High-performance multiphoton excitation microscope using SLM

Observation of deep region of the biological sample by two-photon excitation microscopy

Biological microscopes are used to observe reflections on the surfaces of biological specimens and to observe the transmission of thin sliced biological specimens. Objective lenses of the microscope are designed to have the best condensing condition and best optical performance in these observations. Also, when observing cells, the organelles inside the cell are close to or nearly colorless to the same color, so in many cases, a fluorescence method is used in which only the subject to be observed is stained with a dye and the fluorescence emitted by it is observed.

Recently, in the field of medical and biological sciences, it is required to directly observe deep parts of biological samples. However, if the observation of the deep position of a thick tissue section using biological microscopy is performed, the quality of the condensation by the objective lens will be reduced by deviating from the above design conditions and by generating optical distortion in the observation object itself. This causes blurring in the acquired fluorescent image, which greatly reduces resolution and contrast.

The two-photon excitation fluorescence microscope, which emerged in 1990, is an epoch-making device that can observe the deep part of a biological sample. This microscope uses the phenomenon of two-photon excitation for the emission of fluorescence. Compared with fluorescence observation using confocal fluorescence microscope, in fluorescence observation by two-photon excitation, ultrashort pulsed light of near infrared with high bio-transparency is used for excitation light used for emission of fluorescence, so observation is possible up to the deep part of biological sample. It also has a high depth resolution because the excitation light emits only near the focal point, which is narrowed down by the objective lens. These features are beneficial for observing deep living biological samples.

Three decades have passed since its appearance, and a variety of peripheral technologies have been in place, such as increasing the output and stability of laser light sources that contribute to higher imaging quality, and developing techniques to make living organisms transparent, which enable us to observe deeper sites. Two-photon excitation fluorescence microscopy has begun to be widely used to elucidate biological functions.

Two-photon excitation fluorescence microscope challenges

However, two-photon excitation fluorescence microscopy also has the following challenges:

(1) Image quality deteriorates due to aberrations generated during deep observation of living biological samples.

(2) Long measurement time due to point scan measurement.

(3) Lower resolution than confocal laser microscopy using visible light lasers.

We aim to contribute to medical and biological research by solving these problems using a spatial light modulator (SLM) and realizing high-precision deep observation.

Initiatives to solve problems using SLM

(1) Problem of deteriorating image quality due to aberrations generated when observing the depth of a biological sample

In two-photon excitation fluorescence microscopy, ultrashort pulsed light is made smaller by an objective lens, and light energy is concentrated in a high density in time and space to emit fluorescence. In the observation of the deep part of the biological sample, optical distortion is generated by deviating from the design condition of the objective lens and by being affected by the refractive index mismatch of the light on the surface and inside of the biological sample, and the light energy density is lowered by the blurring of the focal point. Therefore, the spatial resolution is reduced, and at the same time, the brightness of the fluorescence is also reduced, which greatly reduces the quality of the image. Such optical distortion is called aberration.

This aberration must be corrected for observation to obtain a high-resolution image even at the depth of the biological sample. When the SLM is used, the wavefront of the excitation light can be actively controlled to concentrate light in the deep part of the biological sample without the effect of aberration. The important thing to do at this time is to know what aberrations are occurring. This is because aberration is affected by the refractive index difference at the refractive index interface, but it is also affected by the shape of the refractive index interface. We have so far developed a method for correcting aberrations caused by refractive index differences and for correcting aberrations generated by the surface shape of the sample and have confirmed that it is also used for biological samples (Fig. 1).

Fig 1. Blood vessel image of transparent mouse brain (depth 1750 µm to 1900 µm)

If correction is made by considering aberrations due to the shape of the biological sample, blood vessels image became clearer (Reference 2).

(a)Applying the aberration correction method we have developed

(b)No aberration correction

(2) Problems with long measurement times

When observing a high-speed phenomenon of a biological sample, the shorter the measurement time, the more advantageous it is. Two-photon excitation fluorescence microscopy uses a galvo scanner, etc., and the system which scans the laser beam and acquires the image is the mainstream. In this scanning imaging, the measurement time is longer compared to the microscope of image illumination. To reduce the measurement time, we have adopted a multi-point simultaneous scanning imaging system, in which the excitation light is branched into multiple points for simultaneous imaging. This multi-point simultaneous scanning imaging was realized by making multiple focal points by controlling the wavefront of the excitation light using the SLM, and by combined it with a multiple location simultaneous detection device, such as a multi-anode photomultiplier tube. We have confirmed that the measurement time can be reduced to one-fourth by branching the excitation light to four points, that there is no effect on the imaging quality, and that deep observation is possible in combination with aberration correction (Fig. 2).

Fig. 2. Fluorescent images obtained by observing Hela cells staining mitochondria with Rhodamine 123

The measurement time has been reduced to 1/4 by performing simultaneous scanning by dividing the excitation light into four sections with SLM.

(3) Lower resolution problem than confocal microscopy using visible light

The resolution of the microscope is higher as the wavelength of the light used is shorter. Two-photon excitation fluorescence microscopy has the advantage of being highly biotransparent and reaching deep region because the wavelength of the excitation light is in the near-infrared region. However, because of its long excitation wavelength, it has a problem of lower resolution compared to confocal microscopy, which is visible light excitation. We are working on ways to increase resolution while maintaining the superiority of deep observations possessed by two-photon excitation fluorescence microscopy. To date, we have developed a method for improving resolution by controlling the intensity distribution of excitation light with the SLM.

Future targets

We are currently working with Hamamatsu University of Medicine on the study of two-photon excitation fluorescence microscopy using the SLM. To date, we have developed aberration corrections suitable for the optical clearing method and conducted observations on the kidney (Fig. 3).

■Images of wild-type (control) and condition-model mouth (HIGA) glomerulus

Renal instrumentation revealed that the protrusions on the vessels in the glomerulus, which were previously confirmed by observation using a conventional electron microscope, were derived from the basement membrane by using a two-photon excitation microscope equipped with the SLM (Reference 5).

■Three-dimensional observation: Mouse renal glomeruli observation cleared by an optical clearing agent

The fixed kidney of mouse was stained, followed by a tissue clearing process and three-dimensional observation. Podocytes* (cells that cover glomeruli) was stained green, blood vessels red, and nuclei blue. In the video, the glomerulus is observed from the surface to the depths while moving the objective lens up and down. By correcting the aberrations caused by the optical clearing agent using an SLM, it is possible to observe them deep even with a commercially available water-immersion objective.

Traditionally, we had made several preparates of kidney by slicing them and viewed them with a light microscope, but slicing had been associated with problems such as deterioration of cross-sectional shape and misalignment. By using the two-photon microscope incorporated with the SLM, it is possible to observe the stereological tissues well, which will make it easier to understand the morphological changes caused by the pathological conditions such as podocyte disorder（Sample was provided by Dr. Alu Konno Department of Microbiology and Immunology, Hamamatsu University School of Medicine）.

*Podocytes are cells that cover the periphery of the glomerulus and regulate filtration.

Two-photon excitation fluorescence microscopy will be used more widely in the future to elucidate biofunctions. The SLM of Hamamatsu Photonics is capable of making microscopes multifunctional and high-performance. We aim to contribute to medical and biological research by improving the wavefront control and device technologies, which are peripheral technologies surrounding the microscope and the SLM, so that phenomena that could not be observed before can be discovered.

Reference

N. Matsumoto, T. Inoue, A. Matsumoto, and S. Okazaki, “Correction of depth-induced spherical aberration for deep observation using two-photon excitation fluorescence microscopy with spatial light modulator,” Biomed. Opt. Express 6(7) 2575-2587 (2015).
N. Matsumoto, A. Konno, T. Inoue and S. Okazaki, “Aberration correction considering curved sample surface shape for non-contact two-photon excitation microscopy with spatial light modulator,” Sci. Rep. 9252 (2018).
N. Matsumoto, A. Konno, Y. Ohbayashi, T. Inoue, A. Matsumoto, K. Uchimura, K. Kadomatsu, and S. Okazaki, “Correction of spherical aberration in multi-focal multiphoton microscopy with spatial light modulator,” 25(6), 7055-7068 (2017).
N. Matsumoto, A. Konno, T. Inoue, K. Watanabe, and S. Okazaki, “Amplitude-modulation-type multi-ring mask for two-photon excitation scanning microscopy,” OSA continuum 4(6), 1696-1711 (2021).
A. Konno, N. Matsumoto, Y. Tomono, and S. Okazaki, “Pathological application of carbocyanine dye-based multicolour imaging of vasculature and associated structures,” Sci. Rep. 10, 12613 (2020).

High-performance multiphoton excitation microscope using SLM

Observation of deep region of the biological sample by two-photon excitation microscopy

Two-photon excitation fluorescence microscope challenges

Initiatives to solve problems using SLM

(1) Problem of deteriorating image quality due to aberrations generated when observing the depth of a biological sample

(2) Problems with long measurement times

(3) Lower resolution problem than confocal microscopy using visible light

Future targets

■Images of wild-type (control) and condition-model mouth (HIGA) glomerulus

■Three-dimensional observation: Mouse renal glomeruli observation cleared by an optical clearing agent

Reference

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