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Multiphoton microscopy
What is multiphoton microscopy?
Multiphoton microscopy is a type of laser scanning fluorescence microscope represented by confocal microscopy. It utilizes long-wavelength, ultrashort pulse lasers and the phenomenon of multiphoton excitation to observe the deep regions of a sample.
The main advantages of multiphoton microscopy are as follows:
- Excitation light is of a long wavelength, allowing observation of deep regions.
- Only fluorescent dye molecules near the focal point are excited, minimizing fading.
- Fluorescence occurs only at the focal plane, eliminating the need for a pinhole and efficiently detecting scattered or refracted fluorescence within the sample.
- Due to its blueshift property, it enables simultaneous observation of multiple wavelengths.
Two-photon microscope image of mouse brain nerve cell
Provided by: Department of Medical Spectroscopy (now BioPhotonics Innovation Laboratory), Hamamatsu University School of Medicine
Figure 1: Optical system schematic of a confocal microscope
Figure 2: Optical system schematic of a multiphoton microscope
What is multiphoton excitation?
Multiphoton excitation refers to the phenomenon where fluorescent molecules are excited by simultaneously absorbing multiple photons. In this section, two-photon excitation, in which two photons are absorbed simultaneously, is used as an example.
Normally, if a fluorescent molecule is excited by 500 nm light, it absorbs a single photon at that wavelength and enters an excited state. However, in the case of two-photon excitation, it absorbs two photons with a wavelength of 1000 nm (which is twice the 500 nm wavelength) and becomes excited. Since the energy intensity of a single photon is inversely proportional to its wavelength, if we consider the energy of a 1000 nm photon as 1, the energy of a 500 nm photon would be 2. By absorbing two 1000 nm photons, the fluorescent molecule reaches the same excitation energy level.
To induce multiphoton excitation, extremely high photon density (intensity) is required momentarily. Therefore, femtosecond-level ultrashort pulse lasers are commonly used. Multiphoton excitation is a rare phenomenon, typically occurring only in the vicinity of the focal point where photons are concentrated by the objective lens.
Figure 3: Concept of two-photon excitation
What is blueshift?
Blueshift is the property where the maximum absorption wavelength of excitation light, for example during two-photon excitation, shifts to shorter wavelengths compared to the ideal wavelength.
For instance, when performing two-photon excitation with 1000 nm excitation light, it would be ideal for the excitation to occur at 500 nm. However, in practice, blueshift can cause excitation at wavelengths below 500 nm. This property allows simultaneous excitation of multiple fluorescent dyes using a single excitation wavelength.
While conventional fluorescence microscopes require multiple light sources or excitation filters to excite different fluorescent molecules individually, the application of blueshift in multiphoton microscopy enables observation of multiple wavelengths simultaneously using a single laser.
Example images
Observation of the mouse brain’s deep regions
Using a Hamamatsu Photonics photomultiplier tube module equivalent to H15460-40
Provided by: Masanori Murayama, Ph.D., RIKEN Center for Brain Science
Observation of glomeruli in pathological mouse models (HIGA)
Three-dimensional fluorescence observation of kidneys removed from each of the HIGA mouse model of IGA nephropathy (left) and wild-type mice (right) using two-photon microscopy. Blue: vascular endothelium, Green: basement membrane, Red: podocyte secondary process. In the HIGA mouse model of IGA nephropathy, the basement membrane between the vascular endothelium and the podocyte secondary process is seen to bulge out like a spherical ball. In wild-type mice, on the other hand, the basement membrane is not bulged.
Provided by: Aru Konno, Associate Professor, Department of Microbiology and Immunology, Hamamatsu University School of Medicine
High resolution observation of Thy1-YFP mouse using SLM two-photon microscopy
A cross-section of a brain sample from a Thy1-YFP mouse was observed with a NA1.1 objective lens for two-photon observation. By correcting astigmatism and coma aberration that exist in the optical system with an SLM, cell bodies can be observed with high resolution.
Provided by: Aru Konno, Associate Professor, Department of Microbiology and Immunology, Hamamatsu University School of Medicine
3D image of brain nerve fibers in Thy1-YFP mice using SLM two-photon microscopy
This image is a 3D reconstruction of a cross-section of a brain sample from a Thy1-YFP mouse, observed with a two-photon NA1.1 objective lens. By correcting astigmatism and coma aberration of the optical system with an SLM, cell bodies can be observed at high resolution and projections of brain nerve fibers can be clearly observed.
Provided by: Aru Konno, Associate Professor, Department of Microbiology and Immunology, Hamamatsu University School of Medicine
Multipoint scanning with SLM
HeLa cells were observed using a laser-scanning two-photon microscope equipped with an SLM. The left image shows four simultaneous excitation points using the SLM, whereas the right image shows a conventional single-point scan. 4-point simultaneous excitation enables 4-fold faster imaging. The SLM also enables us to obtain images comparable to single-point scanning even with four-point scanning by aligning the intensities among multiple points. A multianode PMT (H12310-40) is used as the detector.
Aberration correction by SLM
3 µm beads encapsulated in epoxy resin were observed using a water immersion objective lens with NA1.0. 200 µm x 200 µm in the XY direction was scanned using a galvanometer scanner, and 1000 images were acquired in the Z direction by moving the objective lens by 1.5 µm. Without SLM correction, spherical aberration occurs due to the difference in refractive index between water and epoxy resin. This causes the focused light shape to elongate vertically at deeper depths, reducing the energy density and making it difficult to observe the beads at deeper depths. On the other hand, SLM correction improves the focusing shape of the excitation light and maintains a high energy density even in deep areas.
Research introduction: High-performance multiphoton excitation microscopy using SLM
By using a spatial light modulator (SLM) to precisely control the wavefront of light, it is possible to manipulate light to improve the performance and functions of an optical system, such as forming multiple focal points to enable simultaneous multipoint observation and correcting optical distortion (aberration), which is a cause of reduced resolution. We aim to incorporate this SLM into a multiphoton excitation fluorescence microscope system to control the wavefront of the excitation laser light to enable highly accurate and simple observation of a living body from the surface to its depths. Currently, we are conducting research and development focused on achieving high precision and functionality using a two-photon excitation fluorescence microscope. We also collaborate with Hamamatsu University School of Medicine to explore fundamental and applied research using this advanced microscope system. Our goal is to contribute to medical and biological research by partnering with various universities in the future.
Recommended products
The H15460-40 is a photomultiplier tube module that employs a GaAsP photocathode photomultiplier tube. It has a wide effective surface area of 14 mm × 14 mm, high gain, and high sensitivity. This makes it an optimal detector for applications such as multiphoton excitation microscopy, where extremely weak light needs to be detected.
Multiphoton microscopy is a technique that allows accurate imaging of deep structures. However, as the depth increases, the fluorescence captured by small detectors is reduced due. The H15460-40 has a significantly larger active area compared to conventional photomultiplier tubes, enabling the capture of the lowest levels of fluorescence that cannot be captured by typical photomultiplier tubes. The H15461-40 contributions to high signal-to-noise ratio (S/N) image acquisition in multiphoton microscopy.
The LCOS-SLM (Liquid Crystal on Silicon - Spatial Light Modulator) is a device that electrically controls the phase of laser light by using a liquid crystal layer between a CMOS chip and transparent electrodes, enabling precise light phase manipulation through voltage application. It is utilized in various applications, including aberration correction, multipoint scanning, and resolution improvement in multiphoton microscopy.
Our photomultiplier tube (PMT) modules modules enhance multiphoton microscopy by delivering exceptional sensitivity and low noise, leading to higher contrast when detecting the faint fluorescence signals. Their fast response time allows faster imaging speeds, capturing dynamic biological processes with precision. Additionally, our PMTs offer a wide spectral response range enabling detection of multiple fluorophores in one sample. This adaptability, combined with reliable performance in demanding research settings, makes Hamamatsu's PMTs invaluable for achieving higher contrast while improving imaging speed in multiphoton microscopy applications.
Our Multi-Pixel Photon Counter (MPPC) modules are ideal for multiphoton microscopy by delivering high photon detection efficiency, and excellent signal-to-noise ratios, ideal for low-light imaging applications. With their solid-state design, MPPCs offer exceptional stability and longevity, providing reliable performance with low dark counts, no damage from high light levels, and high gain. Their compact, robust structure allows easy integration into microscope setups, enhancing sensitivity for capturing fine details, and fast biological processes in deep tissue imaging. Additionally, the gain mechanisms of the MPPC leads to a low excess noise factor allowing photon number resolution to be achieved. This makes Hamamatsu’s MPPC modules a powerful choice for achieving high-resolution, precise results in multiphoton microscopy applications.
MEMS mirrors enhance multiphoton microscopy by enabling precise, fast scanning capabilities with high stability. These mirrors offer high-speed scanning, essential for capturing dynamic biological events, while maintaining durability and low power consumption. This combination of compactness, agility, efficiency, and stability makes Hamamatsu’s MEMS mirrors a powerful addition for researchers seeking sharper, high-quality images in multiphoton microscopy.
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