While dozens of human ailments are now identified as "protein aggregation diseases", aggregation by itself does not
seem to be a clear determinant of the toxicity. The structural transformation that accompanies the initial steps of
aggregation may be an even more important aspect controlling the biological effects of these protein particles. For this,
the key is to develop appropriate fluorescent biomarkers which can probe both aggregation and conformation at low
physiological concentrations. Using Alzheimer's amyloid beta (Aβ) as a model system, we have developed probes
suitable for the application of Fluorescence Correlation Spectroscopy (FCS, which reports aggregation) and Förster
Resonance Energy Transfer (FRET, which reports conformational changes) techniques. To diagnose these changes in
the cerebrospinal fluid of Alzheimer's patients, we are now designing better single molecule detection devices. Here we
report a confocal device with a 4π collection geometry, which detects more than 0.5 million photons per second from a
single rhodamine B molecule in aqueous solution, which to our knowledge is the highest sensitivity achieved so far
with such devices. This allows us to perform quick and sensitive antibunching measurements which report the
aggregate mass and fluorophore lifetime of Aβ oligomers.
Instruments with single-molecule level detection capabilities can potentially benefit a wide variety of fields, including medical diagnostics. However, the size, cost, and complexity of such devices have prevented their widespread use outside sophisticated research laboratories. Fiber-only devices have recently been suggested as smaller and simpler alternatives, but thus far, they have lacked the resolution and sensitivity of a full-fledged system, and accurate alignment remains a critical requirement. Here we show that through-space reciprocal optical coupling between a fiber and a microscope objective, combined with wavelength division multiplexing in optical fibers, allows a drastic reduction of the size and complexity of such an instrument while retaining its resolution. We demonstrate a 4×4×18 cm3 sized fluorescence correlation spectrometer, which requires no alignment, can analyze kinetics at the single-molecule level, and has an optical resolution similar to that of much larger microscope based devices. The sensitivity can also be similar in principle, though in practice it is limited by the large background fluorescence of the commonly available optical fibers. We propose this as a portable and field deployable single molecule device with practical diagnostic applications.
Coupling three-photon microscopy with automated stage movement can now produce a live high resolution
map of the neurotransmitter serotonin in a single cross section of the whole rat brain. Accurate quantification of these
serotonin images demands appropriate spectral filtering. This requires one to consider that the spectral characteristics of
serotonin show a remarkable variation as it non-covalently associates with different molecules, as we discuss here. Also
it is known that serotonin emission changes when it forms a covalent adduct with para-formaldehyde. This provides a
potential route for producing a whole brain serotonin map using multiphoton microscopy in a fixed rat brain. Here we
take the initial step showing that multiphoton microscopy of this adduct can quantitatively image chemically induced
changes in serotonin distribution.