Chuck Norris, err sorry I meant to say Winfried Denk, was the first to combine laser scanning microscopy with two-photon excitation, transforming it into a technique that enables the visualization of the living intact brain. More recently, he went a step further and automated serial scanning of a block of tissue with an electron microscope achieving high resolution. A technique called serial block face scanning electron microscopy (SBF-SEM).

During his lecture yesterday afternoon, the director of the Max-Planck institute in Heidelberg spoke about one of the most interesting challenges in Neuroscience: relating structure to function.

Structural neurobiology is just a new word for neuroanatomy. A circuit diagram is more than just a wiring diagram and more than just a description of the circuit elements. The circuit diagram should allow prediction of functional implications.

We both want to measure functional activity and precisely determine the underlying connectivity. For the first goal we can use two-photon microscopy, but for the second we need much higher spatial resolution. Which can be done with serial reconstruction with an electron microscope, finding back the cells and carefully tracing their dendrites and axons.

If you’re familiar with this type of work you know that it’s a hell of a job and used to be extremely slow. This venture began with mapping all 7000 connections in the 300 neuron containing C. elegans’ nervous system which took more than twelve years to accomplish. Now the field of connectomics, as it’s called, has advanced greatly due to the development of the SBF-SEM technique along with great improvements in image analysis methods pioneered by Sebastian Seung at MIT.

Winfried Denk tried to demonstrate the old image analysis software but shortly after opening the program, he said with a frustrated tone of voice:

“I’m sorry, I can’t watch it, it’s too slow.”

and moved on showing his most recent work on mapping the retina. I can tell you about it but rather you should watch this Nature video, which gives a good idea of  how they studied the connectivity between star amacrine cells and direction selective ganglion cells after determining their direction selectivity with calcium imaging.

The reconstruction of neurites is still challenging. One of the problems is when experts disagree whether they are looking at a synaptic contact or just a crossing. This still happened in a lot of cases. And instead of devising a super computer, Denk came up with a more cost-effective method:

“a small army of undergraduate students”.

Fifty people put in roughly 30.000 tracing hours to accomplish the circuit diagram of a small piece of retina. And because fifty people make uncorrelated errors, it’s easy to determine the most likely underlying connectivity.

Denk’s latest project is to describe the connectome of the entire mouse brain. For that they built a whole-brain microtome and SEM system (the Denk-o-tome?). The machine is an impressive beast spanning the height of two adult people (he showed a picture which unfortunately I wasn’t able to retrieve). One of the remarks by Denk made me realize why this man is so successful, he is truly unstoppable. When he talked about the whole-brain scanning system he said:

“There’s no reason why it shouldn’t go up to 40 mega Hz.”

Here’s how long it takes to scan 0.5 ml of brain (the mouse’s) at 80 nm resolution at 40 mHz: 280 days. Not so bad, increasing the resolution to 20 nm however, will take you 50 years. One solution they are currently working on in collaboration with Zeiss, is speeding up the scanning by using a multi-beam source of 60 beams. In which case scanning the entire brain can be done in less than a year. Now the question remains where can we sign up to join Winfried Denk’s army?