Caught in the act: intravital multiphoton microscopy of host-pathogen interactions

Abstract

Intravital multiphoton microscopy provides a unique opportunity to discover and characterize biological phenomena in the natural context of living organisms. Here we provide an overview of multiphoton microscopy with particular attention to its application for studying host-pathogen interactions.

Figure 1. One (A) and Two (B) -Photon Excitation of a Fluorophore

(A) During single-photon excitation, a fluorophore or fluorescent protein (FP) is excited by an incoming photon to a higher vibrational energy level (the excited state, depicted by the top black lines). In less than a picosecond, the fluorophore undergoes vibrational relaxation to the lowest-energy excited state and finally emits fluorescence as it is returning to its unexcited ground state. (B) When photon densities are high, two photons can combine energy to cause FP transition to the excited state. Vibrational relaxation and fluorescence emission proceeds as if a single photon had excited the FP. Due to the use of mode-locked lasers, the two photons need to excite the FP typically have identical energies equal to half the energy need for 1P excitation, although any two wavelengths summing to the appropriate energy will result in excitation.

Figure 2. Two-Photon Fluorophore Excitation Is Limited to the Focal Plane

During multiphoton excitation, short, high-intensity pulses are delivered to the tissue or cells being examined. Because two photons must be absorbed within an attosecond to accomplish fluorophore excitation, this is only accomplished within the focal plane. Cells outside of the focal plane are not excited, and a traditional confocal pinhole is eliminated during this mode of microscopy.

Figure 3. Routes of Infection Determine Imaging Location

Vaccinia virus expressing nuclear-localized green fluorescent protein was delivered to mice through intradermal, subcutaneous, or intraperitoneal injection, and the relevant areas were imaged. Vaccinia virus-infected cells are shown in green. The signal from second harmonic generation is shown in blue. Subcutanous injection in the flank results in virally infected cells in the inguinal lymph node or fat pad, while intradermal injection produces skin infection. Intraperitoneal injection results in infection of peritoneal muscle and ovaries.