Multipolar microscopy is an advanced imaging technique that uses fluorescence to create detailed, three-dimensional images of cells and subcellular structures deep inside living tissues. This method has become a valuable tool in biological and medical research, offering a window into the complex processes of life. By visualizing tissues in their natural state, researchers can gain insights into everything from brain function to the progression of diseases.
The Principle of Multiphoton Excitation
To understand multipolar microscopy, it is helpful to first consider traditional fluorescence microscopy. In the conventional method, a single, high-energy particle of light, or photon, strikes a fluorescent molecule, called a fluorophore. This collision boosts an electron within the fluorophore to a higher energy state. As the electron returns to its stable, ground state, it releases a photon of a different color, which is the light that forms the image. This process works well for thin samples but struggles with thicker tissues.
Multiphoton microscopy operates on a different principle, first theorized by Maria Goeppert-Mayer in her 1931 doctoral dissertation. Instead of one high-energy photon, it uses two or more lower-energy photons that arrive at the fluorophore at virtually the same instant. These lower-energy photons, in the infrared spectrum, are less likely to be scattered by the tissue. For excitation to occur, the photons must be concentrated in both time and space, a condition achieved by using a rapidly pulsing laser focused to a very fine point.
The combined energy of these two photons is sufficient to excite the fluorophore, causing it to emit a light particle just as it would in conventional fluorescence. The probability of two photons being absorbed simultaneously is extremely low unless they are packed into an incredibly small volume. This means that fluorescence is effectively restricted to the tiny focal point of the laser. This localized excitation is the basis for the technique’s major advantages in imaging deep within living specimens.
Key Modalities of Multipolar Microscopy
The multiphoton principle gives rise to several imaging methods, known as modalities. Each modality generates a signal based on different interactions between light and the tissue, providing unique types of information. By combining these, scientists can build a more complete picture of the sample they are studying.
The most widely used modality is Two-Photon Excitation Fluorescence (2PEF). This method is analogous to standard fluorescence microscopy because it relies on the same types of fluorescent labels, or fluorophores, that are introduced into the sample to tag specific molecules or structures. When two photons excite a fluorophore, it emits a fluorescent signal that reveals the location and concentration of the tagged element, providing specific chemical information about the cell.
A second modality is Second-Harmonic Generation (SHG). Unlike 2PEF, SHG does not require external dyes. It arises from the interaction of laser light with highly organized biological structures that lack a central point of symmetry, known as non-centrosymmetric structures.
When focused laser light passes through materials like collagen or muscle myosin, the tissue itself generates a signal. It converts two incoming infrared photons into a single new photon with double the energy and half the wavelength. This SHG signal provides detailed structural information, allowing researchers to visualize the architecture of tissues like the collagen network.
Advantages for Deep Tissue Imaging
The way multiphoton microscopy generates a signal provides several advantages for researchers. These benefits stem from the localized excitation and use of longer-wavelength light, resulting in high-resolution images deep within living specimens.
A primary advantage is increased penetration depth. The infrared light used in multiphoton systems scatters less within biological tissue compared to the visible or ultraviolet light used in other techniques. This reduced scattering allows the laser light to travel deeper into a sample, enabling clear imaging hundreds of micrometers below the surface.
The technique also significantly reduces phototoxicity and photobleaching. Because fluorescence excitation is confined to the focal volume, the surrounding tissue is spared from damaging laser light. In other microscopy forms, the entire cone of light can cause damage and fade fluorescent molecules over time, a process called photobleaching. Limiting excitation this way minimizes stress on living cells, allowing for longer observation.
The method also provides intrinsic optical sectioning. Since the signal is only produced at the focal plane, there is no out-of-focus light that needs to be filtered out. This capability eliminates the need for a pinhole, which is required in confocal microscopy, improving the efficiency of signal collection and contributing to brighter images.
Applications in Scientific Research
The capabilities of multipolar microscopy have made it an indispensable tool across numerous fields of scientific inquiry. Its ability to image deep within intact, living tissue provides dynamic views of biological processes as they unfold.
In neuroscience, multiphoton microscopy is used to study the activity of individual neurons deep inside the brain of a living animal. Researchers can visualize changes in calcium levels, which are indicators of neural firing, or observe the physical growth and retraction of dendritic spines—the tiny structures involved in forming synaptic connections. This provides insight into learning, memory, and the progression of neurological disorders.
Developmental biologists use the technique to watch the movement of cells during embryogenesis. Because of its low phototoxicity, researchers can track cell migration, division, and differentiation in a developing embryo for hours or even days without harming it. This allows for the creation of detailed “fate maps” that show how a single fertilized egg develops into a complex organism.
The modality of Second-Harmonic Generation is useful in cancer research. The collagen fibers that make up the extracellular matrix surrounding a tumor change their organization as the cancer becomes more invasive. SHG microscopy can visualize the structure of this collagen network without any stains or labels. By analyzing the alignment and density of these fibers, researchers can gain clues about a tumor’s potential to metastasize.