We demonstrate a home-made two-photon laser scanning microscopy (TPLSM) with a light stimulus system. In this system, the femtosecond pulses are produced by a picosecond fiber laser with pulse width compression. A laser diode serves as stimulus, which modulated by an AOM and coupled into the same light path of a femtosecond laser. The control signal of AOM and trigger signal of two scanning mirrors are synchronized by a FPGA board. With modulating the intensity of CW laser at precise point in one scanning frame, any pattern of light stimulus can be delivered to the sample in real time.
Mouse is one of the most common animal models used in retinal researching, and obtaining its fundus images in vivo have significant significance for finding out formation and development mechanism of retinopathy, resolution and field-of-view are the key in fundus imaging. In this paper, we present an ophthalmoscope based on line scanning confocal technology, which utilizes one-dimension scanning line beam to increase image resolution and frame rate, it could capture mouse fundus at 1730*1730 μm field of view and 24 fps frame rate, retinal capillaries and vessels could be distinguished through reflectance and fluorescent images.
Decorrelation-based OCTA is a widely used optical coherence tomography angiography method which utilizes OCT intensity information. However, cardiac and respiratory motions in animals are seriously degrade image quality. These kind of bulk motion is periodic, and its C-Scan (slow scan) direction component hinder motion correction because of scan position and OCT structure’s change. Some correction methods were proposed, but vasculature information will be lost when larger bulk motion occur. Here we demonstrate a correction method which uses stitch scan protocol in C-Scan direction, and sets a threshold to the maximum value of normalized cross-correlation among repeated B-Scan intensity signal to exclude false OCT B-scans. Result of In vivo imaging experiment for mice indicates that our method can reserve whole vasculature information and effectively improve image quality.
We design a real-time laser stimulus system for laser confocal scanning microscope. By introducing the FPGA and AOM to achieve high speed modulation of a scanning laser, we can adjust the laser lighting area freely. For reducing the size of the optical path, we use MEMS-mirror instead of traditional fast and slow axis mirrors. The size of MEMS-mirror is 1.5 mm diameter and the scanning frequencies are set 16 kHz and 12 Hz at the fast and slow axis, respectively. Our system is capable of delivering stabilized large stimulus pattern (up to 500 x 500 pixels) to the biological tissues.
Phase instability is a serious problem in swept-source optical coherence tomography (OCT) with polygon tunable lasers; however, these devices have additional issues. We found that polygon tunable lasers also have fluctuations in output power and sweep range: the former creates artifacts that may impair the recognition of sample information, and the latter reduces the interference signal utilization during phase correction. We demonstrate a method that uses the calibration signal to quantify these problems and improve system stability and image quality. The proposed amplitude correction and phase correction methods are used to eliminate vertical artifacts and improve the resolution of OCT flow and intensity images while reducing the phase error.