Light Sheet Microscopy (SPIM)

Unlike conventional widefield or confocal microscopes that illuminate the entire sample volume, light sheet microscopy uses a thin sheet of laser light to excite fluorescence in a single plane of the specimen.
A second objective, positioned orthogonally, collects the emitted fluorescence. By scanning the light sheet through the sample, the microscope builds up a three-dimensional image stack.

This geometry provides several major advantages:

• Only the imaging plane is illuminated, drastically reducing photobleaching and phototoxicity.
• Fast volumetric imaging, as whole planes are captured simultaneously rather than point by point.
• Exceptional signal-to-noise ratio, since out-of-focus fluorescence is largely eliminated.

In simple terms: SPIM offers gentle, fast, and deep imaging — the perfect balance for observing life in motion.

In biophotonics research, SPIM has become indispensable for studying living organisms over extended timescales, providing insights that were previously impossible due to photodamage or limited imaging depth.

Its unique advantages include:

• Minimal photodamage and bleaching — only the observed plane is exposed to light.
• Reduced background signal — because non-imaged regions are not illuminated.
• Fast acquisition — enabling volumetric imaging of dynamic processes.
• High optical sectioning capability — resulting in crisp, 3D fluorescence maps.
• Compatibility with large, optically cleared or living samples.

This makes light sheet microscopy ideal for imaging whole embryos, developing tissues, organoids, and neuronal networks — systems that are too delicate for traditional high-intensity laser scanning microscopy.

Lasers are the illumination engine of every light sheet microscope. Their beam quality, stability, and tunability directly determine image quality and system performance.

Key requirements for SPIM lasers include:

• Excellent beam pointing and spatial mode quality for creating a uniform, thin light sheet.
• Low intensity noise (RIN) to achieve high signal-to-noise fluorescence detection.
• Stable, precisely controlled power output to prevent photodamage and ensure reproducibility.
• Multiple excitation wavelengths to match a wide range of fluorescent dyes and proteins.
• Compact, reliable, and fiber-coupled configurations for easy integration and alignment-free operation.

Modern light sheet systems often need to excite several fluorophores simultaneously or sequentially — each with a specific excitation wavelength.
TOPTICA’s multi-laser engines combine violet, visible, and near-infrared diode lasers into one compact, modular platform.

Instead of relying on multiple standalone lasers, all wavelengths are:

• Precisely beam-combined into a single optical output.
• Fiber-coupled directly to the microscope, ensuring perfect spatial overlap.
• Actively stabilized for constant power and long-term operation.

This approach provides several key advantages:

• Simplified optical alignment – all laser lines remain coaxial and stable over time.
• Fast switching between wavelengths – ideal for multi-color fluorescence imaging.
• Consistent illumination intensity – essential for quantitative imaging.
• Space- and cost-efficiency – one laser engine replaces several discrete sources.

With one light engine spanning from violet (405 nm) through visible (488, 561, 594 nm) to near-infrared (640–780 nm), users can cover virtually all common fluorescent dyes and proteins.

Fiber coupling is a cornerstone of reliable and flexible SPIM operation.
TOPTICA’s single-mode polarization-maintaining fiber coupling ensures:

• Perfect Gaussian beam profiles for optimal light-sheet formation.
• Long-term stability without realignment, even after months of use.
• Plug-and-play integration with commercial and custom microscope setups.

Combined with COOL AC technology —  and active temperature and power stabilization — TOPTICA’s laser engines offer true hands-off operation for long-term imaging experiments.
Researchers can focus on biological discovery, not laser maintenance.

Compared to confocal or widefield microscopy, light sheet microscopy dramatically reduces photodamage and photobleaching, enabling long-term observation of living systems.
Because illumination is limited to a thin plane:

• Cells remain healthy and active for extended imaging sessions.
• Background fluorescence and noise are minimized.
• Data volumes are reduced, accelerating processing and analysis.

Light sheet microscopy has illuminated some of the most exciting frontiers in modern biology:

• Embryonic development in zebrafish and Drosophila: Scientists visualized cell movements and gene expression in real time, revealing how tissues and organs form during early development (Keller et al., Science, 2008).
• Brain mapping in transparent mice: Whole-brain light sheet imaging enabled complete neuronal reconstruction at subcellular resolution, advancing connectomics research.
• Organoid and cancer research: Long-term imaging of growing organoids provided insights into tumor evolution, metastasis, and drug response under physiological conditions.
• Plant biology: SPIM has been used to visualize nutrient transport and root growth with unprecedented clarity in living plants.

These breakthroughs underline one key fact: gentle, volumetric imaging is essential for understanding life in motion.

Light Sheet Microscopy embodies the essence of biophotonics — using gentle light to explore living systems in all their complexity.
With multi-wavelength laser engines, fiber-coupled precision, and hands-off stability, TOPTICA’s laser systems empower scientists to observe, quantify, and understand life as it happens.

TOPTICA Photonics:
Delivering the light that reveals the living world — one plane at a time.