Imaging T cells in real time is Grizzly

Part of my job is to discuss animal work as it relates to MS.

Some of you aren't interested in the science and it is often years before animal work reaches the clinic.

Imaging cells in real time has provided many insights into biology and is something that just can't be done in humans.

The advent of two photon microscopy has allowed to to be done.

Finsh your Breakfast lunch or Tea(Dinner) before reading this.

Two-photon excitation microscopy is a fluorescence imaging technique that allows imaging of living tissue up to a depth of about one millimeter. Being a special variant of the multiphoton fluorescence microscope, it uses red-shifted excitation light which can also excite fluorescent dyes. However for each excitation, two photons of the infrared light are absorbed. Using infrared light minimizes scattering in the tissue. Due to the multiphoton absorption the background signal is strongly suppressed. Both effects lead to an increased penetration depth for these microscopes. However, the resolution remains diffraction-limited. Two-photon excitation can be a superior alternative to confocal microscopy due to its deeper tissue penetration, efficient light detection and reduced phototoxicity. The concept of two-photon excitation is based on the idea that two photons of comparably lower energy than needed for one photon excitation can also excite a fluorophore in one quantum event. Each photon carries approximately half the energy necessary to excite the molecule. An excitation results in the subsequent emission of a fluorescence photon, typically at a higher energy than either of the two the two excitatory photons.

Haghayegh Jahromi N, Tardent H, Enzmann G, Deutsch U, Kawakami N, Bittner S, Vestweber D, Zipp F, Stein JV, Engelhardt B. A Novel Cervical Spinal Cord Window Preparation Allows for Two-Photon Imaging of T-Cell Interactions with the Cervical Spinal Cord Microvasculature during Experimental Autoimmune Encephalomyelitis. Front Immunol. 2017 Apr 11;8:406. doi: 10.3389/fimmu.2017.00406. eCollection 2017.

T-cell migration across the blood-brain barrier (BBB) is a crucial step in the pathogenesis of experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). Two-photon intravital microscopy (2P-IVM) has been established as a powerful tool to study cell-cell interactions in inflammatory EAE lesions in living animals. In EAE, central nervous system inflammation is strongly pronounced in the spinal cord, an organ in which 2P-IVM imaging is technically very challenging and has been limited to the lumbar spinal cord. Here, we describe a novel spinal cord window preparation allowing to use 2P-IVM to image immune cell interactions with the cervical spinal cord microvascular endothelium during EAE. We describe differences in the angioarchitecture of the cervical spinal cord versus the lumbar spinal cord, which will entail different hemodynamic parameters in these different vascular beds. Using T cells as an example, we demonstrate the suitability of this novel methodology in imaging the post-arrest multistep T-cell extravasation across the cervical spinal cord microvessels. The novel methodology includes an outlook to the analysis of the cellular pathway of T-cell diapedesis across the BBB by establishing visualization of endothelial junctions in this vascular bed.

The images are publically available and shows you what is done.
Gross isn't it.

This grizzy set-up is the stuff of anti-vivisectionist, but the animal is anaesthetised and will never wake-up

This allows you to monitor cells entering the CNS is the living animal and so can tell you how it is done...and how to stop it.