Two-photon characterisation of long-Stokes-shift dye ATTO 490LS for single-laser multicolour imaging

This study characterizes the two-photon excitation properties of the long-Stokes-shift dye ATTO 490LS in *Drosophila* brains, demonstrating its suitability for single-laser, multicolour two-photon imaging alongside Alexa Fluor 488.

The Gist

Imagine you are trying to take a high-resolution photograph of a tiny, bustling city inside a fruit fly's brain. To see the details without damaging the city, scientists use a special kind of "night vision" camera called two-photon microscopy. This camera uses invisible infrared light (like a laser) to make specific parts of the brain glow.

However, there's a catch: most of these microscopes only come with one laser color (like a single flashlight). If you want to see two different things at once—for example, the "roads" (neurons) and the "traffic" (brain activity)—you usually need two different flashlights. If you only have one, the colors often get mixed up, and you can't tell them apart.

This paper is about a clever new tool that solves this problem. Here is the breakdown:

1. The Problem: One Flashlight, Two Jobs

Think of the microscope's laser as a single flashlight that shines a specific color of light (920 nm).

• The Green Light: Scientists often use a sensor called GCaMP (or Alexa Fluor 488) that glows green when neurons are active. It's like a traffic light turning green when cars are moving.
• The Missing Red Light: To see the structure of the brain (the roads), they need a red glow. Usually, red dyes need a different flashlight to light them up. If you try to use the green flashlight on a red dye, it either doesn't work or the colors blend into a muddy mess.

2. The Hero: ATTO 490LS (The "Shape-Shifter")

The researchers tested a special dye called ATTO 490LS. This dye is a "Long-Stokes Shift" (LSS) molecule.

• The Analogy: Imagine a magical chameleon. You shine a blue light on it, and it doesn't just reflect blue; it absorbs the blue energy, loses a little bit of it as heat (like a runner getting tired), and then jumps up and glows a completely different color, like bright red.
• The Magic: Because it changes its color so drastically (from the invisible laser light to a bright red glow), there is almost no overlap. The microscope can easily tell the difference between the "green traffic" and the "red roads" even though they were both lit up by the same single flashlight.

3. The Experiment: Finding the Right Key

The team had to figure out exactly how to "tune" their single flashlight to make this chameleon dye glow.

• They tried shining the laser at different "frequencies" (like tuning a radio).
• They found that while the dye loves a 780 nm frequency, it also works surprisingly well at 940 nm.
• Why this matters: Most standard microscopes in labs are already set to 920 nm (a very common setting for seeing green brain activity). The researchers discovered that ATTO 490LS glows brightly enough at 920 nm to be useful.

4. The Result: A Clearer Picture

They tested this on a fruit fly brain.

• They painted the brain's structure with the new red dye (ATTO 490LS).
• They painted the active neurons with the standard green dye (Alexa Fluor 488).
• They turned on the single 920 nm laser.
• The Outcome: The microscope successfully separated the signals. It saw the green traffic lights and the red roads clearly, without them bleeding into each other.

Why This is a Big Deal

• No New Hardware Needed: You don't need to buy a second, expensive laser. You can use the microscope you already have.
• Better Brain Mapping: Scientists can now watch where neurons are (red structure) and what they are doing (green activity) at the exact same time.
• Future Potential: This opens the door for studying complex brain circuits in living animals without needing complicated, multi-laser setups.

In short: The researchers found a special "magic paint" that lets a single-laser microscope see two distinct colors at once, turning a blurry, mixed-up image into a sharp, multi-colored map of the brain.

Technical Summary

1. Problem Statement

• Limitation of Current Systems: Many laboratories, particularly those studying Drosophila (fruit flies), utilize two-photon microscopy systems equipped with a single fixed-wavelength laser (typically 920 nm). This setup is ideal for exciting green fluorescent proteins (like GCaMP or GFP) but limits the ability to perform multicolour imaging because standard red fluorophores often require different excitation wavelengths.
• Spectral Overlap: Conventional dyes often have excitation and emission spectra that overlap significantly with the excitation laser or other fluorophores, leading to high background noise (autofluorescence) and crosstalk, making simultaneous detection difficult.
• Knowledge Gap: While the long-Stokes-shift (LSS) dye ATTO 490LS is known for its large separation between excitation (496 nm) and emission (661 nm) under single-photon conditions, its two-photon excitation properties had not been characterized. It was unknown if it could be efficiently excited by a 920 nm laser, which is the standard for green sensor imaging.

2. Methodology

The authors employed a combination of spectral characterization and biological imaging to validate the dye's utility:

• Biological Model: They used ex vivo brains from Drosophila melanogaster with the genotype elav-Gal4>UAS-TID-Dlg1.
  • Labeling Strategy: They utilized the TurboID proximity labeling system. The bait protein (TurboID-Dlg1) biotinylates nearby proteins. Streptavidin-conjugated ATTO 490LS was used to label these biotinylated structures (red channel).
  • Dual Labeling: To test duplex imaging, they simultaneously stained for the c-Myc tag on the bait protein using an anti-c-Myc primary antibody and an Alexa Fluor 488 secondary antibody (green channel).
• Spectral Characterization (Leica TCS SP8 DIVE):
  • Excitation Scan: Scanned excitation wavelengths from 680 nm to 1200 nm to identify optimal two-photon excitation peaks for ATTO 490LS.
  • Emission Scan: Fixed excitation at identified peaks (780, 920, 940, 1040 nm) and scanned emission from 500 nm to 780 nm to determine the emission spectrum.
• Imaging Setup (Thorlabs Microscopes):
  • Used two different Thorlabs two-photon microscopes (A-SCOPE and B-SCOPE) equipped with fixed 920 nm fibre lasers.
  • Detection: Simultaneous detection using two Photomultiplier Tubes (PMTs):
    • Green Channel: 500–550 nm (for Alexa Fluor 488).
    • Red Channel: 570–640 nm (for ATTO 490LS).
  • Power/Gain: Tested at various laser powers (3% to 8%) and detector gains to optimize signal-to-noise ratio.

3. Key Contributions

• First Characterization of ATTO 490LS for Two-Photon Imaging: The study provides the first empirical data on the two-photon excitation and emission properties of ATTO 490LS.
• Validation of Single-Laser Multicolour Imaging: It demonstrates that a single 920 nm laser can simultaneously excite both a green fluorophore (Alexa Fluor 488) and a red LSS dye (ATTO 490LS) with distinct, non-overlapping emission signals.
• Protocol Development: The paper establishes a robust protocol for using streptavidin-conjugated ATTO 490LS in conjunction with TurboID proximity labeling in Drosophila brains.

4. Key Results

• Excitation Spectrum:
  • ATTO 490LS exhibits three two-photon excitation peaks at 780 nm, 940 nm, and 1040 nm.
  • While 780 nm yielded the highest intensity, 940 nm (and by extension, the commercially available 920 nm) showed significant sensitivity (approx. 3–4 fold higher than baseline autofluorescence).
  • Excitation at 920 nm generates significantly less tissue autofluorescence compared to 780 nm.
• Emission Spectrum:
  • Under two-photon excitation at 780, 920, 940, and 1040 nm, ATTO 490LS consistently emits with a peak at 640 nm.
  • This confirms a massive Stokes shift, allowing clear separation from the 920 nm excitation source and the green emission of Alexa Fluor 488.
• Duplex Imaging Performance:
  • Successful simultaneous imaging of ATTO 490LS (red) and Alexa Fluor 488 (green) was achieved using a single 920 nm laser.
  • Specificity: Distinct anatomical structures were differentially labeled:
    • Ellipsoid body: Labeled only by ATTO 490LS.
    • γ-lobe of the mushroom body: Labeled only by Alexa Fluor 488.
    • α/β-lobes and antennal lobes: Double-labeled.
  • No significant spectral crosstalk was observed between the green and red channels when using appropriate bandpass filters.

5. Significance and Future Implications

• Lowering Technical Barriers: This work enables laboratories with "legacy" single-laser (920 nm) systems to perform high-quality multicolour two-photon imaging without the need for expensive laser upgrades or complex multi-laser setups.
• Functional + Structural Imaging: The compatibility of ATTO 490LS with standard 920 nm excitation allows for the simultaneous imaging of:
  • Functional signals: Green calcium indicators (e.g., GCaMP) to monitor neuronal activity.
  • Structural/Reference signals: Stable red labeling (via ATTO 490LS) to visualize anatomy or serve as a denominator for normalization.
• Future Applications: The authors propose engineering ATTO 490LS conjugates for specific tags (HaloTag, SNAP-tag) to enable in vivo chemogenetic labeling in transgenic lines. This would facilitate the dissection of neural circuit function in behaving animals by combining functional calcium imaging with stable structural markers.

In summary, the paper validates ATTO 490LS as a powerful tool for expanding the capabilities of standard single-laser two-photon microscopes, enabling robust, high-sensitivity, multicolour imaging in neuroscience research.