How Large Format Lenses Revolutionize ExA-SPIM Lightsheet Microscopy

The Allen Institute for Neural Dynamics, a division of the Allen Institute, is renowned for its ambitious projects aimed at understanding the complexities of the brain. Their scientists tackle fundamental questions about how the brain develops, functions, and is affected by disease. To do this, they often need to push the boundaries of existing technology, or even invent new tools. The development of the ExA-SPIM microscope is a prime example of this innovative spirit.

Imagine an optical lens, meticulously engineered by Schneider-Kreuznach to find the smallest imperfections on electronic components during manufacturing. Now, envision this same high-precision technology playing a crucial role in helping scientists observe the brain's incredibly detailed structure. This is precisely what's happening with the groundbreaking "ExA-SPIM" microscope, where our industrial lens is enabling researchers to see tissue at the nano-scale, offering unprecedented views of entire mouse brains.

The Challenge: Seeing the Brain's Intricate Network
The brain is an astonishingly complex organ. The mouse brain, though only the size of a jellybean, contains nearly 80 million neurons, each forming thousands of connections. Understanding how these neurons are wired and how they communicate requires imaging them with high magnification and clarity, but also over very large areas. This presents a significant challenge for traditional microscopy illumination techniques.

Light-Sheet (SPIM) Microscopy: A Smarter Illumination Strategy
Conventional light microscope illumination often bathes the entire sample in light. While simple, this can lead to blurry images from out-of-focus light and can also damage delicate biological samples, especially when using intense fluorescence excitation.

Light-sheet microscopy, also known as SPIM (Selective Plane Illumination Microscopy), offers a more elegant solution. In SPIM microscopy, a thin, focused sheet of light selectively illuminates only a narrow plane within the sample. The microscope’s detection optical path is positioned perpendicular to this light sheet, capturing an image of only the illuminated plane. By systematically moving the sample through this plane of light, or vice-versa, a series of 2D images are acquired. These images are then computationally combined to reconstruct a sharp, three-dimensional view of the sample. This method significantly reduces photobleaching and phototoxicity, providing clearer images of thick tissue.

ExA-SPIM: Pushing the Boundaries with Expansion
The "ExA" in ExA-SPIM stands for "Expansion-Assisted." Scientists at the Allen Institute have incorporated a clever chemical technique called expansion microscopy. Before imaging, the brain tissue is infused with a special gel that causes it to swell uniformly, like carefully inflating a complex, miniature balloon. This physical magnification makes the incredibly fine neuronal structures—axons, dendrites, and even smaller synaptic components—effectively larger and thus easier to resolve with a light microscope.

While expansion is a brilliant way to achieve nano-scale effective resolution, it also creates a new set of challenges. The once small brain tissue becomes a much larger, more fragile, and mostly water-based hydrogel. Imaging such a large, delicate sample at high resolution is where conventional SPIM microscopy systems, with their standard objectives, often hit a wall. They typically lack the combination of a large field of view, long working distance, and high optical quality needed for these expanded samples.

^{Picture of neurons of a mouse brain.}

What is the advantage of our large format lens:

• Massive Field of View (FOV): Our lens, designed for inspecting relatively large electronic components with precision, offers an exceptionally large FOV of 10.6×8.0 mm². For the ExA-SPIM, this means it can capture a significantly larger area of the expanded brain in a single image frame. The impact is transformative: an entire 3x expanded mouse brain can be imaged in as few as 15 tiles. A conventional SPIM setup might require over 400 tiles to cover the same volume at a similar resolution, drastically increasing imaging time and data processing complexity.
• High Resolution Across the Field: Despite its large FOV, the lens maintains a respectable Numerical Aperture (NA) of 0.305. This NA is crucial for collecting enough light and resolving the fine details that become visible after tissue expansion. Combined with 3x expansion, the ExA-SPIM system achieves an effective optical resolution of approximately 0.5 µm laterally and 1 µm axially – truly nano-scale detail.
• Generous Working Distance: Expanded tissues are bulky. The Schneider-Kreuznach lens provides a working distance of around 35 mm. This ample space is essential to accommodate the physically larger sample and the liquid immersion chamber it resides in, without the risk of collision or optical compromise.
• Optimized for Liquid Immersion: The Allen Institute team ingeniously adapted our lens for direct liquid immersion. Since expanded tissues are hydrogels (mostly water), imaging them while immersed in a medium with a similar refractive index minimizes optical aberrations (distortions) that would otherwise degrade image quality, especially deep within the tissue. This ensures that the fluorescence signals are captured with maximum clarity.
• Superior Etendue: Etendue is an optical parameter that describes a system's ability to accept light. It's a product of the area and the solid angle over which it can collect light (related to FOV and NA). Our lens boasts a high etendue (G = 19.65 mm²), meaning it's exceptionally efficient at gathering light and information over its large field, enabling fast and high-resolution volumetric imaging.
• Industrial Precision, Biological Insight: Designed for metrology where accuracy is paramount, our lens exhibits minimal distortion and is highly corrected for chromatic and spherical aberrations. This ensures that the structural information captured from the brain is a faithful representation, crucial for accurate neuronal tracing and network reconstruction.

The Large-Format Camera – A Perfect Partner:

To fully leverage the capabilities of our large-format lens, a correspondingly large and fast camera sensor is required. The ExA-SPIM system uses a Vieworks VP-151MX camera, which incorporates the Sony IMX411 sensor.

• High Pixel Count: This sensor is an industrial marvel, boasting 151 megapixels (14,192 x 10,640 pixels). This massive pixel array is perfectly suited to digitize the large image projected by our lens without sacrificing detail.
• Speed and Synchronization: A key feature of the Sony IMX411 is its single-sided rolling shutter. This, combined with clever engineering, allows for very fast read-out of pixel data. Critically, this rolling shutter can be precisely synchronized with an "axially-swept" light-sheet. Instead of the light-sheet being static, it can be rapidly scanned (swept) through the focus of the detection objective, and the camera's rolling shutter follows this sweep. This technique, pioneered in other structured illumination microscopy methods, effectively improves axial resolution and image quality without the need for complex deconvolution, and the ExA-SPIM camera handles this at incredible speeds – up to 946 megavoxels per second.
   

^{Comparison of a standard microscope lens and a high magnification large format lens.}
 

^{This TITANITE lens has been specially developed for light-sheet microscopy and comes with a dipping cap.}