Intravital microscopy (IVM) is a newer approach for the imaging of living tissues and organs in live animals. A wide variety of organs can be imaged in rodents–eyes, kidneys, brain, lymph nodes, and gut, to name a few. Imaging the organ at the site (in situ)  provides more physiologically relevant results.

IVM coupled with multiphoton microscopy enables greater penetration of tissues with lower background when compared to confocal microscopy. In brief, the sample is placed on an inverted microscope to enable manipulation of the dissected tissues for exposure of the site of interest. Fluorescent dyes are used to observe and track cells and subcellular structures. Read more for an in depth explanation of IVM.1 In this article, I touch on the intravital imaging of the popliteal lymph node, the lymph node on the hind leg of the mouse. The popliteal lymph node is a good choice for imaging as it is far enough away from the vital organs that imaging instabilities are minimized by the breathing of the mouse.2

Mind the Anesthesia

There are several different anesthetics you can use  and your choice depends on your requirements/conditions of the experiment. Two commonly used ones are a ketamine/xylazine mixture and isoflurane gas.

An advantage of the ketamine mixture is its  ease of use—however, an overdose can cause death. So, it is very important to administer the drug according to the weight of the mouse. Ketamine is better suited for shorter-term imaging (a couple of hours).

Isoflurane is a great choice for longer duration imaging of up to several hours. However, be sure to consider the complications from all the tubing and the gas tank required for the continuous administration of the gas.

Once you have administered the anesthetic, make sure the mouse is deep asleep before making the first cut. To check this, firmly pinch the toes. A mouse that is completely asleep will not respond to the pinch. Furthermore, continuously monitor the mouse and maintain the anesthesia. You do not want the mouse waking up mid-imaging!

Developing Your Surgery Skills

Patience is a virtue here, as you will most probably not get the surgery right (assuming you are not a pro at animal surgeries!) the first, second, or third time. Experts from other labs have told me that a few months of sustained practice is more the norm than the exception. But, once you get the hang of it, you will be much faster at the first cut-to-microscope step.

Steady hands and sharp instruments definitely help! Do not use forceps with even the slightest bent at the tip. Crooked ends can rupture nearby blood vessels while you are gently dissecting out the fat surrounding the lymph node. Also, refer and stick to your approved animal protocol when performing the various steps of the surgery. This will also help to avoid any unpleasant interactions with IACUC.

Control the Temperature

The temperature surrounding the tissue should be at or close to 37oC. T cell motility, for example, is highly temperature-dependent. You can continuously monitor the temperature using a thermocouple probe. In addition, you can make use of  a black imaging chamber to house the microscope. This helps reduce temperature fluctuations  as well as  prevents stray light from getting to the imaging area.

Keep in mind that the objective lens of the microscope acts as a heat sink. It is therefore important to let the whole system stabilize for a few minutes. This will reduce drifts during imaging.

The Microscope is Key for Intravital Microscopy

Due to the thickness of the sample to be imaged (compare taking a movie using an entire organ as the sample versus just a layer of cells in that organ), the microscopy you use  is critical. Multiphoton microscopy is usually the imaging method of choice, which enables imaging at depths greater than 300 um. It involves the use of two excitation photons at longer wavelengths than the emitted light that together excite the sample.

You can use a tunable laser at a range of wavelengths, which enables the imaging of fluorescent probes like DAPI, GFP, and mCherry. Some probes, like mCherry, have two excitation peaks—820 nm and 1010 nm, meaning it shines brightest at two different wavelengths. This duality can be exploited for imaging with another probe, for example CFSE which excites at 820 nm, using a single laser.

Remember the less laser power used for imaging, the better. This ensures the fluorescence doesn’t bleach out in thinner samples.


For a beginner, intravital imaging can be challenging. But the perseverance definitely pays off. For me, it is well worth the effort when I see the beautiful fluorescent image on the computer screen!


  1. Squier, JA and Hoover, EE (2013). Advances in multiphoton microscopy technology Nat Photonics. 7(2):93.
  2. Mempel, TR (2010) Single-cell analysis of cytotoxic T cell function by intravital multiphoton microscopy. Methods Mol Biol. doi: 10.1007/978-1-60761-461-6_12