Multiphoton microscopy

What is multiphoton microscopy?
What is multiphoton excitation?
What is blueshift?
Example images
Featured product: Photomultiplier tube module H15460-40 / H15461-40
Featured product: LCOS-SLM
Research introduction: High-performance multiphoton excitation microscopy using SLM
Multiphoton microscopy related products
Other imaging techniques

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 microscopic image of mouse brain neurons

Two-photon microscope image of mouse brain nerve cell

Provided by: Department of Medical Spectroscopy (now BioPhotonics Innovation Laboratory), Hamamatsu University School of Medicine

Optical system schematic of a confocal microscope

Figure 1: Optical system schematic of a confocal microscope

Optical system schematic of a multiphoton 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.

Concept of two-photon excitation

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.

Featured product: Photomultiplier tube module H15460-40 / H15461-40

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, making 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 imaging of deep structures. However, as the depth increases, the fluorescence captured by small detectors is reduced due to scattering effects caused by internal structures. The H15460-40 has a significantly large light-receiving surface compared to conventional photomultiplier tubes, enabling the capture of fluorescence in a range that cannot be captured by typical photomultiplier tubes and contributing to high signal-to-noise ratio (S/N) image acquisition.

Photomultiplier tube module H15460-40 (Voltage output)

Photomultiplier tube module H15461-40 (Voltage output)

Featured product: LCOS-SLM

The LCOS-SLM (Liquid Crystal on Silicon - Spatial Light Modulator) is a device that allows electrical control of the phase of laser light. It consists of a structure where a liquid crystal is sandwiched between a CMOS chip with pixel electrodes arranged in a two-dimensional pattern and transparent electrodes deposited on a glass substrate. Digital images output from a PC are converted to analog signals by a dedicated driving circuit and applied with voltage to the pixel electrodes on the CMOS chip. By tilting the liquid crystal molecules through this voltage, the refractive index of the liquid crystal changes, allowing precise control of the phase of light irradiated onto each pixel. The LCOS-SLM, capable of high-precision control of the light phase, finds applications ranging from research purposes such as optical vortex generation to industrial applications like aberration correction in microscopy and fine code marking through multipoint branching.

For two-photon microscopy, the LCOS-SLM is used for studies for the following applications:

Aberration correction based on the shape of biological specimens.
Multipoint simultaneous scanning using branched excitation light.
Improved resolution through controlled excitation light spots.

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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.

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