High-accuracy fluorescence anisotropy microscopic imaging device and method

Abstract

The invention relates to a high-accuracy fluorescence anisotropy microscopic imaging device and method. The imaging method comprises the following steps of: on a reflective fluorescence microscope, leading lights emitted by a light source to pass through a polarizer, an excitation filter plate and a spectroscope, and then focusing the lights on a sample; leading fluorescence reflected by the sample to pass through an objective lens, the spectroscope and an emission filter plate, and then imaging on an image plane of a CCD (Charge-Coupled Device) camera through an adjustable angle analyzer; controlling the adjustable angle analyzer to rotate around an optical axis of an imaging optical path step by step by using a computer, wherein when the adjustable angle analyzer rotates by an angle every time, the CCD camera shoots a frame of polarized fluorescence image, M images are totally shot; and finally calculating fluorescence anisotropic images from the M images by using Fourier transform.The imaging method is characterized in that the measuring resolution ratio of the fluorescence anisotropy is greatly improved by establishing a single-channel fluorescence analyzing optical path; andthe microscopic imaging system has higher stability and better anti-jamming capability.

Description

technical field [0001] The invention relates to a method and an instrument for fluorescent microscopic imaging, in particular to a device and a method for performing high-precision fluorescent anisotropic microscopic imaging on biological samples by means of a reflective wide-field fluorescent microscope. Background technique [0002] Optical microscopy imaging technology can observe living cells in the normal state of the sample, keeping the real environment of the sample to the greatest extent. However, due to the limitations of Abbe's principle, it is impossible to use conventional wide-field microscopy imaging techniques to study the interaction between molecules at the nanometer scale. Fluorescence anisotropic microscopic imaging technology is based on the optical phenomenon of fluorescence polarization. It can perform functional imaging of the interaction between fluorescent molecules at the nanometer scale. It has strong signal selectivity. Compared with other functio...

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Problems solved by technology

However, as described in "Imaging lifetime and anisotropy spectrum in the frequency domain," in Journal of Microscopy-Oxford. vol. 234, 2009, pp. 80-88, the traditional fluorescence polarization microscopy imaging method is used Observations on different samples show that the light intensity spectrum distribution of rhodamine 6G and 59% glycerol solution is uniform, but in the fluorescence anisotropy diagram of fluorescence polarization, noise can be clearly seen

And for observing the one-dimensional spatial distribution light intensity spectrum of rhodamine 6G and 50% glycerol solution and 670nm fluorescent sphere, the corresponding spectral line positions can be clearly distinguished from the light intensity spectrum, but in the anisotropy spectrum, the noise Extremely obvious, it is difficult to identify the position of the fluorescent ball

In "Wide-field time-resolved fluorescence anisotropy imaging (TR-FAIM): Imaging the rotational mobility of a fluorophore," in Review of Scientific Instruments. vol. 74, 2003, pp. 182-192 As mentioned in , for CHO cells expressing erbB1–eGFP fluorescence image and anisotropy image spectrum, the internal details of the cell can be resolved on the light intensity image, but the interior details of the cell can hardly be resolved on the anisotropy image of the fluorescence polarization details, indicating that the resolution of existing fluorescence anisotropy microscopy imaging methods is not enough to support finer observation of cells

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