transmission characteristics of the optics and the inner filter properties of the tissues. 25 Thus, an excitation Ͼ840 nm (ie, 420-nm single photon) was required to detect the collagen SHG. The elastin fluorescence emission was found to shift with excitation frequency, suggesting multiple chromophores within elastin.…”

Improved intravital microscopy via synchronization of respiration and holder stabilization

“…1 Tissue movement, however, is unavoidable as it is the result of vital physiological processes such as respiration and cardiovascular activity. Movement arising from respiration is particularly extensive and has effects on each and every organ of the body and every organ of the body such as tibila anterior, 2 lung, 3 kidney, 4 spinal cord, 5 and brain. 6,7 Because the time taken to acquire an image is typically the same as or longer than a breathing cycle (approx.…”

“…400 ms), respiration can have a very detrimental effect on image acquisition, causing severe image distortions and unstable imaging conditions. 2 Faster image acquisition would be one way to overcome this problem, but would likely come at the expense of image quality (high signal-to-noise ratios and/ or reduced image sizes). Recently, considerable research has been directed towards developing strategies to overcome motion artifacts.…”