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Light Sheet Microscopy: What Is It & How Does It Work?

Light Sheet Microscopy: What Is It & How Does It Work?

In the 17th century, Robert Hooke used an improved compound microscope to examine thin slices of cork and other plant tissues. He observed small compartments in its structure, which he coined "cells." Soon after, Hooke published a book titled Micrographia, which illustrated his findings using the microscope.

His discovery marked the beginning of cell biology and demonstrated the power of microscopy in revealing the basic building blocks of life. Not long after his discovery, another scientist, Antonie van Leeuwenhoek, observed hidden, microscopic organisms, which would turn out to be bacteria and protozoa, unlocking even more information about the cellular world.

Advances in microscopy and microscopes provide a window into a tiny world, allowing scientists and researchers to visualize and study the intricate details of living organisms and their structures at scales that we can’t observe with the naked eye. These observations lead to groundbreaking discoveries regarding biological processes, cellular organization, and the functioning of living systems.

These advances include a wide range of microscopy techniques, including optical, confocal, super-resolution, and electron microscopy.

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Optical microscopy encompasses bright-field, phase contrast, and fluorescence, which uses fluorescent probes to visualize specific molecules within cells. Confocal microscopy improves optical sectioning and resolution by eliminating out-of-focus light, while super-resolution techniques surpass the diffraction limit to achieve nanoscale imaging. Electron microscopy uses electrons instead of light to visualize ultrastructural details in transmission electron microscopy (TEM) and produce 3D surface images in scanning electron microscopy (SEM).

Light sheet microscopy, the focus of our post, is an advanced imaging technique that has gained popularity in a variety of research fields. If you’re reading this post, it’s probably because the method has recently come onto your radar, and you are curious to learn more. Maybe you want to acquire a light-sheet microscope for your company’s research and development efforts.

In this article, you’ll get an introduction to light sheet microscopy, including its principles, applications, advantages, and more. Read on to explore the fascinating world of light sheet microscopy.

What Is Light Sheet Microscopy?

Light sheet microscopy, also known as selective plane illumination microscopy (SPIM), is a powerful imaging technique that enables high-resolution 3D imaging of biological samples with minimal photo-induced damage and exceptional spatial resolution. Its unique capabilities enable detailed visualization and analysis, fostering breakthroughs in a variety of research areas.

It offers several advantages over traditional microscopy methods, making it a valuable tool for studying dynamic biological processes, capturing a wide field of view, and achieving superior spatial resolution.

The fundamental principle of light sheet microscopy involves illuminating the sample with a thin sheet of laser light composed of photons, typically generated by a specialized optical setup. This perpendicular illumination scheme allows for efficient excitation and fluorescence collection across the entire field of view, preserving fine details and enhancing spatial resolution.

Light sheet microscopy's capabilities extend beyond spatial resolution. It can also reduce photodamage, enabling long-term live imaging experiments while preserving sample viability. Additionally, it supports high-speed imaging of dynamic processes with minimal phototoxicity and photobleaching.

By imaging the entire field of view, the technique provides excellent optical sectioning and detailed 3D representations of biological structures, further enhancing the spatial resolution and facilitating comprehensive analysis.

Furthermore, light sheet microscopy's versatility extends to multidimensional imaging, allowing the combination of modalities such as fluorescence, phase contrast, or differential interference contrast (DIC). This combination of techniques further enhances the understanding of complex biological systems within the expansive field of view, providing valuable insights with remarkable spatial resolution.

It is also well known for its excellent axial resolution. By selectively illuminating the specific plane within the sample, the technique minimizes out-of-focus light and reduces blurring along the optical axis. This optical sectioning capability, combined with high numerical aperture objective lenses and advanced detection systems, allows for improved axial resolution and the ability to resolve fine structures within the sample.

How Does Fluorescence Play a Role in Light Sheet Microscopy?

Fluorescence and fluorescence imaging play a crucial role in light sheet microscopy, as it allows researchers to visualize and study biological structures and processes at high resolution and in three dimensions.

In light sheet fluorescence microscopy (LSFM), the laser light is used to selectively illuminate a specific plane within the specimen, typically generated by directing the laser through a cylindrical lens that focuses the light into a thin sheet.

The illuminated plane is imaged by a separate objective lens perpendicular to the light sheet. The resulting fluorescence emission from the illuminated plane is then collected and detected by a camera or other imaging device.

When the photons from the light sheet interact with the fluorescent molecules within the sample, they excite the molecules, causing them to emit fluorescent photons. These emitted photons are then collected by the detection optics and converted into an electrical signal, forming the basis of the acquired image.

Fluorescent molecules are used to label specific structures or molecules of interest within the specimen. These fluorescent labels absorb light at a particular wavelength and then re-emit light at a longer wavelength. The emitted fluorescence is typically at a lower energy (i.e., longer wavelength) than the absorbed excitation light, allowing for selective detection of the fluorescence emission while rejecting the excitation light.

How Does Light Sheet Microscopy Work? Step-By-Step Overview

Here's a general overview of how light sheet microscopy is used to capture high-resolution, three-dimensional images of biological samples. It involves several key steps:

  1. Sample preparation: The biological sample of interest must be appropriately prepared for imaging. Preparation may involve labeling specific structures or molecules within the sample with fluorescent markers or probes. The choice of fluorophores depends on factors such as excitation and emission wavelengths, brightness, photostability, and compatibility with the sample.
  2. Light sheet generation: A thin sheet of laser light photons is generated to selectively illuminate a specific plane within the sample by passing a laser beam through a cylindrical lens. The lens shapes the laser light into a sheet-like form, which is then directed toward the sample.
  3. Orthogonal arrangement of optics: The illumination and detection optics are arranged orthogonally to optimize imaging quality. The illumination optics include a laser source, a cylindrical lens, and any necessary optical components to shape and direct the light sheet onto the sample. The detection optics consist of an objective lens that collects the fluorescence emitted from the illuminated plane and directs it toward a detector, such as a camera.
  4. Sample mounting and positioning: The biological sample is typically mounted on a coverslip, which is a thin, transparent piece of glass or plastic. The coverslip holds the sample in place and provides a flat surface for imaging. It ensures that the sample is positioned properly and minimizes the distance between the specimen and the objective lens, optimizing image quality and reducing aberrations. The sample is placed so the plane of interest aligns with the light sheet.
  5. Imaging process: The imaging process begins once the sample is appropriately positioned. The thin light sheet selectively illuminates the targeted plane within the sample, exciting the fluorophores attached to specific structures or molecules. The fluorophores absorb the excitation light and emit fluorescence at a longer wavelength.
  6. Fluorescence detection: The fluorescence emitted by the illuminated plane is collected by the objective lens in the detection optics. The objective lens focuses the emitted fluorescence onto a detector, such as a camera or a photomultiplier tube (PMT). The detector captures the emitted fluorescence and converts it into an electrical signal.
  7. Image acquisition: The electrical signals from the detector are processed and transformed into digital images. These images represent a two-dimensional cross-section of the sample at the illuminated plane. The imaging system can capture images rapidly, allowing for real-time or time-lapse imaging of dynamic biological processes.
  8. Scanning or movement for 3D imaging: To obtain a three-dimensional representation of the sample, multiple images at different planes need to be acquired. This can be achieved through sample scanning, light sheet scanning, or a combination of both, as described in the previous response. The sample or the light sheet is moved to sequentially illuminate and capture images at different depths within the sample.
  9. Image reconstruction: The acquired images from different planes are computationally processed and reconstructed to generate a three-dimensional image of the sample. This reconstruction process aligns and combines the images to represent the spatial structure and organization of the specimen in three dimensions.
  10. Data analysis and visualization: The reconstructed three-dimensional image can be further analyzed and visualized using various software tools and algorithms. Researchers can explore the intricate details of the sample's structure, study dynamic processes, measure distances, quantify fluorescence intensities, and extract valuable information about the biological system under investigation.

What are the Components of a Light Sheet Microscope?

What equipment do you need to perform light sheet microscopy, and what are its components? Various manufacturers offer light sheet microscopes and specialized imaging instrumentation that you can purchase or lease to perform the technique in your laboratory. The key components include the following:

Illumination Module

The illumination module generates a thin laser light sheet that selectively illuminates the sample. It includes components such as:

  • Laser Source: A laser source provides a coherent light beam of the desired wavelength for excitation. Depending on the specific experimental requirements, the laser can be a solid-state laser, a gas laser, or other types.
  • Beam Expander: A beam expander can be used to adjust the laser beam's diameter and improve its spatial quality, ensuring proper illumination.
  • Shaping Optics: The laser beam is directed through shaping optics, such as cylindrical or cylindrical lens pairs. These optics transform the laser beam into a thin sheet of light suitable for illuminating the sample.
  • Spatial Light Modulator: A spatial light modulator (SLM) may be employed in some advanced light sheet microscopy setups; however, it is optional. An SLM is a device that can modify the phase or amplitude of the laser beam, allowing for precise control and shaping of the light sheet. It enables more flexible and customizable light sheet profiles, such as Bessel beams or structured illumination patterns.

Sample Holder

A sample holder positions and immobilizes the biological sample during imaging. Depending on the nature of the sample being imaged, it can be a specialized chamber, a sample stage, or a microfluidic device.

Detection Module

The detection module collects and detects the fluorescence emitted by the illuminated sample plane. It includes components such as:

  • Objective lens: An objective lens with a high numerical aperture (NA) collects the emitted fluorescence from the sample. It is usually positioned orthogonally to the light sheet to collect the fluorescence signal efficiently.
  • Tube lens: The tube lens focuses the collected fluorescence onto a detector, ensuring proper imaging and magnification.
  • Filters: Filters are utilized to separate the fluorescence emission from the excitation light and other background signals. These include emission filters, dichroic mirrors, and bandpass filters, selected based on the specific fluorophores used in the experiment.
  • Detector: The detector captures the emitted fluorescence and converts it into an electrical signal for imaging. Standard detectors used in light sheet microscopy include scientific-grade cameras, photomultiplier tubes (PMTs), or hybrid detectors.

Imaging System

The imaging system acquires and processes the fluorescence signals from the detector, generating images of the sample. It can include:

  • Camera control and image acquisition: Software or hardware control systems manage camera settings and image acquisition parameters, enabling real-time or time-lapse imaging.
  • Data processing and reconstruction: Image processing algorithms and software are used to process and reconstruct the acquired images into three-dimensional datasets, enabling visualization and analysis.

Other Setups & Specialized Imaging Systems for Light Sheet Microscopy

In addition to the essential components mentioned earlier, specialized imaging systems and microscope setups, including adaptive optics, are explicitly designed for light sheet microscopy to enhance its capabilities and performance.

These advanced configurations, such as adaptive optics, cater to specific research needs, offering improved image quality, resolution, and depth penetration. Adaptive optics technology can be integrated into light sheet microscopy setups to correct optical aberrations, ensuring precise focusing and minimizing signal degradation.

A few examples of specialized configurations include:

  • Dual-sided light sheet microscopy: In this setup, two opposing light sheets are used to simultaneously illuminate the sample from both sides. Dual-sided illumination improves imaging uniformity, reduces shadowing effects, and enhances imaging speed, especially when combined with adaptive optics for precise light sheet alignment.
  • Lattice light sheet microscopy: Lattice light sheet microscopy (LLSM) employs an optical lattice pattern created by multiple intersecting laser beams. This technique offers improved resolution, reduced photo-bleaching, and phototoxicity. The integration of adaptive optics in LLSM can further enhance the precision and quality of imaging.
  • Swept confocally aligned planar excitation (SCAPE) microscopy: SCAPE microscopy combines light sheet illumination with confocal imaging and utilizes adaptive optics to optimize the light sheet alignment. This configuration enables high-speed volumetric imaging with optical sectioning capability and reduced out-of-focus background noise.
  • Digital scanned laser light sheet fluorescence microscopy (DSLM): DSLM employs a digitally scanned laser beam controlled by galvanometric mirrors or acousto-optic deflectors. Adaptive optics can play a role in DSLM by optimizing the laser beam's scanning parameters, ensuring precise control over the light sheet's shape, position, and thickness.
  • Multiview light sheet microscopy: This setup captures multiple views of the sample simultaneously by imaging it from different angles. By integrating adaptive optics into multi-view configurations, researchers can correct for optical distortions and aberrations, resulting in improved image quality and more accurate 3D reconstructions.

Incorporating adaptive optics into these specialized configurations enhances the precision, resolution, and image quality of light sheet microscopy, further expanding its capabilities for studying complex biological systems.

Real-World Applications of Light Sheet Microscopy

Light sheet microscopy’s versatility has led to applications in the life sciences, including:

  • Cell imaging
  • Developmental biology
  • Plant biology
  • Neurobiology and neuroscience
  • Organoid imaging
  • Immunology
  • Cancer research
  • Whole-organ imaging

The technique’s features and use of fluorescence make it ideal for imaging large and delicate specimens, such as living organisms and developing embryos, as well as for visualizing cellular structures and subcellular details with exceptional clarity—enabling the study of individual cells, their organelles, and their interactions within complex biological systems.

Furthermore, light sheet microscopy plays a significant role in the imaging of cleared tissue samples. Cleared tissue techniques render biological samples transparent, allowing for enhanced light penetration and imaging of intact tissues. The combination of light sheet microscopy and cleared tissue imaging enables researchers to visualize complex anatomical structures and organ development in three dimensions, providing valuable insights into tissue architecture and intercellular interactions.

Another key application to highlight is the high-resolution imaging of neurons, their connectivity, and dynamic activities within the brain. By selectively illuminating specific planes within cleared brain tissue, light sheet microscopy allows researchers to visualize and study neuronal circuits, map connections, and observe neuronal dynamics in three dimensions. This application has significantly contributed to advancing our understanding of neural architecture, function, and diseases related to the nervous system.

What are the Advantages of Light Sheet Microscopy

There are several benefits to using light sheet microscopy. These include:

Rapid Acquisition of 3D Images

One of the main benefits of light sheet microscopy is its ability to rapidly acquire high-resolution 3D images of large biological specimens while minimizing phototoxicity. Since only the illuminated plane is captured by the detector, the technique eliminates the need for scanning or mechanical movement of the sample, reducing the duration of light exposure and minimizing photodamage.

Faster Imaging Speeds & Shorter Acquisition Time

The elimination of scanning and mechanical movement and the reduction of time exposed to light results in speedier imaging speeds and reduced acquisition times compared to traditional confocal or widefield microscopy. Moreover, acquiring multiple planes over time enables the reconstruction of 4D datasets, capturing dynamic processes in living samples without causing significant phototoxic effects.

Excellent Optical Sectioning

Another advantage of light sheet microscopy is its excellent optical sectioning capability. By illuminating a thin optical slice at a time, out-of-focus light and phototoxic effects are significantly reduced, resulting in an improved signal-to-noise ratio and higher resolution in the acquired images. This optical sectioning ability is particularly beneficial when imaging thicker samples or tissues, where light scattering and absorption can limit the achievable image quality and contribute to phototoxicity with other techniques.

Capable of Imaging Thicker Samples

This technique allows the imaging of thicker samples or tissues using selective plane illumination and a thin optical section. It minimizes light scattering and absorption by confining the excitation light to a specific plane, resulting in improved resolution and reduced background signal. The reduced light exposure also minimizes photodamage, allowing for more precise imaging of thick samples without compromising their integrity.

Ability to Label Specific Targets

One of the key advantages of fluorescence in light sheet microscopy, specifically, is the ability to label particular targets, such as proteins or cellular structures, with different fluorescent probes or markers. This enables researchers to visualize and distinguish other components within the specimen while minimizing phototoxicity. By using different fluorophores with distinct emission spectra, multiple targets can be simultaneously imaged and separated using appropriate filters or spectral detection techniques, further enhancing the imaging capabilities while minimizing the impact on the sample.

3D Imaging

The use of fluorescence in light sheet microscopy enables imaging in three dimensions. Moving the specimen relative to the light sheet allows multiple planes within the specimen to be sequentially illuminated and imaged, reconstructing a complete three-dimensional dataset. This provides detailed spatial information about the specimen, allowing researchers to study its structure, dynamics, and interactions with minimal phototoxicity concerns.

Long-Term Imaging of Living Specimens

Fluorescence in light sheet microscopy also enables imaging of living specimens over extended periods. Researchers can monitor and study dynamic events in real-time while mitigating phototoxicity effects by using appropriate fluorescent probes that are minimally toxic and do not perturb the biological processes of interest.

Physiological Preservation & Minimization of Photodamage

The high-speed imaging capability of light sheet microscopy, combined with fluorescence, allows for the capture of rapid biological processes with minimal photodamage to the specimen, facilitating long-term imaging experiments and preserving the physiological conditions of the sample.

In Summary

Light sheet microscopy is a technique that uses a thin sheet of laser light to illuminate specific planes within a sample selectively. 

This enables high-resolution, three-dimensional imaging of biological specimens with reduced phototoxicity and improved imaging speed. Its key features include excellent optical sectioning, fast acquisition, and the ability to image living samples. 

By studying complex biological systems and processes in their native three-dimensional context, light sheet microscopy provides valuable insights across various research areas.

With its high-resolution, three-dimensional imaging capabilities, light sheet microscopy has the potential to advance scientific understanding and drive discoveries in various fields. It supports breakthroughs in neuroscience, developmental biology, immunology, and other areas by visualizing neuronal circuits, embryogenesis, immune cell interactions, and dynamic processes. Non-invasively imaging living specimens and generating multidimensional datasets contributes to a deeper understanding of biological phenomena.

Ultimately, light sheet imaging has the power to uncover new insights and catalyze scientific discoveries across disciplines. Its ability to capture detailed three-dimensional information while minimizing phototoxicity positions it as a transformative tool for advancing our understanding of complex biological systems and processes.

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You can maximize your research potential with the latest light sheet microscopy and fluorescence microscopy technology through Excedr's flexible leasing options. Learn more about our cost-effective solutions that you can use to secure cutting-edge equipment without straining your budget.

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