Two-photon imaging within the murine thorax without respiratory and cardiac motion artifact

Abstract

Intravital microscopy has been recognized for its ability to make physiological measurements at cellular and subcellular levels while maintaining the complex natural microenvironment. Two-photon microscopy (TPM), using longer wavelengths than single-photon excitation, has extended intravital imaging deeper into tissues, with minimal phototoxicity. However, due to a relatively slow acquisition rate, TPM is especially sensitive to motion artifact, which presents a challenge when imaging tissues subject to respiratory and cardiac movement. Thoracoabdominal organs that cannot be exteriorized or immobilized during TPM have generally required the use of isolated, pump-perfused preparations. However, this approach entails significant alteration of normal physiology, such as a lack of neural inputs, increased vascular resistance, and leukocyte activation. We adapted techniques of intravital microscopy that permitted TPM of organs maintained within the thoracoabdominal cavity of living, breathing rats or mice. We obtained extended intravital TPM imaging of the intact lung, arguably the organ most susceptible to both respiratory and cardiac motion. Intravital TPM detected the development of lung microvascular endothelial activation manifested as increased leukocyte adhesion and plasma extravasation in response to oxidative stress inducers PMA or soluble cigarette smoke extract. The pulmonary microvasculature and alveoli in the intact animal were imaged with comparable detail and fidelity to those in pump-perfused animals, opening the possibility for TPM of other thoracoabdominal organs under physiological and pathophysiological conditions.

Figure 1

Representative frames of time-series TPM images in the intact rat showing FITC-labeled (green) pulmonary microvasculature in low magnification ×20 (A) and high magnification ×60 (B–D). Note detailed microvasculature morphology of medium- and small-sized arterioles (white arrowheads, A), venules (white bidirectional arrows, C and D), and capillaries (yellow arrows) surrounding normal alveolar airspaces. Nuclei are stained with intravenous Hoechst (blue), and circulating cells appear as black streaks within the vessels. Scale bars: 75 μm (A) and 25 μm (B–D).

Figure 2

Three-dimensional reconstruction of FITC-labeled microvasculature (green) surrounding normal alveolar airspaces (dark regions) imaged in intact preparations (A and C) are comparable to pump-perfused preparations (B and D). Nuclei are stained blue with intravenous Hoechst. Scale bars = 25 μm. Note that the capillary circulation (A–D) and larger venules (C and D) are visualized by TPM with similar detail in the two preparations.

Figure 3

Gated imaging eliminates motion for maximum clarity in three-dimensional TPM reconstructions. A: Comparison between ungated (left) and gated (right) image acquisition of an identical field of view showing FITC-labeled (green) alveolar microvasculature in the intact rat. Reconstructions in the x-z orientation correspond to the indicated slice regions (B–E) from the 3 dimensional images. Nuclei are stained with intravenous Hoechst (blue). Scale bars = 25 μm.

Figure 4

Capturing real-time development of pulmonary edema using TPM of the lung microvasculature in the intact rat. Three-dimensional reconstruction of FITC-labeled vessels (green) surrounding alveoli without (A) and with Rho-6G–labeled leukocytes (red, B–F) imaged before (A and B) and after (C–F) intravenous administration of PMA (1 mmol/L, 0.10 mg/kg). Nuclei are stained with intravenously administered Hoechst (blue). Note increasing leukocyte sequestration in the capillaries at 5 minutes (C; arrow) and plasma extravasation (asterisks) into airspaces at 9 minutes (D), 12 minutes (E), and 30 minutes (D) post-PMA administration. D and E depict an identical field of view. Scale bars = 25 μm. Representative image of n = 3 animals. G: Quantification of leukocyte adherence in vivo in response to PMA (1 mmol/L in dimethylformamine, 100 μL/kg, i.v; 5 to 20 minutes) or soluble cigarette smoke extract (100%, 2 mL/kg i.v; 20 to 30 minutes) administration compared to untreated animals (n = 2 rats). Mean ± SD; *P < 0.001 versus control; n = 5 to 19 time-lapse movies.