Adapting Upright Light Sheet Fluorescence Microscopy for Imaging at Air-Liquid Interface

This paper presents a custom device that enables robust light sheet fluorescence microscopy imaging of biological specimens requiring an air-liquid interface, demonstrated through successful applications on mouse salivary glands, human skin cultures, and fruit fly brains.

The Gist

The Big Problem: The "Swimming Pool" vs. The "Breathing Room"

Imagine you are a scientist trying to take a high-definition, 3D movie of a tiny living thing (like a developing organ or a fruit fly's brain) to see how it grows and moves.

For years, the best tool for this job has been a Light Sheet Microscope. Think of this microscope like a very gentle, super-fast camera that shines a thin sheet of light through a specimen, taking pictures slice-by-slice without hurting it with too much heat or light.

But there's a catch: To use this microscope, the specimen usually has to be completely submerged in a pool of water (or culture medium), like a fish in a tank. The camera lenses also have to dip into the water to get a clear picture.

This works great for fish, tadpoles, or cells in a petri dish. But it fails miserably for things that need to breathe air.

• Skin cells need air on top and water on the bottom.
• Lungs need air to function.
• Adult fruit flies will drown if you put them underwater.

If you try to force these air-breathing samples into the "swimming pool" microscope, they die, or they stop working properly. Scientists have been stuck with blurry, low-quality cameras for these specific samples because the high-tech "pool" microscope couldn't be used.

The Solution: The "Dry Dock" for Microscopes

The authors of this paper built a clever gadget called the LSFM-ALI device.

Think of it like a dry dock for a ship. A dry dock is a place where a ship can be lifted out of the water so workers can fix the hull, but the ship is still supported and accessible.

This new device creates a tiny, custom "dry dock" inside the microscope's water bath:

1. The Base: It's a stainless steel plate that fits into the microscope.
2. The Wall: A silicone ring creates a barrier.
3. The Air Pocket: Inside the ring, there is a small pocket of air.
4. The Bridge: A special, see-through membrane (like a very thin, invisible plastic wrap) separates the air pocket from the water below.

How it works:

• The sample (like a piece of skin or a fly) sits in the air pocket, so it can breathe.
• The bottom of the sample touches the water (through the membrane), so the microscope lenses can dip in and take crystal-clear pictures from below.
• A tiny tube pumps fresh air into the pocket so the sample doesn't suffocate or get too hot.

It's like building a tiny, transparent diving bell where the top is open to the sky, but the bottom is submerged in the ocean, allowing a diver (the microscope) to look up at a swimmer (the sample) without drowning them.

What They Tested It On (The "Proof of Concept")

The team tested this "dry dock" on three very different types of "swimmers" that usually can't use this microscope:

1. The Growing Salivary Gland (The "Tree" in the Water)

• The Sample: A tiny mouse salivary gland growing in a dish. These glands branch out like trees.
• The Challenge: They need air on top to grow correctly.
• The Result: The device let the gland breathe while the microscope filmed it for hours. They saw cells dividing and immune cells zooming around to clean up debris, all in 3D.

2. Human Skin (The "Two-Faced" Layer)

• The Sample: A culture of human skin cells. Real skin has a "basement" (bottom) in fluid and a "roof" (top) exposed to air.
• The Challenge: If you put the whole thing in water, the top layer dies. If you look from the top with a regular lens, the image is blurry.
• The Result: They cut the skin culture, flipped it upside down, and stuck it to the device. The bottom touched the water (for the camera), and the top stayed in the air (for the skin). They could see the inner workings of the skin cells moving and stretching in high definition.

3. The Adult Fruit Fly (The "Drowning Fly")

• The Sample: An adult fruit fly. You can't put a fly underwater to look at its brain; it will drown.
• The Challenge: How do you look at a fly's brain without killing it?
• The Result: They made a tiny hole in the fly's head, exposed the brain, and placed the fly in the device. The fly's body stayed in the air pocket (so it could breathe), while only the exposed brain dipped into the water (so the microscope could see it). They watched the fly's brain cells change shape over many hours, something that was impossible before.

Why This Matters

Before this invention, scientists had to choose between high-quality imaging (using the light sheet microscope) or keeping the sample alive (by keeping it in air). They couldn't have both.

This new device is like a universal adapter. It allows scientists to use the most powerful, gentle, and fast cameras on a huge variety of living things that were previously "off-limits."

• For doctors: It could help us understand how skin heals or how lungs develop.
• For biologists: It lets us watch the brains of living animals change in real-time.
• For everyone: It proves that with a little bit of engineering creativity (a ring, some silicone, and a pump), we can break down the barriers between different ways of studying life.

In short: They built a bridge between the world of water and the world of air, so our best microscopes can finally take a good look at the things that need to breathe.

Technical Summary

1. The Problem

Light Sheet Fluorescence Microscopy (LSFM) is the gold standard for gentle, high-speed, volumetric imaging of biological specimens. However, upright LSFM configurations (where excitation and detection objectives are dipped into a medium from above) face a critical physical constraint: they require the specimen to be fully submerged in imaging media.

This limitation excludes a vast class of biological models that require an Air-Liquid Interface (ALI) for viability and proper development, including:

• Epithelial tissues: Skin, lungs, and airways, which require the apical surface to be exposed to air while the basal surface remains in medium.
• Organ explants: Such as embryonic mouse salivary glands, which need gas exchange for branching morphogenesis.
• Non-aquatic whole organisms: Adult Drosophila melanogaster (fruit flies), which cannot survive immersion but require high-resolution imaging of internal structures like the brain.

Existing solutions (e.g., standard confocal microscopy) often lack the speed, optical sectioning, or low phototoxicity of LSFM, or they suffer from refractive index mismatches and condensation issues when imaging through air.

2. Methodology: The LSFM-ALI Device

The authors designed and fabricated a modular device, the LSFM-ALI, to create a stable air pocket within a bath of imaging media, compatible with upright LSFM systems (specifically the MOSAIC microscope at Janelia Research Campus).

Key Design Features:

• Structure: A stainless-steel base holds a silicone spacer sealed with silicone sealant. A plastic ring sits within the spacer, creating a central circular opening where the ALI is formed.
• Membranes: The central opening is covered by a membrane (gas-permeable, fluid-permeable, or impermeable) depending on the sample. This allows the sample to be sandwiched or mounted such that one side faces air and the other faces liquid.
• Modularity: The device features interchangeable plastic rings (varying inner diameters: 1mm, 5mm, 10mm) and silicone spacers (varying heights: 4mm, 5mm, 6mm) to accommodate different sample sizes and volumes.
• Active Airflow: To prevent CO2 buildup and ensure respiration for long-term imaging (especially for live animals), the device includes inlet and outlet ports connected to a peristaltic pump for continuous fresh air circulation.
• Mounting: The device is secured to the microscope's sample holder using existing tightening screws, allowing for easy removal and sample mounting prior to insertion.

3. Key Contributions

• Hardware Innovation: The creation of a robust, open-source device that enables upright LSFM on samples requiring an air-liquid interface.
• Versatility: Demonstration of the device across three distinct biological use cases:
  1. Ex vivo Organ Explants: Mouse embryonic salivary glands (mostly submerged, air-exposed top).
  2. Epithelial Cultures: Human epidermal equivalents (mostly in air, medium-exposed bottom).
  3. In vivo Organisms: Adult Drosophila brains (whole animal in air, brain exposed to medium).
• Open Science: Full 3D design files and raw image data are made publicly available to facilitate adaptation to other upright LSFM systems.

4. Results

The authors successfully demonstrated live, multi-color, volumetric, time-lapse imaging across all three use cases:

• Application 1: Mouse Salivary Glands

  • Setup: Glands were sandwiched between gas-permeable membranes.
  • Findings: The system supported development over multiple days. Researchers observed robust cell division (H2B-EGFP) and rapid 3D migration of immune cells clearing debris. Multi-color imaging (Keratin14-mStayGold and NLS-mScarlet-I3) revealed that keratin filaments trail the nucleus during migration through confined intercellular spaces.

• Application 2: Human Epidermal Equivalent

  • Setup: N/TERT-2G keratinocytes grown on PTFE Transwell membranes were inverted and mounted. The basal side faced the objectives through the PTFE membrane (refractive index matched to water).
  • Findings: The system allowed high-resolution imaging of cell shape changes in basal and suprabasal layers. It captured rapid dynamics of the Endoplasmic Reticulum (ER) network during cell body extension, which would be difficult to capture with conventional microscopy due to phototoxicity or resolution limits.

• Application 3: Adult Drosophila Brains

  • Setup: Flies were immobilized in aluminum foil with the dorsal head cuticle dissected to expose the brain. The fly's body remained in an air pocket with active airflow, while the exposed brain was submerged in medium.
  • Findings: Long-term imaging (>12 hours) was achieved without anesthetizing the fly with CO2 (which the airflow prevented). Researchers visualized the structural remodeling of small lateral ventral neurons (sLNvs) and surrounding glial processes over circadian cycles, observing the extension of neuronal processes in real-time.

5. Significance and Limitations

Significance:

• Expanding the Scope of LSFM: This work bridges a critical gap, allowing the application of high-speed, low-phototoxicity LSFM to tissues and organisms that were previously inaccessible to upright light sheet systems.
• Biological Insight: It enables the study of dynamic processes (e.g., branching morphogenesis, neuronal plasticity, immune cell migration) in their native physiological environments (ALI) rather than forced submerged conditions.
• Blueprint for Adaptation: The modular design serves as a template for adapting other upright LSFM systems (e.g., iSPIM, diSPIM, LLSM) to ALI samples.

Limitations:

• Sample Preparation: Mounting is non-trivial and often requires custom iteration for each sample type.
• Optical Trade-offs: Imaging through permeable membranes causes slight losses in resolution, light collection efficiency, and working distance.
• Penetration Depth: Scattering and absorption still limit effective imaging depth to approximately 50–100 µm, regardless of the ALI setup.
• Mechanical Stability: The air perfusion system can cause pulsatile movement of the membrane, potentially complicating imaging if not managed carefully.

In conclusion, the LSFM-ALI device represents a significant hardware innovation that democratizes high-performance volumetric imaging for a broader range of biological models, particularly those requiring gas exchange or existing in non-aquatic environments.