Two-Color STED Microscopy of Living Synapses Using A Single Laser-Beam Pair

The advent of superresolution microscopy has opened up new research opportunities into dynamic processes at the nanoscale inside living biological specimens. This is particularly true for synapses, which are very small, highly dynamic, and embedded in brain tissue. Stimulated emission depletion (STED) microscopy, a recently developed laser-scanning technique, has been shown to be well suited for imaging living synapses in brain slices using yellow fluorescent protein as a single label. However, it would be highly desirable to be able to image presynaptic boutons and postsynaptic spines, which together form synapses, using two different fluorophores. As STED microscopy uses separate laser beams for fluorescence excitation and quenching, incorporation of multicolor imaging for STED is more difficult than for conventional light microscopy. Although two-color schemes exist for STED microscopy, these approaches have several drawbacks due to their complexity, cost, and incompatibility with common labeling strategies and fluorophores. Therefore, we set out to develop a straightforward method for two-color STED microscopy that permits the use of popular green-yellow fluorescent labels such as green fluorescent protein, yellow fluorescent protein, Alexa Fluor 488, and calcein green. Our new (to our knowledge) method is based on a single-excitation/STED laser-beam pair to simultaneously excite and quench pairs of these fluorophores, whose signals can be separated by spectral detection and linear unmixing. We illustrate the potential of this approach by two-color superresolution time-lapse imaging of axonal boutons and dendritic spines in living organotypic brain slices

Chronically CNS-Injured Adult Sensory Neurons Gain Regenerative Competence upon a Lesion of Their Peripheral Axon

Several experimental manipulations result in axonal regeneration in the central nervous system (CNS) when applied before or at the time of injury [1-6] but not when initiated after a delay [5-10], which would be clinically more relevant. As centrally injured neurons show signs of atrophy and degeneration [11-13], it raises the question whether chronically injured neurons are able to regenerate. To address this question, we used adult rodent primary sensory neurons that regenerate their central axon when their peripheral axon is cut (called conditioning) beforehand but not afterwards. We found that primary sensory neurons express regeneration-associated genes and efficiently regrow their axon in cell culture two months after a central lesion upon conditioning. Moreover, conditioning enables central axons to regenerate through a fresh lesion independent of a previous central lesion. Using in vivo imaging we demonstrated that conditioned neurons rapidly regrow their axons through a fresh central lesion. Finally, when single sensory axons were cut with a two-photon laser, they robustly regenerate within days after attaining growth competence through conditioning. We conclude that sensory neurons can acquire the intrinsic potential to regenerate their axons months after a CNS lesion, which they implement in the absence of traumatic tissue

Detecting fluorescent protein expression and co-localisation on single secretory vesicles with linear spectral unmixing

Many questions in cell biology and biophysics involve the quantitation of co-localisation and the interaction of proteins tagged with different fluorophores. However, the incomplete separation of the different colour channels due to the presence of autofluorescence, along with cross-excitation and emission "bleed-through" of one colour channel into the other, all combine to render the interpretation of multi-band images ambiguous. Here we introduce a new live-cell epifluorescence spectral imaging and linear unmixing technique for classifying resolution-limited point objects containing multiple fluorophores. We demonstrate the performance of our technique by detecting, at the single-vesicle level, the co-expression of the vesicle-associated membrane protein, VAMP-2 (also called synaptobrevin-2), linked to either enhanced green fluorescent protein (EGFP) or citrine [a less pH-sensitive variant of enhanced yellow fluorescent protein (EYFP)], in mouse cortical astrocytes. In contrast, the co-expression of VAMP-2-citrine and the lysosomal transporter sialine fused to EGFP resulted in little overlap. Spectral imaging and linear unmixing permit us to fingerprint the expression of spectrally overlapping fluorescent proteins on single secretory organelles in the presence of a spectrally broad autofluorescence. Our technique provides a robust alternative to error-prone dual- or triple colour co-localisation studies