Intravital microscopy is a high-resolution imaging technique based on laser-scanning two-photon and confocal microscopy, which can enable a dynamic 3D cellular-level imaging of various biological processes in a living animal
Utilizing a custom-built integrative intravital laser-scanning two-photon and confocal microscopy system, various internal organs and tissues of live mouse model can be visualized at cellular level by detecting fluorescence emitted from individual cells or microenvironment (Fig. 1). The major features of confocal and two-photon microscopy are summarized in Table 1. In both types of microscopy, image is created by observing fluorescence emitted from a fluorophore, which is categorized as fluorescence microscopy. In fluorescence microscopy, only cellular-level objects of interests those are tagged by fluorophore are visible from an otherwise black background, thereby readily achieving high contrast and sensitivity even in
In both types of microscopy, a tightly focused laser beam is scanned in two-dimensional Raster-pattern to achieve high-resolution optically sectioned imaging of live tissue. This tight focus is typically created by using high numerical aperture (NA) objective lens. The higher the NA of objective lens and the shorter the wavelength of light for excitation, the smaller the spot becomes to the diffraction-limit that is calculated to 0.61λ/NA in radius; λ is the wavelength of the light. Although this diffraction-limited spot at the focal plane enables a highly localized fluorescence signal generation needed for a high-resolution imaging, still the excitation of fluorophore occurs non-selectively throughout the converging and diverging beam path at out-of-focal plane. Therefore, in confocal microscopy, a pinhole is placed in front of the detector at a position optically-conjugated to the focal plane and the pinhole aperture is aligned with the focus, which implements a highly effective rejection spatial filter to block the fluorescence from our-of-focus plane. It makes the fluorescence arise from the focus exclusively pass the pinhole aperture to the detector, thereby achieving optical sectioning.
On the other hand, two-photon microscopy achieves optical sectioning based on the nonlinear optical process, two-photon absorption. It is a phenomenon occurs when two seperate photons arrive at the same fluorophore at the same time within ~0.5 fs time difference and are absorbed together to combine their energy to excite the fluorophore. To achieve enough photon flux required for two-photon excitation, mode-locked Ti:Sapphire fs-pulse laser generating ultrashort pulse with 50-200 femtoseconds pulsewidth at about 80 MHz repetition rate is commonly used for two-photon microscopy. In addition, the high photon density sufficient for two-photon absorption is achieved only at the very small volume at the focus. Thereby, the two-photon excited fluorescence is generated only from the focal plane, which facilitates intrinsic sectioning without additional components such as pinhole in confocal microscopy. De-scanning is the back-propagation of the fluorescence through the laser scanner optics. It is required only for confocal microscopy to implement stationary focus at the pinhole optically conjugated with 2D scanning beam inside the sample.
For the high NA objective lens, the working distance defined by the distance between the objective lens and the focus is typically in the range of several hundreds of micrometers to 1 mm, thereby a surgical procedure is normally required to obtain a high-resolution image from internal visceral and thoracic organs. After the access to the organ and tissue surface, the confocal microscopy typically achieves non-invasive imaging depth up to 100-150 μm depending on type of tissue. Soft tissue normally provides more imaging depth than hard tissue. Blood-rich tissue has tendency to limit the imaging depth to less value than other tissue. In comparison, two-photon microscopy can achieve improved imaging depth in various tissue than the confocal microscopy, as it uses longer wavelength at near infrared (NIR) band from 700 nm to 1000 nm where the degree of light scattering limiting the imaging depth is greatly reduced than that in the visible wavelength band from 360 nm to 640 nm used in the confocal microscopy. Additionally, two-photon microscopy can detect second harmonic generation (SHG) signals, which are optical signals generated when two photons of one wavelength are converted to a single photon of half the wavelength. As this process is purely optically-driven process, it does not require exogenous fluorescence labeling and enabled label-free imaging. Currently, SHG imaging is widely used to visualize collagen in the biological tissue.
During last decade, intravital microscopic imaging of various internal organs including liver,5-7 spleen8 , pancreas9 , kidney10 , small intestine,11,12 colon,13,14 lung,15,16 heart,17 prostate,18 mesentery19 and brain20 was achieved (Fig. 1). Fig. 2A shows the preparation of live mouse for the intravital imaging of liver.5-7 After anesthetization, small incision was made on skin and peritoneum to expose the left lobe of liver. The cover glass attached with a customized heater and commercial thermometer was positioned to gently press the exposed left lobe to reduce the liver movement. Additionally, a commercial homeothermic system composed of rectal probe for body temperature monitoring and feedback-controlled heating pad was used to maintain the body temperature of the anesthetized at 36°C during the intravital imaging. Fig. 2B shows the microscopic-scale images obtained from the liver prepared for intravital imaging. Nucleus of individual hepatocyte and hepatic sinusoid was clearly visualized. The most distinct advantage of intravital microscopy is its capability of providing longitudinal view of disease progression in cellular-level with repeated intravital imaging of single animal over time by saving the animal after each imaging session. Fig. 2C, D shows the intravital imaging of the liver showing the progression of nonalcoholic hepatic steatosis in the nonalcoholic fatty liver disease (NAFLD) mouse model induced by feeding a methionine and choline-deficient (MCD) diet over 3 weeks.21,22 In each image session, liver sinusoid endothelial cells were labeled
To conclude, intravital imaging and