Take a look behind the scenes of an article recently published in Nature Methods entitled “jYCaMP: an optimized calcium indicator for two-photon imaging at fiber laser wavelengths” as we invite first author Manuel Alexander Mohr to shed light on this recent research.
Please can you give us a short summary of the new indicator presented in your Nature Methods article, “jYCaMP: an optimized calcium indicator for two-photon imaging at fiber laser wavelengths”?
jYCaMP1 is a yellow fluorescent genetically encoded calcium indicator (GECI) with a redshifted excitation spectrum compared to its green fluorescent parent GCaMP. It inherits highly optimized Ca2+ affinity and excellent performance in vitro and in vivo from its parent, which has been engineered for many years. Its yellow excitation spectrum enables two-photon excitation at >1000 nm with low-maintenance fiber lasers that are widely available at smaller sizes, much higher powers and – importantly – drastically reduced costs compared to traditional titanium-sapphire lasers.
What inspired you to develop jYCaMP?
At Janelia (VA, USA) we have been asked to develop such ‘yellow GCaMPs’ for several reasons ranging from spectral multiplexing with differently-coloured fluorescent proteins and indicators to applications in advanced non-linear microscopy. However, most often, labs would like to make use of existing inexpensive fiber lasers (>1000 nm), or accessory beams from fiber-laser pumped optical parametric amplifiers, for two-photon calcium imaging.
Frustratingly, previous ‘yellow’ GCaMP variants had only a small spectral shift: they were still green, and therefore excited poorly above 1000 nm.
How does it compare with other options? Do you envisage yellow becoming the new green?
jYCaMP1 markedly outperforms jGCaMP7 in vivo at two-photon excitation wavelengths typical of fiber lasers. Moreover, due to its red-shifted excitation and overall excellent in vivo performance, jYCaMP can be co-excited together with an RFP-based sensor and hence presents a great choice for dual-color two-photon calcium imaging.
To name a few reasons, jYCaMP might be right for a researcher if:
- They are looking for an indicator that has less spectral overlap with the green/red fluorescence channel.
- They do multi field of view in vivo calcium imaging, high-speed microscopy, or other advanced two-photon techniques that requires large pulse energies.
- They want to do single-laser two-color imaging alongside red GECIs.
- They want to save some serious money when buying their next two-photon laser –
jYCaMP works well with inexpensive fiber-lasers.
Other potential applications might include compact optical systems with miniaturized low-cost lasers, or multiplexing many microscopes using a single high-power source.
I certainly hope that yellow will become the new green! As a matter of fact, several other prominent tool development labs have already started developing yellow sensors and/or shifting the existing ones towards yellow. I am very excited to see what’s to come!
Do you have any tips or tricks for someone looking to utilize this indicator?
jYCaMP1 is comparable in speed and overall in vivo performance to GCaMP6s or jGCaMP7s, which many experimenters are already familiar with. For most labs familiar with GCaMP, the implementation should be very straightforward.
We also report jYCaMP1s, which has slower kinetics and might be of interest in some specific settings, where imaging speed is limiting, or short-term calcium integration is desired, such as in small cellular compartments like axonal boutons or dendritic spines.
Plasmids and AAVs for Cre-dependent and Cre-independent “straight” expression of jYCaMP1, jYCaMP1s as well as axon-targeted jYCaMP1  are conveniently available from addgene.
If you are reading this, you are likely using our sensors and have ideas on how to make them even better, so please do let us know – we love feedback!
What impact do you hope this development will have in the lab?
I hope that many labs will find this new tool helpful in their scientific endeavors. I hope that our work will start a trend towards more yellow sensors and more cost-efficient two-photon microscopes. Reducing the cost of microscopes could significantly impact labs and core facilities with limited funding. While jYCaMP was initially designed for use in neuroscience labs, I hope it will find acceptance also in other fields of biological investigation, where calcium dynamics are of importance.
Have you as-yet observed anything exciting using this?
jYCaMP1s enabled us to record calcium signals from thousands of presynaptic boutons simultaneously with spatially overlapping postsynaptic dendrites labeled with the red indicator jRGECO. In the video you can see what the results from such an experiment look like. We image the light blue-colored thalamocortical axonal boutons (which are actually yellow due to jYCaMP) simultaneously with the red postsynaptic dendrites labeled with the red indicator jRGECO in the visual cortex of a living mouse while it is watching a video on a screen. Bright flashes correspond to activity in the respective location.
Finding connections between axons and dendrites in this way has been very hard in the past because most pairs are not connected and it was hard to excite two different colors of indicators at once. jYCaMP and jRGECO are now the best way we know to do these types of experiments, and we can detect coactive pairs of axons and dendrites much more easily.
What are you hoping to do next in this area?
jYCaMP was just our first foray into improving indicators specifically for in-vivo two-photon imaging. In the past, we’ve always developed indicators under one-photon illumination, and hoped that they would perform well under two-photon. But it turns out that one-photon and two-photon properties are not always the same. The Podgorski lab and others are now screening indicators under two-photon illumination to make them brighter and more sensitive for the toughest in vivo experiments.
Manuel Mohr, PhD, is trained as a biotechnologist and protein engineer. His passion lies in building molecular tools for systems neurobiology.
Mohr received a Bachelor’s degree in Molecular Biotechnology from Technical University Munich (TUM) in Germany and a Master’s degree in Biotechnology and Systems Biology from ETH Zurich (Switzerland). He conducted his PhD research in the nano-bio-imaging lab of Periklis Pantazis at ETH Zurich, and in the protein engineering groups of Eric Schreiter and Loren Looger at the HHMI Janelia Research Campus (VA, USA) as a Janelia graduate research fellow, where he learned and engaged in fluorescent protein engineering for applications in development and neurobiology.
As a Postdoctoral Researcher in the lab of Xiaoke Chen at Stanford University (CA, USA) he is spearheading the Chen lab’s virus design and molecular tool development efforts. In collaboration with imaging experts at the UC Berkeley (USA), he combines these novel tools with next-generation multi-photon imaging techniques to unravel functional circuit architecture in the mouse spinal cord.