in vivo imaging

The development of modern ultrafast lasers and transgenic fluorescent proteins provides the means to monitor (sub)cellular events in vivo. The transgenically fluorescent mice developed in 2000 by Sanes & Lichtman have proved invaluable for this type of work. Two-photon microscopy allows us to see quite deeply into the brain at chosen intervals without disturbing it. An example from our own recent work is the image (below) of eGFP-labelled neurons in the neocortex from the Sanes & Lichtman M-line mouse.

Such single images are very striking, but it is repetitive in vivo fluorescent imaging that is potentially much more informative. This method was pioneered in 2001-2 by three labs (Hyman/Bacskai, Svoboda and Gan). This approach is just like taking daily (or weekly) photographs of a plant coming into bloom: time-lapse snap-shots enable you to construct a picture of the build up of a complex system step by step, in real time. We are currently applying this method to murine models of Alzheimer's disease, in order to study the causes and consequences of neurodegeneration during disease progression. Using the window implantation method, developed in Karel Svoboda's lab, we can re-image layer 5 pyramidal neurons in the cortex of a living mouse. We use the H-line mouse in which these neurons are intensely labeled with yellow fluorescent protein:

We also use the thinned skull method developed by Wen-Biao Gan's lab to perform repetitive imaging of the same neurons.

Images A, B, C, and D show re-imaging of 16 synapses (in these examples, boutons) at 2 week intervals in a male H-line mouse. Also apparent are some dendritic segments decorated with spine heads. (Scale bar 20 microns.)

Each approach has strengths and weaknesses. Windows are a major surgery, cause gliois and eventually go cloudy. However clear images can be taken numerous times (between 10-20) over a period of 3-6 months. Thinned skull only allows imaging of layers 1-3 but is less invasive. Typically only four imaging sessions are possible but these can be spaced by more than one year.