Multiphoton microscopy imaging technology: polarization-resolved second-harmonic generation microscopy and its image processing
In nonlinear optical microscopes, second harmonic generation (SHG) imaging is commonly used to observe endogenous fibrous structures, and the intensity of SHG largely depends on the relative angle between the polarization direction of the incident beam and the orientation axis of the target molecule. Therefore, polarization based SHG imaging (P-SHG) can obtain structural information of target molecules by analyzing the functional relationship between SHG signal intensity and the polarization state of the incident beam. It is now used as an important tool for medical and biological analysis.
Simple SHG images can be obtained through traditional two-photon excitation fluorescence microscopy (TPM). Most TPM systems still use a single beam scanning method based on a moving mirror, whose time resolution depends on the physical movement speed of the mirror. In order to achieve faster imaging, the TPM system can also adopt a multi beam scanning method (Figure 1A), one of which is to use a rotating disk scanning unit. This unit consists of a coaxial micro lens turntable and a pinhole turntable, with the micro lenses and pinholes on each turntable corresponding one-to-one.
When the laser passes through the micro lens turntable, the wavefront covers multiple micro lenses. Different micro lenses focus different parts of the wavefront to different positions and pass through corresponding pinholes, forming multiple micro beams. These micro beams hitting the sample can simultaneously excite multiple signals. These signals return along the microscope system and pass through the pinhole again, and are finally reflected by the dichroic mirror between the two turntables into the detection device. However, the commonly used mode-locked titanium sapphire laser as a light source has insufficient energy, which limits the number of excitation beams and results in a small effective field of view (FOV) for TPM (TPM-SD) using a rotary scanning unit.
Ai Goto et al. aimed to achieve high-speed P-SHG imaging with a large field of view (FOV) using the TPM-SD system. Therefore, a Yb based laser source with higher peak power was introduced into the TPM-SD system.
This is a schematic diagram of the TPM-SD system they developed. The light source of the system is a Yb based laser, which generates femtosecond pulses with a center wavelength of 1042 nm, an average power of 4 W, a pulse width of 300 fs, and a repetition rate of 10 MHz. The system first adjusts the laser power through a half wave plate and a Glan laser polarizer, and then expands the beam through a beam expander. The expanded beam is introduced into the turntable scanning unit, and multiple micro beams coming out of the scanning unit are focused on multiple points of the sample through a water immersion objective lens. In order to adjust the polarization state of the light beam at the objective lens, a half wave plate and a quarter wave plate are placed on the optical path of the excitation beam.
