Objective  Optical microscopy technology is to observe and record images of the microstructure of objects at a scale indistinguishable from the human eye, and has become an important tool for human observation of the microscopic world. Among them, electron microscopy has a resolution that breaks the limit of optical diffraction and can reach the nanometer level. However, it needs to provide a vacuum environment for electron acceleration, so it is not conducive to the observation of living samples. Optical microscopes are easy to operate and inexpensive, and are widely used in scientific research, industry, medicine and other fields. Conventional light microscopes rely on the contrast produced by differences in the optical properties of the sample for imaging, and do not require labeling or staining, but do not have sufficient specificity. In contrast, nonlinear optical microscopy not only realizes specificity imaging, but also has higher imaging depth and resolution. Among them, fluorescence microscopy technology with the help of fluorescent probes to label different components within the biological sample, through the detection of fluorescence signal to achieve imaging of the labeled components in the sample, to obtain its distribution within the sample. TPF (two-photon fluorescence) microscopic imaging technology based on the nonlinear effect of two-photon absorption, the fluorescence signal will not be excited outside the focal plane, and therefore has a high spatial resolution. TPF microscopy mostly uses infrared wavelength light source, which has lower phototoxicity and photobleaching to biological samples and higher imaging depth. In summary, this paper builds a TPF microscopy system based on femtosecond pulsed light source to study the imaging performance of the system on biological samples.

  Methods  We build a TPF microscopic imaging system (Fig.1), using a Ti: sapphire femtosecond laser as the excitation laser source, with a central wavelength of 800 nm, a repetition frequency of 80 MHz, and a pulse width of 100 fs. The scanning optical path of the system was formed by lens and a scanning oscillator to complete the collimation, beam reduction, and two-dimensional deflection of the excitation light. The fluorescence signal is converted into an electrical signal and processed by a computer to obtain the imaging results.

  Results and Discussions   The output spectrum of the femtosecond laser, and the fluorescence spectrum of rhodamine B were obtained using a TPF spectroscopic measurement system (Fig.2). The central wavelength of the femtosecond laser was 1 030 nm, and the half-height width of the spectrum was 14.47 nm. While the spectral range of the fluorescence covered from 620 nm to 710 nm, the intensity increased steeply from 620 nm, with a peak at 630 nm, and then the intensity decreased slowly with increasing wavelength, so the laser could effectively excite the TPF signal of the sample. The relationship between the two-photon fluorescence intensity and the excitation pulse power was analyzed by adjusting the power of the excitation pulse (Fig.4). The fluorescence intensity was linearly related to the square of the excitation power in the region of different concentrations of the samples. The ratio coefficient of fluorescence intensity to the square of the excitation power was larger in the region with higher concentration for the same excitation power. The fluorescence intensity distribution of the samples within 0-14 μm depth was obtained by 3D TPF microscopic imaging experiments of mouse brain sections (Fig.5). It was obtained that the gray matter portion within the mouse brain sample was located in the superficial layer within 6 μm of the sample, while the white matter portion was more widely distributed longitudinally. The depth distribution curves of the fluorescence intensity of different tissues were obtained by curve fitting, which led to an imaging depth of 12.9 μm for the system. The intensity distribution curves of the narrow slits of multiple samples were plotted, and by analyzing the minimum distance that the imaging system could resolve, a lateral resolution of at least 2.25 μm was derived.

  Conclusions  A femtosecond laser was used as the excitation laser source. The fluorescence spectra of the rhodamine B solution samples were measured under excitation at 800 nm. Thus a detection window of 636-703 nm was selected for subsequent microscopic imaging experiments. TPF microscopic imaging experiments of mouse brain sections stained by rhodamine B were carried out to obtain the fluorescence intensity distribution of biological samples in the depth of 0-14 μm by tomography imaging. After three-dimensional reconstruction of the images, it was concluded that the gray matter portion within the mouse brain sample was located in a shallow layer within 6 μm of the sample, while the white matter portion was more widely distributed longitudinally, while the gray matter of the mouse brain had higher fluorescence intensity and had a higher density than the white matter portion. The experimental results demonstrate that the constructed microscopic imaging system has excellent spatial resolution and imaging depth, with an imaging depth of 12.9 μm and a lateral resolution of at least 2.25 μm.