CN118011614A - Dynamic speckle wide-field fluorescence imaging system based on random structured light illumination - Google Patents

Abstract

The invention provides a dynamic speckle wide-field fluorescent imaging system based on random structured light illumination. The method is characterized in that: the laser beam output by the laser forms a structural light stripe containing a speckle pattern through a grating and a scatterer, the structural light stripe is projected onto a sample to be detected through a microscope objective, the position of the grating is sequentially changed, the scatterer is continuously moved after the grating is moved to one position, a plurality of speckle illumination fluorescent images which dynamically change are recorded by a CMOS camera, and a fluorescent image with high three-dimensional spatial resolution is obtained through a four-step phase shift method and a root mean square algorithm. The method combines the structured light illumination and the dynamic speckle illumination, realizes a dynamic speckle wide-field fluorescent imaging system based on random structured light illumination, can acquire a high-resolution fluorescent tomographic image of a three-dimensional structure of a sample to be detected, has the characteristics of high three-dimensional spatial resolution, simple structure, low cost, simple and convenient operation and the like, and has wide application prospects in a plurality of research fields such as biology, medicine, life science and the like.

Description

Dynamic speckle wide-field fluorescence imaging system based on random structured light illumination

Technical Field

The present invention relates to a dynamic speckle wide-field fluorescence imaging system based on random structured light illumination, which combines structured light illumination and dynamic speckle illumination methods together, and projects structured light stripes containing speckle patterns onto a biological sample to be tested through a microscope objective lens, and changes the position of the grating in sequence. When the grating moves to a position, the scatterer is continuously moved, and a CMOS camera records multiple dynamically changing speckle illumination fluorescence images. A fluorescence image with high three-dimensional spatial resolution is obtained through a four-step phase shift method and a root mean square algorithm. The system can be used for high spatial resolution microscopic imaging of the three-dimensional structure of a living single cell or biological tissue to obtain clearer tomographic resolution images, and belongs to the field of biophotonics.

Background technique

The 21st century is the century of life sciences. People's understanding of life begins with cells, and gradually unfolds from macro to micro, from the whole to the part, from individuals to organs, tissues, cells, organelles, and even the basic substances that make up life - molecules. As the basic unit of life structure and function, in-depth research on cells is the key to revealing the mysteries of life phenomena, conquering diseases and transforming life.

The emergence of optical microscopy technology plays a vital role in the understanding of the genetics, development and physiological functions of organisms, as well as pathology, pharmacology, etc., which are the basis of medicine, and agricultural breeding, etc., and the observation of single cells. Optical microscopy technology refers to the technology that can obtain a magnified image of a tiny sample after passing through one or more lenses through the visible light that passes through the sample or reflected from the sample. The obtained image can be observed directly with the eyepiece, or recorded with a digital image detector, and can also be displayed and analyzed on a computer, making research more convenient.

Traditional wide-field fluorescence microscopes focus the excitation light through the objective lens and collect the fluorescence signal of the sample for imaging. It is not difficult to observe its lighting method. Although the light on the focal plane is the strongest, the samples above and below it will also be illuminated, resulting in the following limitations: additional phototoxicity is introduced, affecting the biological activity of the sample and even causing cell death; interference signals outside the imaging focal plane enter the image, resulting in reduced image resolution and contrast.

In recent years, fluorescence microscopy and laser scanning confocal microscopy structured light illumination imaging have become important tools for studying cells. Fluorescence microscopy uses ultraviolet light as a light source and uses it to illuminate the object to be inspected, causing the object to emit fluorescence. Finally, the shape and specific position of the object can be observed under a microscope. The spatial resolution of a fluorescence microscope is determined by the Abbe diffraction limit. This Abbe diffraction limit is suitable for oblique uniform illumination, far-field optical illumination and detection, and linear absorption and emission regions. It has a wide range of applications in cell biology. It also has the characteristics of high specificity, high spatial resolution, and three-dimensional tomography capabilities. However, exogenous fluorescent labels will inevitably affect the properties and life activities of the cells themselves, and may even produce phototoxicity and photobleaching of the fluorophore itself, which are problems that cannot be avoided by fluorescence microscopy.

The emergence of optical microscopes with tomographic resolution capabilities has epoch-making significance for the development of microscopic imaging technology. The emergence of this microscope enables optical continuous sectioning and three-dimensional structural reconstruction of cells and tissues, providing an important tool for research. Laser scanning confocal fluorescence microscopy is based on fluorescence microscopy imaging with the addition of a laser scanning device, using ultraviolet or visible light laser fluorescent probes, and using a computer for image processing. It can not only observe fixed cells and tissue sections, but also observe and detect the structure, molecules, and ions of living cells in real time and dynamically. Compared with ordinary wide-field fluorescence microscopy imaging methods, its advantage is that it can effectively improve the spatial resolution and imaging quality of images, and has the ability of deep resolution and three-dimensional tomographic imaging. The disadvantages are that the system structure is complex, the cost is high, and the imaging speed is slow.

In recent years, the emergence of dynamic speckle microscopy has solved this problem. This imaging method not only has tomographic resolution capabilities, but also uses a wide-field imaging method, does not require complex equipment, has fast imaging speed and low cost.

Although dynamic speckle microscopy has the ability of tomographic resolution, in order to obtain the three-dimensional structure of the object to be measured, it is necessary to obtain multi-layer tomographic images, which requires continuous adjustment of the focus position of the microscope, which is very inconvenient to operate. The present invention uses continuous adjustment of the position of the grating to achieve multi-layer tomographic images, and to achieve this goal, it is necessary to use the four-step phase shift method in structured light illumination. Structured light illumination is a lighting method that changes the spatial structure of the illumination light. Usually, the structured light of the illumination is a carrier frequency stripe. This lighting method can be used for the measurement of angles, lengths, vibrations, etc., and is widely used in three-dimensional imaging. This special lighting method forms moiré patterns to make visible some high-resolution information that cannot be distinguished under conventional lighting methods.

The grating projection measurement method is a type of structured light illumination and is also a representative three-dimensional visual technology developed in recent years. In the actual experimental process, a grating is inserted into the illumination light path. The illumination light is modulated by the grating and projected onto the sample through the objective lens. In this way, the sample is illuminated by the modulated light at the focal plane and is not affected even when it is far away from the focal plane. Finally, the fluorescence information generated by the modulated light is received by the CCD through the imaging system, and then the spatial domain and frequency domain are changed through Fourier transform to obtain an image. This measurement method has the advantages of non-contact, low cost and high precision. The most commonly used algorithm in the digital grating projection measurement method is the phase shift algorithm. The present invention adopts the four-step phase shift method in the dependent algorithm, which has the advantages of high resolution, high precision, simple algorithm and fast processing speed. In recent years, it has been widely used in the field of optical three-dimensional measurement and imaging.

The present invention relates to a dynamic speckle wide-field fluorescence imaging system based on random structured light illumination. The system adopts a four-step phase shift method in structured light illumination. A grating is required in the system. The grating rotates four times, each time by 90 degrees and reaches a stable state. Then, a wide-field fluorescence microscopy technique with dynamic speckle illumination is used to obtain a tomographic image of the cell. By obtaining cell tomographic images at different angles, a high-resolution three-dimensional cell structure image is finally obtained.

Summary of the invention

The purpose of the present invention is to provide a dynamic speckle wide-field fluorescence imaging system based on random structured light illumination, which has the advantages of fast imaging speed, high resolution, simple structure, and no need for external markers.

The object of the present invention is achieved in that:

When the sample to be tested is illuminated by dynamic speckle, the fluorescent marker dye in the cell is excited and emits fluorescence, which is collected by the detection objective lens and transmitted to the CMOS camera to achieve cell imaging. The present invention adopts a four-step phase shift method, which is divided into four times, each time with an interval of 90° to move the position of the grating 5, respectively. Pictures of cells were captured with a CMOS camera at four different phase shift positions.

In equations (3)-(6), A(x,y) is the background light intensity; B(x,y) is the reflectivity of the object surface; f is the spatial frequency of the grating fringes; is the principal phase value truncated at (-π, +π].

In the dynamic speckle wide-field fluorescence imaging system based on random structured light illumination, when the laser beam passes through a grating 5 and a constantly changing scatterer 7, a series of randomly changing speckle patterns will be formed on the back focal plane of the apochromatic microscope objective 15, and these speckle patterns are used to illuminate the cells to be tested 16. The fluorescence signals generated by the cells to be tested 16 after being excited are divided into two sources, one is the fluorescence signal from the focal plane of the field of view of the apochromatic microscope objective 15, and the other is the background fluorescence signal from outside the focal plane of the field of view of the apochromatic microscope objective 15. As the illumination speckle pattern changes, the fluorescence intensity of the granular distribution generated by the speckle illumination changes dramatically in the focal plane of the apochromatic microscope objective 15, but changes slowly outside the focal plane of the field of view. This signal feature is the basis for realizing tomographic imaging. The CMOS camera 11 records a series of changing speckle patterns on a specific layer of the cells to be tested 16, and a special algorithm can be used to extract the fluorescence signal of this layer, thereby having a tomographic imaging function.

In formula (2), N is the number of images in the image sequence, _Ii is the intensity of the i-th image, and I _Rms is the root mean square image of the N images collected, that is, tomographic imaging. Generally, N is 50-60 to obtain a relatively clear fluorescence tomographic image.

A dynamic speckle wide-field fluorescence imaging system based on random structured light illumination, characterized in that: the system mainly consists of a laser light source 1; lenses 2, 3, 8, 9; micro-displacement stages 4, 6; gratings 5; scatterers 7; computers 10; CMOS cameras 11; imaging lenses 12; filters 13; dichroic mirrors 14; apochromatic microscope objective lenses 15; cells to be measured 16; and a stage 17. In the system, the laser light beam emitted by the laser light source 1 is expanded by lenses 2 and 3 and then projected onto the grating 5, and then forms structured light stripes containing a speckle pattern through the scatterer 7, and then expands through lenses 8 and 9, and forms an image of the speckle pattern on the rear focal plane of the apochromatic microscope objective lens 15 after being reflected by the dichroic mirror 14, and forms full-field illumination on the cells to be measured 16 through the apochromatic microscope objective lens 15; the position of the grating 5 is changed by moving the micro-displacement stage 4 to realize the four-step phase shift method, and the micro-displacement stage 6 is moved at each phase shift position to change the position of the scatterer 7 The position of the structured light stripes containing the speckle pattern projected on the cell to be tested 16 changes, thereby realizing random structured light illumination; the fluorescence signal generated by the random structured light excitation is collected by the apochromatic microscope objective 15, the background noise is eliminated by the dichroic mirror 14 and the filter 13, and the fluorescence signal is recorded by the CMOS camera 11 through the imaging lens 12. The fluorescence signal of the cell to be tested or the biological tissue in the focal plane is extracted by the computer 10 using the four-step phase shift method and the root mean square algorithm, thereby obtaining a three-dimensional structure fluorescence tomography image with high spatial resolution of the sample to be tested.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a schematic diagram of the structure of a dynamic speckle wide-field fluorescence imaging method and system based on random structured light illumination.

Figure 2 shows the four-step phase shift method.

Figure 3 shows the four-step phase shift method after adding speckle.

FIG4 is a schematic diagram of dynamic speckle illumination wide-field fluorescence microscopy technology.

Explanation of the reference numerals: 1-laser light source; 2-lens; 3-lens; 4-micro-displacement stage; 5-grating; 6-micro-displacement stage; 7-scatterer; 8-lens; 9-lens; 10-computer; 11-CMOS camera; 12-imaging lens; 13-filter; 14-dichroic mirror; 15-apochromatic microscope objective; 16-cell to be tested; stage 17.

Detailed ways

The present invention will be further described below in conjunction with specific embodiments.

A dynamic speckle wide-field fluorescence imaging system based on random structured light illumination, characterized in that: the system mainly consists of a laser light source 1; lenses 2, 3, 8, 9; micro-displacement stages 4, 6; gratings 5; scatterers 7; computers 10; CMOS cameras 11; imaging lenses 12; filters 13; dichroic mirrors 14; apochromatic microscope objective lenses 15; cells to be measured 16; and a stage 17. In the system, the laser light beam emitted by the laser light source 1 is expanded by lenses 2 and 3 and then projected onto the grating 5, and then forms structured light stripes containing a speckle pattern through the scatterer 7, and then expands through lenses 8 and 9, and forms an image of the speckle pattern on the rear focal plane of the apochromatic microscope objective lens 15 after being reflected by the dichroic mirror 14, and forms full-field illumination on the cells to be measured 16 through the apochromatic microscope objective lens 15; the position of the grating 5 is changed by moving the micro-displacement stage 4 to realize the four-step phase shift method, and the micro-displacement stage 6 is moved at each phase shift position to change the position of the scatterer 7 The position of the structured light stripes containing the speckle pattern projected on the cell to be tested 16 changes, thereby realizing random structured light illumination; the fluorescence signal generated by the random structured light excitation is collected by the apochromatic microscope objective 15, the background noise is eliminated by the dichroic mirror 14 and the filter 13, and the fluorescence signal is recorded by the CMOS camera 11 through the imaging lens 12. The fluorescence signal of the cell to be tested or the biological tissue in the focal plane is extracted by the computer 10 using the four-step phase shift method and the root mean square algorithm, thereby obtaining a three-dimensional structure fluorescence tomography image with high spatial resolution of the sample to be tested.

In the system, the laser beam emitted by the laser light source 1 is expanded by lenses 2 and 3 and then projected onto the grating 5. Then, the position of the grating is changed every 90° to complete the four-step phase shift method. Then, the beam is expanded by lenses 8 and 9, and after being reflected by the dichroic mirror 14, an image is formed on the rear focal plane of the apochromatic microscope objective 15. The apochromatic microscope objective 15 forms full-field illumination on the cell to be measured 16. The CMOS camera 11 records the four images formed by the four-step phase shift method, and then reconstructs them through a computer algorithm to form a high spatial resolution cell image.

Claims (3)

1. A dynamic speckle wide-field fluorescence imaging system based on random structured light illumination, characterized in that: the system mainly consists of a laser light source 1; lenses 2, 3, 8, 9; micro-displacement stages 4, 6; gratings 5; scatterers 7; computers 10; CMOS cameras 11; imaging lenses 12; filters 13; dichroic mirrors 14; apochromatic microscope objective lenses 15; cells to be measured 16; and a stage 17. In the system, the laser light beam emitted by the laser light source 1 is expanded by lenses 2 and 3 and then projected onto the grating 5, and then forms structured light stripes containing a speckle pattern through the scatterer 7, and then expands through lenses 8 and 9, and forms an image of the speckle pattern on the rear focal plane of the apochromatic microscope objective lens 15 after being reflected by the dichroic mirror 14, and forms full-field illumination on the cells to be measured 16 through the apochromatic microscope objective lens 15; the position of the grating 5 is changed by moving the micro-displacement stage 4 to realize the four-step phase shift method, and the micro-displacement stage 6 is moved at each phase shift position to change the position of the scatterer 7 The position of the structured light stripes containing the speckle pattern projected on the cell to be tested 16 changes, thereby realizing random structured light illumination; the fluorescence signal generated by the random structured light excitation is collected by the apochromatic microscope objective 15, the background noise is eliminated by the dichroic mirror 14 and the filter 13, and the fluorescence signal is recorded by the CMOS camera 11 through the imaging lens 12. The fluorescence signal of the cell to be tested or the biological tissue in the focal plane is extracted by the computer 10 using the four-step phase shift method and the root mean square algorithm, thereby obtaining a three-dimensional structure fluorescence tomography image with high spatial resolution of the sample to be tested. 2. The dynamic speckle wide-field fluorescence imaging system based on random structured light illumination according to claim 1. The structured light illumination microscopic imaging system mainly comprises a laser light source 1; lenses 2, 3, 8, 9; a micro-displacement stage 4; a grating 5; a computer 10; a CMOS camera 11; an imaging lens 12; a dichroic mirror 14; an apochromatic microscope objective 15; and a cell to be measured 16. In the system, the laser light beam emitted by the laser light source 1 is expanded by lenses 2 and 3, projected onto the grating 5 to generate structured light stripes, then expanded by lenses 8 and 9, reflected by the dichroic mirror 14, and formed an image on the rear focal plane of the apochromatic microscope objective 15. The apochromatic microscope objective 15 forms a structured light full-field illumination on the cell to be measured 16. By sequentially changing the position of the grating, the structured light stripes projected on the sample to be measured are phase-shifted, thereby realizing a structured light illumination fluorescence microscopic imaging method based on a four-step phase shift method. 3. The dynamic speckle wide-field fluorescence imaging system based on random structured light illumination according to claim 1. In order to increase the tomographic imaging depth of the structured light illumination fluorescence microscopy imaging method, a scatterer 7 mounted on a micro-displacement stage 6 is added behind the grating 5. The structured light stripes generated by the laser beam through the grating 5 pass through the scatterer 7 to generate structured light stripes with a speckle pattern, and an image of the structured light stripes with a speckle pattern is formed on the rear focal plane of the apochromatic microscope objective 15, and full-field illumination is formed on the cell to be tested 16 through the apochromatic microscope objective 15. When the grating 5 moves to a specific position under the control of the micro-displacement stage 4 and reaches a stable state, the position of the scatterer 7 is changed by the micro-displacement stage 6, so that the structured light stripes with a speckle pattern projected on the cell to be tested 16 can be changed, and the fluorescence signal generated by the fluorophore in the sample to be tested is collected by the apochromatic microscope 15, and a plurality of fluorescence images are recorded by the CMOS camera 11. The position of the grating 5 is adjusted in sequence, and the scatterer 7 is continuously moved at each specific position to change the structured light stripes with speckle patterns projected on the sample to be tested, so as to excite the fluorophores in the sample to be tested to generate fluorescence signals. Since the intensity of the fluorescence signal generated by the excitation near the focal plane of the sample to be tested changes most dramatically under the wide-field illumination condition of the structured light stripes with dynamically changing speckle patterns, the fluorescence signal near the focal plane can be extracted by using the root mean square algorithm, thereby increasing the tomographic depth of the structured light illumination fluorescence microscopy imaging, realizing a dynamic speckle wide-field fluorescence imaging system based on random structured light illumination, and obtaining a three-dimensional high-resolution fluorescence tomographic image of the biological cells or tissues to be tested.