Optical sectioning is a microscopy technique that creates clear, high-resolution images of specific planes deep within a sample. It achieves this by selectively eliminating blurry light from regions above and below the focused area. This allows for revealing internal structures without physically cutting the specimen, benefiting fields like biology and materials science.
Overcoming Blurry Images
Traditional widefield microscopy challenges imaging thick specimens. Light illuminates the entire sample, and the detector captures light from all depths simultaneously. This means light from both in-focus and out-of-focus planes contributes to the final image.
The light from out-of-focus regions appears as a hazy background, obscuring fine details within the focal plane. This is similar to trying to read a book through a frosted window. This out-of-focus blur makes it difficult to discern internal structures clearly and hinders accurate three-dimensional reconstructions of the sample.
The Core Principle of Optical Sectioning
Optical sectioning techniques address out-of-focus blur by selectively collecting light from a very thin plane, often called an “optical slice” or “virtual slice.” This process rejects light from out-of-focus regions from reaching the detector.
The mechanism relies on manipulating the microscope’s point spread function (PSF). The PSF describes how a microscope images a point of light. Optical sectioning methods narrow this PSF in the Z-axis (depth) direction, ensuring only light from the chosen focal plane contributes to the image, thereby improving resolution.
Common Optical Sectioning Methods
Optical sectioning techniques achieve high-resolution imaging within thick samples. Each method employs unique optical principles to isolate the signal from a specific focal plane.
Confocal microscopy
Confocal microscopy employs a pinhole to block out-of-focus light. A focused laser beam scans the specimen point by point. Emitted fluorescence from the focal plane passes through a pinhole in front of the detector, blocking light from outside this plane and ensuring a clear, optically sectioned image. By acquiring multiple optical sections at different depths, three-dimensional reconstructions of the sample can be generated.
Multiphoton microscopy
Multiphoton microscopy utilizes longer-wavelength light, in the infrared spectrum, for excitation. Instead of a single photon, two or more photons simultaneously excite fluorophores only at the precise focal point where photon density is highest. This localized excitation reduces photodamage to the specimen and allows for deeper penetration into thick biological tissues.
Light sheet microscopy
Light sheet microscopy illuminates a thin plane of the sample with a sheet of laser light. A detection objective positioned perpendicularly to this light sheet captures emitted fluorescence only from the illuminated plane. This orthogonal arrangement minimizes photobleaching and phototoxicity, enabling faster image acquisition and extended observation of living samples while maintaining high spatial and temporal resolution.
Impact Across Fields
Optical sectioning techniques have impacted various scientific disciplines by enabling researchers to visualize structures in three dimensions with clarity.
In biology, these methods allow for detailed imaging of live cells, complex tissues, and developing organisms, providing insights into dynamic cellular processes and tissue architecture. Neuroscience has benefited from the ability to visualize neural networks and monitor brain activity in living specimens.
Developmental biologists use optical sectioning to track cell lineages and observe organ formation during embryogenesis. Materials scientists also employ these techniques to analyze the internal structures of non-biological materials, aiding in the development of new materials with desired properties. In medicine, optical sectioning contributes to diagnostics and a deeper understanding of disease progression by providing high-resolution images of biological samples.