What is intravital imaging?

Intravital microscopy is a method of imaging tissues or cells in an animal (typically a mouse) while it is still living.  This is done by creating an imaging or vacuum window in an anesthetized animal.  Each vacuum window has been developed and optimized for different organs.  This technique is particularly advantageous because it allows visualization of cellular movement and interactions in the natural environment of a whole organism rather than an artificial culture.  However, imaging in a live animal presents other challenges and limitations, which are not major concerns in explant imaging. 

Our experimental set up for intravital imaging can be found here

Sedin, John et al. (2019) Frontiers in Immunology.  

What is explant imaging?

Explant microscopy is a method of imaging live tissues or cells outside of the host animal.  This is typically done by removing a whole organ from an animal and then sectioning the sample into ~100-500um slices by using a tissue slicer.  The slices are cultured and kept alive during imaging by using a perfusion chamber, which is a small incubation dish that allows control of media added to the sample while imaging.  This technique not only leads to long term imaging of the live sample, but also permits visualization of cellular movement and migration within the original tissue microenvironment.  However, the artificial culture is not completely representative of the conditions in a living body.

Our experimental set up for explant imaging can be found here

PeCon GmbH.


Both intravital and explant imaging techniques primarily use 2-photon microscopy for the best, most consistent results.

What is 2-photon microscopy?

2-photon or multiphoton imaging is a fluorescence microscopy method that can penetrate tissues at greater depths and with less light scatter when compared with other types of traditional fluorescencemicroscopy (i.e. confocal).  This is particularly important when imaging handling live animals, whole organs, and thick tissue slices.

How does 2-photon microscopy work?

In normal fluorescence microscopy, lasers use a single photon in the visible spectrum (400nm-700nm) to excite a desired fluorophore.  The excitation photon has a shorter wavelength (and therefore higher energy) than the wavelength of the emission photon.  Since the wavelength is short and high energy, a single photon is enough to excite the fluorophore to a higher, but less stable, energy state.  Once in the excited state, the fluorophore returns to its original energy state by letting off energy in the form of a photon, which is the emitted light we can measure using detectors such as photomultiplier tubes (PMTs).  The emitted photon is always a slightly longer wavelength and lower energy than the excitation photon because some energy is always lost due to processes such as vibrational relaxation.  The difference between the excitation and emission wavelengths for a fluorophore is called the Stokes shift.

In 2-photon imaging, lasers use two photons in the infrared spectrum (~700nm-1000nm) to excite a desired fluorophore.  The excitation photon has a longer wavelength (and therefore lower energy) than that of the emission photon from the fluorophore.  Since these wavelengths are long and low energy, a single photon is not enough to excite the fluorophore to a higher energy state.  This is because a photon with a wavelength of 800nm has half the amount of energy as a photon with a wavelength of 400nm.  Therefore, the fluorophore must absorb two long wavelengths simultaneously to reach the same higher energy state.  Once there, the fluorophore behaves the same as in single photon microscopy and lets off energy in the form of a photon to drop back down to its more stable, lower energy state.  Like traditional fluorescence microscopy, 2-photon microscopy also uses detectors such as PMTs to measure and quantify these emitted photons.

What are the advantages and disadvantages of 2-photon microscopy?

2-photon microscopy is especially advantageous when imaging live tissues and samples.  But, it can also be a double-edged sword because some of the key features that make 2-photon microscopy so innovative and successful can also leads to serious drawbacks. 

Longer Wavelengths: 

Longer wavelengths have less energy than shorter wavelengths and are therefore less damaging to tissues.  This allows live cells and samples to be viewed for longer periods of time with less phototoxic and photobleaching effects.  Additionally, using infrared lasers instead of lasers in the visible spectrum decreases light scattering within a tissue.  All of these factors allow 2-photon microscopy to penetrate tissues at least 5 times deeper than other fluorescence microscopy techniques such as confocal.  

However, since 2-photon lasers emit photons at a lower energy, the absorption is much less when compared to single photon excitation.  To combat this, the infrared lasers are often used at very high intensities which can damage the tissues and lead to extreme photobleaching of the dyes and samples.  In addition, 2-photon imaging typically produces images at a lower resolution compared with single photon imaging due to the longer wavelengths used.   

Simultaneous Absorption:

The need for simultaneous absorption of two photons helps restrict excitation to the focal plane where the laser and photon density is high enough for the fluorophore.  This helps subdue background signal by limiting off-target fluorescence and leads to a sharper image.  Additionally, simultaneous absorption helps limit tissue damage to the areas surrounding the focal plane which are not being imaged.  

However, since simultaneous absorption is so restricted even at the focal plane, high laser intensities are often used.  This leads to the same issues stated above: severe phototoxicity and photobleaching.

Which microscopes in the BIDC are capable of 2-photon imaging?

Nikon A1R:

The Nikon A1R is an upright 2-photon laser scanning microscope.  Designed for intravital and explant deep tissue imaging, this microscope includes a perfusion system with an in-line stage heater and an anesthesia setup.  The Nikon A1R utilizes one MaiTai Deep See IR laser and an ultra-sensitive gallium arsenide phosphide non-descanned detector (GaAsP NDD) to take sharp images deep within a sample.  The 2-photon microscope is built to accurately image in three channels (Hoechst, FITC, TRITC).  Additionally, the Nikon A1R features a 1K resonant scanner capable of imaging at either an ultrahigh-resolution of up to 4096 x 4096 pixels (15 frames per second) or at an ultrahigh-speed of up to 420 frames per second (512 x 32 pixels).  This hybrid scanner makes it possible to use one microscope in order to visualize clear images with fine details as well as capture quick cell interactions and movements deep within a tissue.  Plus, the ultrahigh-speed scanner images with a short dwell time which limits photobleaching, phototoxicity, and sample damage.  

This instrument uses the NIS-Elements  software.  More information can be found here.

Analyzing images from the Nikon A1R can be performed by using the image analysis software Imaris .  NIS-Elements exports images as .nds files, which can be quickly converted to Imaris files using the Imaris File Converter .  Images and movies are then easily imported into Imaris and ready for further processing.  The BIDC holds two Imaris licenses that are available on our Analysis Stations.  Anyone can use our stations by signing up and booking time on the equipment.  A more detailed work through of image analysis can be found here

A1R Filter Set: 


Generation 3 (“Gen 3”):

The "Gen 3" is the third generation of custom upright 2-photon laser scanning microscopes designed and built by the BIDC.  Constructed for intravital and explant deep tissue imaging, this microscope includes a perfusion system with an in-line stage heater and an anesthesia setup.  The “Gen 3” claims two IR lasers: an 18W Coherent Chameleon Vision II (680nm - 1080nm) and an 8W MaiTai Ti-Saphire (710nm - 920nm).  The instrument features an impressive array of 6 filter sets and 6 H9433MOD photomultiplier tubes (PMTs), which have a high signal to noise ratio.  This set up allows for the simultaneous detection of violet, blue, green, yellow, red, and far red dyes at video rate.  The 6 filter sets are as follows: dichroic 458 (transmitted 417/60), dichroic 490 (transmitted 475/23), dichroic 520 (transmitted 510/42), dichroic 568 (transmitted 542/27), dichroic 650 (transmitted 607/70), and dichroic 725 (transmitted 675/67).  The custom microscope makes it possible to design and image a remarkably large and reliable panel in live tissues.

This instrument is controlled by a specialized Micro-Manager  plugin called µMagellan.  More information about µMagellan can be found in the article here, and the µMagellan user guide can be found here

Similar to the Nikon A1R, analyzing images from “Gen 3” can also be performed by using Imaris .  However, µMagellan exports data as a TIF and must be converted to an Imaris file by using a FIJI  plugin called Imaricumpiler.  Then, the data can be imported into Imaris and analyzed.  For more details, please refer to our Analysis Stations and Image Analysis pages. 

Gen 3 Filter Set:

Gen 3 Microscope Layout: