Long-Stokes-Shift mScarlet3 as a Structural Marker for Two-Photon Imaging

This paper introduces transgenic Drosophila expressing a long-Stokes-shift mScarlet3 variant that enables simultaneous two-photon imaging of anatomical landmarks and green calcium sensors using a single 920-nm laser, thereby reducing equipment complexity and cost while minimizing channel crosstalk.

The Gist

Imagine you are trying to watch a play inside a tiny, dark theater (a fruit fly's brain). You have two main goals:

1. Watch the actors move: You want to see when the neurons (the actors) "fire" or get excited. Scientists use a special green glow-in-the-dark paint (called GCaMP) for this. When a neuron fires, it turns bright green.
2. See the stage: You also need to know where the actors are standing and how the theater is built. For this, you need a structural marker, like a red glow-in-the-dark paint, to outline the buildings and streets.

The Old Problem: Two Lasers, One Messy Stage
In the past, to see both the green actors and the red stage, scientists needed two different flashlights (lasers).

• One flashlight (920 nm) was needed to make the green paint glow.
• A completely different flashlight (around 1040 nm) was needed to make the red paint glow.

This was like trying to film a movie with two different camera crews, two different sets of lights, and two different schedules. It was expensive, complicated, and the extra light could actually hurt the delicate actors (phototoxicity). Plus, if the stage moved slightly between the two shots, the picture would be blurry.

The New Solution: The "Magic Chameleon" Paint
This paper introduces a new, super-smart red paint called LSSmScarlet3. Think of it as a "Long-Stokes-Shift" chameleon.

In the world of light, "Stokes shift" is just a fancy way of saying: "How far apart are the color of the light you shine in, and the color of the light that bounces back?"

• Old red paints were picky. You had to shine a specific red light on them to make them glow red.
• LSSmScarlet3 is a chameleon. You can shine the green light (the 920 nm laser used for the actors) on it, and it will happily absorb that energy and bounce back a red glow.

Why is this a game-changer?

1. One Laser, Two Colors: Now, scientists only need one flashlight (the 920 nm laser). They shine it on the brain, and poof! The neurons glow green, and the structural markers glow red, all at the exact same time.
2. No Cross-Talk: Imagine trying to listen to a violin (green) while a trumpet (red) plays nearby. If they are too close in pitch, you can't tell them apart. This new red paint is so good at shifting its color that the red signal stays perfectly in the "red channel" and doesn't leak into the "green channel." It's like the trumpet is playing in a completely different room, so the violinist can be heard clearly.
3. Simpler and Cheaper: Scientists don't need to buy a second expensive laser or build a complex machine. They can use the standard equipment they already have.

The Experiment
The researchers built a special line of fruit flies that carry the instructions to make this new red paint in their brains.

• They tested it in a petri dish (HEK293T cells) and saw that the red paint glowed brightly under the green laser.
• They tested it in live fly brains alongside the green calcium sensors.
• The Result: They could see the brain's architecture (red) and the neurons firing (green) simultaneously, perfectly aligned, using just one laser beam.

The Bottom Line
This paper is like inventing a new type of paint that lets you see the whole picture at once without needing extra tools. It makes studying the brain simpler, cheaper, and less stressful for the tiny creatures being studied, allowing scientists to finally watch the "actors" perform on their "stage" in perfect harmony.

Technical Summary

1. Problem Statement

• Limitation of Current Dual-Channel Imaging: In neuroscience, researchers often need to simultaneously monitor neural activity (using green genetically encoded calcium indicators like GCaMP) and anatomical structure (using red fluorescent proteins like tdTomato or mCherry) to identify cell types and correct for motion.
• Hardware Complexity: Standard red fluorescent proteins (RFPs) typically require excitation wavelengths around 1040 nm, while green sensors (GCaMP) require ~920 nm. This necessitates complex, expensive multi-laser two-photon (2P) microscope setups.
• Consequences: Multi-laser systems increase equipment costs, system complexity, and phototoxicity (due to sequential scanning or higher total power). There is a need for a red structural marker that can be efficiently excited by the standard 920 nm laser used for GCaMP, enabling single-laser dual-color imaging.

2. Methodology

The authors developed and characterized a transgenic Drosophila line expressing a codon-optimized variant of LSSmScarlet3 (Long-Stokes-Shift mScarlet3).

• Construct Generation:
  • The LSSmScarlet3 coding sequence was codon-optimized for Drosophila.
  • It was cloned into a UAS vector (pJFRC81-10XUAS-IVS-Syn21-GFP-p10) to create the UAS-DmLSSmScarlet3 construct.
  • Transgenic flies were generated by injecting the construct into yw flies at the attP40 site.
• Spectral Characterization:
  • Tissue: Fixed Drosophila brains expressing LSSmScarlet3 (driven by MB247-Gal4) were imaged on a Leica TCS SP8 DIVE multiphoton microscope.
  • Excitation Scan: Excitation wavelengths were tested at 860 nm, 940 nm, and 1040 nm.
  • Emission Scan: Emission spectra were recorded for each excitation wavelength.
• In Vitro Validation (HEK293T Cells):
  • Cells were transfected with pcDNA-DmLSSmScarlet3.
  • Imaging was performed on an A-SCOPE system using a single 920 nm laser.
  • Detection was split between a green PMT (500–550 nm) and a red PMT (570–640 nm) to measure channel crosstalk at varying laser powers.
• In Vivo Dual-Channel Imaging:
  • Flies co-expressing GCaMP8m (green) and LSSmScarlet3 (red) in the mushroom body (driven by MB247-Gal4) were imaged.
  • A single 920 nm laser (FEMTO3D Atlas microscope) was used to excite both fluorophores simultaneously.
  • Signals were separated using spectral filtering (Green PMT for GCaMP, Red PMT for LSSmScarlet3).

3. Key Contributions

• Development of a Transgenic Fly Line: The creation of a ready-to-use Drosophila transgenic line expressing a codon-optimized LSSmScarlet3.
• Single-Laser Dual-Color Solution: Demonstration that LSSmScarlet3 can be robustly excited at 920 nm (the standard wavelength for GCaMP), eliminating the need for a second laser (typically 1040 nm).
• Validation of Spectral Separation: Proof that the large Stokes shift of LSSmScarlet3 allows for minimal crosstalk between the green (GCaMP) and red (structural) channels when using standard bandpass filters.

4. Key Results

• Excitation Spectrum: LSSmScarlet3 in Drosophila tissue showed a peak excitation at 940 nm, with local maxima at 860 nm and 1040 nm. Crucially, at 920 nm, the fluorescence intensity was approximately 75% of the peak intensity (99 vs. 132 arbitrary units), confirming efficient excitation by standard 2P lasers.
• Emission Spectrum: The emission peak was centered around 620 nm regardless of the excitation wavelength.
• Channel Crosstalk (In Vitro):
  • In HEK293T cells excited at 920 nm, the red PMT detected robust LSSmScarlet3 signals at low laser power.
  • The green PMT detected no signal below 50% laser power.
  • Significant crosstalk into the green channel only occurred above 60% laser power, indicating that with appropriate gain settings, the channels are well-separated.
• In Vivo Co-Imaging: Simultaneous imaging of GCaMP8m and LSSmScarlet3 in the live mushroom body of flies was successful. Both signals were clearly resolved using a single 920 nm laser, with the red channel providing clear anatomical landmarks (mushroom body lobes) without interfering with the calcium signal.

5. Significance and Impact

• Simplified Experimental Setup: This approach allows researchers to perform dual-channel functional-structural imaging using a single 920 nm laser, significantly reducing equipment costs and complexity.
• Reduced Phototoxicity: By enabling simultaneous scanning rather than sequential scanning (required for different wavelengths), the method reduces the total light dose delivered to the sample, minimizing phototoxicity.
• Improved Temporal Resolution: Simultaneous acquisition ensures perfect temporal registration between the functional (calcium) and structural data, which is critical for motion correction and precise anatomical mapping.
• Broad Applicability: The high brightness of LSSmScarlet3 makes it suitable not only for structural markers but also for fusion proteins with subcellular localization signals or monitoring transcription levels, provided expression levels are titrated to avoid PMT saturation.
• Resource Availability: The authors have made the transgenic fly line, construct sequences, and data/code publicly available (Zenodo), facilitating immediate adoption by the neuroscience community.

In summary, this paper presents a practical and validated solution for high-quality, single-laser two-color two-photon imaging in Drosophila, bridging the gap between functional activity monitoring and anatomical context.