When we hear about imaging that allows scientists to see the inner
workings of the brain, we first think about colossal advances in recent years
in functional magnetic resonance imaging (fMRI). But another type of imaging,
inspired by lasers that produce illumination brighter than the surface of the
sun, holds even greater promise—and is expected to figure prominently
in the multi-billion dollar Brain
Research through Advancing Innovative Neurotechnologies (BRAIN) Initiative. In this month’s Cerebrum article, “Imaging the Neural Symphony,” a scientist who has helped pioneer
this new form of technology—two-photon microscopy—writes about the development,
current capabilities, and enormous promise that will permit neuroscientists go
where they have never gone before.
did you meet Winifred Denk, a pioneer of two-photon microscopy?
I met Denk when
I was working on my thesis research on a completely different topic. I used a
tool that he had developed year earlier to measure very, very tiny movements of
biological structures. I used the tool, a kind of interferometer, to measure
the movement of the molecules that produce movement in muscles and in neurons,
so-called molecular motors.
received my Ph.D. I joined Denk and others at Bell Laboratories, among other
things to work on new applications for two-photon microscopy. My lab has been
developing applications for two-photon microscopy ever since.
Since lasers are able to produce a brightness that exceeds
all other light intensities in the known universe, including illumination light
more intense than the surface of the sun, can there be further development of
lasers as it applies to two-photon microscopy?
Yes. We’d like to see lasers, for example, that radiate further
in the infrared region of the spectrum. Their light would penetrate even
further into tissue and you might see deeper and more clearly. The problem is
that water starts to absorb the light and then heats the brain; but there are slight
dips in water absorption at specific wavelengths of the spectrum. We call these
dips “windows of transparency.” And so the race is on to make powerful,
efficient turn-key lasers that operate within these windows of transparency.
Can two-photon microscopy be used to
study neurological conditions?
microscopy is mostly used in model systems. Many scientists in academia and various
companies use two-photon microscopy to study disease progression in model
systems and the response of disease progression to various treatments.
two-photon microscopy in humans would require a different contrast mechanism,
one that did not rely on adding fluorescent substances. Or perhaps fluorescent
substances could be added in a fully non-invasive way. In the future it might
be possible to use viral vectors to deliver fluorescent probes (i.e. gene
therapy), but that’s a very slowly moving field. Also, such an application of
two-photon microscopy would have to be clinically justified.
Does two-photon microscopy have any other
Attempts are underway to use two-photon microscopy in dermatology
and in the diagnosis of muscular disorders.
website includes a list of tools and databases you’ve made available to others,
and you write that the work on the Janelia Research Campus is highly
collaborative. Is there anyone else outside the building that is collaborating
with you to advance the research?
what we do is quite collaborative. In brain research, we don’t have a strong
commercial driver for technology development, which is little bit different
from molecular biology, which directly feeds into diagnostics for cancer and
infectious diseases and where billion-dollar companies fine-tune technology. We
have to collaborate and use our resources efficiently. Folks are fairly open
about what they're doing and in which direction they are working. There is
overlap and competition, but on the whole, it’s a collaborative and
constructive climate. We make an effort to distribute technologies we develop
quickly and without strings attached.
At Janelia we
have a major collaborative effort to develop molecules that report neuronal
function. We do this in a way that is complimentary to what others are doing.
For example, we are trying to develop high-throughput screening, which most university
labs are poorly set up for. The GCaMP6 probes I mention in the article are a
product of this pipeline.
that report neuronal function are getting better and better. In turn we are
getting more ambitious about the number of neurons that we think can image at
the same time in an animal. We started with tens of neurons ten years ago, then
hundreds five years ago. Now we can image tens of thousands of neurons
simultaneously. For that we need microscopes that have larger fields of view to
see multiple parts of the brain simultaneously. We hope to determine how groups
of neurons “collaborate” across the brain. Two or three laboratories in the
country are developing similar types of microscopes, but with slightly
there a role that two-photon microscopy is playing is the Human Connectome
Project (HCP) and the federal BRAIN Initiative?
HPC is really about
analyzing the brain using MRI machines. One voxel in the fanciest MRI machine still
contains approximately 100,000 neurons. So this is a coarse-scale analysis of
the brain. With optical methods we are trying to do things at a different scale,
the single neuron, albeit still in model systems. We want to track activity at
the level of individual neurons and connections between them rather than at the
level of hundreds of thousands of neurons.
The Obama brain
initiative has generated quite a bit off activity through funding. Two-photon
microscopy and novel molecular sensors of activity play a prominent role in the
BRAIN initiative. I would think that one-third of the funded proposals involve
two-photon imaging or related methods as a major component.
might be accomplished with more funding?
analogy for the state of brain research might be cancer research in the 1970s. Nixon
rang in the war on cancer, but this largely failed. Scientists decided that they
needed to take a step back and figure out how the cell cycle works and how
cells control growth, the processes that go wrong in cancer. Fast forward to
the present and we now have quite a few rationally developed medicines for
cancer. Progress has been large and palpable.
Neuroscience has to go through those same steps. We really need to
understand basic processes of neuronal information processing. How do neurons
process information at the level of large ensembles, and what goes wrong with
information processing when neurons get sick. Once we understand these
underlying basic processes and principles, we will be in a much better position
to develop treatments for disorders of the brain.