Frequency-shift super-resolution microimaging method and device based on evanescent field illumination

Abstract

The invention discloses a frequency-shift super-resolution microimaging method based on evanescent field illumination. The method comprises the steps that 1) incident illumination light obliquely irradiates a substrate medium interface, is totally reflected and generates an evanescent field; 2) the evanescent field is used for illuminating the surface of a sample; a strength image of the surface of the sample is received by a microscope; 3) the strength image is subjected to Fourier transformation; a corresponding frequency spectrum is obtained and is restored; a corresponding frequency spectrum restoring image is obtained; 4) the direction of the incident illumination light is changed for many times around the sample till the direction of the incident illumination light covers 0-360 DEG; frequency spectrum restoring images in different directions are obtained; 5) the frequency spectrum restoring images in the different directions are stacked; a complete high frequency spectrum image is obtained; and 6) the complete high frequency spectrum image is subjected to inverse Fourier transformation; and a super-resolution microscopic image of the observed sample is obtained. The invention further discloses a frequency-shift super-resolution microimaging device based on the evanescent field illumination.

Description

technical field

[0001] The invention belongs to the field of microscopic observation and measurement, and in particular relates to a frequency-shifting super-resolution microscopic imaging method and device based on evanescent field illumination. Background technique

[0002] Nanotechnology and biotechnology are the fastest-growing and hottest scientific fields in the 21st century. Nanotechnology has a wide range of applications, including imaging, measurement, processing, manipulation, etc. within the scale of 1-100nm. Many important organisms such as glucose, antibodies, viruses, etc. are in this scale range, and the need to study these tiny objects has driven the development of high-resolution microscopy. In turn, the development of super-resolution microscopy has also promoted the progress of the entire life sciences. A major advantage of light microscopy over other microscopy techniques is the ability to study living cells in their natural state. Since the world's fi...

Examples

Embodiment 1

[0066] Such as figure 1 As shown, the device of the frequency-shifting super-resolution microscopic imaging system based on evanescent field illumination includes: a light source group 1, a prism 2, a microscope 3, a computer 4, and a sample 5.

[0067] The light source group 1, the prism 2, the microscope 3 and the sample 5 are located on the coaxial optical path. The incident illumination light emitted by a single sub-light source 6 in the light source group 1 is totally reflected in the prism 2 to produce an evanescent field to illuminate the sample 5 . The generated light intensity distribution is collected and analyzed by the microscope 4 in the far field. Wherein, the incident illumination light refers to visible monochromatic linearly polarized collimated light with the same polarization direction and a wavelength in the range of 380-780 nm.

[0068] Fundamentally speaking, the incident illumination light is provided by LED7, in order to meet the requirements of the a...

Embodiment 2

[0086] Except in the specific embodiment of the present inventionfigure 1 In addition to the structural principle diagram provided in , the present invention can also improve the lighting method under the premise of ensuring the same principle.

[0087] Such as Figure 8 Shown is a structural principle diagram of the improved illumination mode of the frequency-shifting super-resolution microscopic imaging device based on evanescent field illumination of the present invention.

[0088] Such as Figure 8 As shown, after improving the illumination mode, the device of the frequency-shifting super-resolution microscopic imaging system based on evanescent field illumination includes: a laser light source 13, a vibrating mirror 14, a focusing mirror 15, a reflecting mirror 16, a total reflection objective lens 17, Slide 18, Microscope 3, Computer 4, Sample 5.

[0089] Laser light source 13, vibrating mirror 14, focusing mirror 15, reflecting mirror 16, total reflection objective le...

Embodiment 3

[0093] In addition to the method described in Embodiment 1 that can improve the illumination method, it can also be improved by using an off-center diaphragm.

[0094] Such as Figure 9 Shown is a schematic diagram of the structure of the evanescent field illumination-based frequency-shifting super-resolution microscopic imaging device of the present invention after using an eccentric diaphragm to improve the illumination mode.

[0095] Such as Figure 9 As shown, after improving the illumination mode, the device of the frequency-shifting super-resolution microscopic imaging system based on evanescent field illumination includes: a laser light source 13, an eccentric diaphragm 19, a total reflection objective lens 17, a glass slide 18, and a microscope 3, Computer 4, Sample 5.

[0096] Laser light source 13, eccentric diaphragm 19, total reflection objective lens 17, glass slide 18, microscope 3, and sample 5 are located on the coaxial optical path. The incident illuminatio...

technical field

[0001] The invention belongs to the field of microscopic observation and measurement, and in particular relates to a frequency-shifting super-resolution microscopic imaging method and device based on evanescent field illumination. Background technique

[0002] Nanotechnology and biotechnology are the fastest-growing and hottest scientific fields in the 21st century. Nanotechnology has a wide range of applications, including imaging, measurement, processing, manipulation, etc. within the scale of 1-100nm. Many important organisms such as glucose, antibodies, viruses, etc. are in this scale range, and the need to study these tiny objects has driven the development of high-resolution microscopy. In turn, the development of super-resolution microscopy has also promoted the progress of the entire life sciences. A major advantage of light microscopy over other microscopy techniques is the ability to study living cells in their natural state. Since the world's fi...

Examples

Embodiment 1

[0066] Such as figure 1 As shown, the device of the frequency-shifting super-resolution microscopic imaging system based on evanescent field illumination includes: a light source group 1, a prism 2, a microscope 3, a computer 4, and a sample 5.

[0067] The light source group 1, the prism 2, the microscope 3 and the sample 5 are located on the coaxial optical path. The incident illumination light emitted by a single sub-light source 6 in the light source group 1 is totally reflected in the prism 2 to produce an evanescent field to illuminate the sample 5 . The generated light intensity distribution is collected and analyzed by the microscope 4 in the far field. Wherein, the incident illumination light refers to visible monochromatic linearly polarized collimated light with the same polarization direction and a wavelength in the range of 380-780 nm.

[0068] Fundamentally speaking, the incident illumination light is provided by LED7, in order to meet the requirements of the a...

Embodiment 2

[0086] Except in the specific embodiment of the present inventionfigure 1 In addition to the structural principle diagram provided in , the present invention can also improve the lighting method under the premise of ensuring the same principle.

[0087] Such as Figure 8 Shown is a structural principle diagram of the improved illumination mode of the frequency-shifting super-resolution microscopic imaging device based on evanescent field illumination of the present invention.

[0088] Such as Figure 8 As shown, after improving the illumination mode, the device of the frequency-shifting super-resolution microscopic imaging system based on evanescent field illumination includes: a laser light source 13, a vibrating mirror 14, a focusing mirror 15, a reflecting mirror 16, a total reflection objective lens 17, Slide 18, Microscope 3, Computer 4, Sample 5.

[0089] Laser light source 13, vibrating mirror 14, focusing mirror 15, reflecting mirror 16, total reflection objective le...

Embodiment 3

[0093] In addition to the method described in Embodiment 1 that can improve the illumination method, it can also be improved by using an off-center diaphragm.

[0094] Such as Figure 9 Shown is a schematic diagram of the structure of the evanescent field illumination-based frequency-shifting super-resolution microscopic imaging device of the present invention after using an eccentric diaphragm to improve the illumination mode.

[0095] Such as Figure 9 As shown, after improving the illumination mode, the device of the frequency-shifting super-resolution microscopic imaging system based on evanescent field illumination includes: a laser light source 13, an eccentric diaphragm 19, a total reflection objective lens 17, a glass slide 18, and a microscope 3, Computer 4, Sample 5.

[0096] Laser light source 13, eccentric diaphragm 19, total reflection objective lens 17, glass slide 18, microscope 3, and sample 5 are located on the coaxial optical path. The incident illuminatio...

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