Multi-photon excitation microscopy in intact animals

Abstract

Two-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 20x 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.

FIGURE 1

Rabbit TPEFM imaging system: Periscope with an Olympus 20X/0.95 NA XLUMPlanFl water immersion objective.

FIGURE 2

Montage of TPEFM images of mouse TA in vivo exhibiting the effect of respiratory motion. Endogenous blue fluorescence is seen in the mouse TA in vivo. Vasculature and nuclei in the mouse TA is seen as fluorescent voids. An example muscle image montage with motion imaged using 710 nm excitation, 12 bit resolution, 20x magnification with 1.2x zoom and blue emission (emission less than 465 nm) monitored at 512 x 512. Images presented were acquired approximately every 8 seconds and had a scan time of 1.97 seconds. Scale bar of 50 microns is shown.

FIGURE 3

TPEFM image of mouse TA in vivo using a gel optical coupling agent. Endogenous blue fluorescence is seen in the mouse TA in vivo. Vasculature in the mouse TA is seen as fluorescent voids. Imaged using 710 nm excitation, 12 bit resolution, 20x magnification and blue emission (emission less than 465 nm) monitored at 1024 x 1024. Scale bar of 50 microns is shown.

FIGURE 4

B-SHG TPEM images of mouse and rabbit TA in vivo. Both skeletal muscle myosin striations and collagen are seen. (A) B-SHG TPEM images of mouse TA in vivo. Imaged using 900 nm excitation, 8 bit resolution and blue emission (410–490 nm) monitored. Image cropped from a 512 x 512 image with pixel resolution of 0.299 x 0.299 microns. Scale bar of 10 microns is shown. (B) B-SHG TPEM images of rabbit TA in vivo. Imaged using 900 nm excitation, 12 bit resolution, 20x magnification, pixel resolution of 0.45 x 0.45 microns and blue emission (435–485 nm) monitored. Scale bar of 50 microns is shown. (C) Montage of B-SHG TPEM images of rabbit TA in vivo. Images presented in 5 micron steps from inside the tissue at a depth of 205 microns from the tissue surface (labeled zero microns) to the tissue surface (labeled 205 microns). Imaged using 900 nm excitation, 12 bit resolution, 20x magnification, pixel resolution of 0.45 x 0.45 microns and blue emission (435–485 nm) monitored. A median (3 x 3) filter was applied. Scale bar of 50 microns is shown.

FIGURE 4

B-SHG TPEM images of mouse and rabbit TA in vivo. Both skeletal muscle myosin striations and collagen are seen. (A) B-SHG TPEM images of mouse TA in vivo. Imaged using 900 nm excitation, 8 bit resolution and blue emission (410–490 nm) monitored. Image cropped from a 512 x 512 image with pixel resolution of 0.299 x 0.299 microns. Scale bar of 10 microns is shown. (B) B-SHG TPEM images of rabbit TA in vivo. Imaged using 900 nm excitation, 12 bit resolution, 20x magnification, pixel resolution of 0.45 x 0.45 microns and blue emission (435–485 nm) monitored. Scale bar of 50 microns is shown. (C) Montage of B-SHG TPEM images of rabbit TA in vivo. Images presented in 5 micron steps from inside the tissue at a depth of 205 microns from the tissue surface (labeled zero microns) to the tissue surface (labeled 205 microns). Imaged using 900 nm excitation, 12 bit resolution, 20x magnification, pixel resolution of 0.45 x 0.45 microns and blue emission (435–485 nm) monitored. A median (3 x 3) filter was applied. Scale bar of 50 microns is shown.

FIGURE 4

B-SHG TPEM images of mouse and rabbit TA in vivo. Both skeletal muscle myosin striations and collagen are seen. (A) B-SHG TPEM images of mouse TA in vivo. Imaged using 900 nm excitation, 8 bit resolution and blue emission (410–490 nm) monitored. Image cropped from a 512 x 512 image with pixel resolution of 0.299 x 0.299 microns. Scale bar of 10 microns is shown. (B) B-SHG TPEM images of rabbit TA in vivo. Imaged using 900 nm excitation, 12 bit resolution, 20x magnification, pixel resolution of 0.45 x 0.45 microns and blue emission (435–485 nm) monitored. Scale bar of 50 microns is shown. (C) Montage of B-SHG TPEM images of rabbit TA in vivo. Images presented in 5 micron steps from inside the tissue at a depth of 205 microns from the tissue surface (labeled zero microns) to the tissue surface (labeled 205 microns). Imaged using 900 nm excitation, 12 bit resolution, 20x magnification, pixel resolution of 0.45 x 0.45 microns and blue emission (435–485 nm) monitored. A median (3 x 3) filter was applied. Scale bar of 50 microns is shown.

FIGURE 5

TPEFM image of rabbit kidney in vivo using a gel optical coupling agent. Endogenous and exogenous fluorescence is seen in the rabbit kidney in vivo using gel coupled imaging. White cells stained with the nuclear stain SytoGreen24 are visible. The rabbit was maintained on jet ventilation with an imaging ventilation pause. The tissue was imaged using 710 nm excitation, 12 bit resolution, 20x magnification, pixel resolution of 0.90 x 0.90 microns and 515–650 nm emission was monitored at 512 x 512. A dust and scratches noise (radius 1, threshold 21) filter was applied. Scale bar of 50 microns is shown.