Scanning device and iscat confocal microscope system

Abstract

To provide a scanning device and an iSCAT confocal microscope system used for observation of an iSCAT confocal microscope.SOLUTION: A scanning device includes: a lens for receiving incident light; a rotary disk having multiple pin holes; and a polarization beam splitter positioned between the lens and the rotary disk. The lens, the polarization beam splitter, and the rotary disk are disposed so as to irradiate a sample with incident light through the lens, the polarization beam splitter, and the rotary disk. The rotary disk and the polarization beam splitter are disposed so as to transmit a return iSCAT signal to the polarization beam splitter through the rotary disk. Multiple pin holes are disposed so as to filter the return iSCAT signal spatially for detection based on confocusing. The polarization beam splitter is disposed so as to guide the return iSCAT signal to an optical path for observing images.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to a scanning device and an interferometric scattering (iSCAT) confocal microscope system.

Background Art

[0002] The iSCAT microscope is a high-sensitivity imaging method that measures the linear scattered light of a sample by interference. The iSCAT microscope can detect the linear scattering field related to the sample by the common-path interference method. However, in the iSCAT microscope, usually, a wide-field imaging with poor axial resolution is used. Therefore, when measuring a complex sample such as a biological cell with an iSCAT microscope, the superposition of scattered signals from various sources, especially those along the optical axis of the objective lens of the microscope, makes data analysis quite complicated.

Summary of the Invention

Problems to be Solved by the Invention

[0003] The present invention provides a scanning device and an iSCAT confocal microscope system used for observation with an iSCAT confocal microscope.

Means for Solving the Problems

[0004] In one aspect of the present invention, there is provided a scanning device used for observation with an iSCAT confocal microscope, including a lens that receives incident light, a rotating disk having a plurality of pinholes, and a polarization beam splitter positioned between the lens and the rotating disk. The lens, the polarization beam splitter, and the rotating disk are arranged to illuminate a sample by passing the incident light through the lens, the polarization beam splitter, and the rotating disk. The rotating disk and the polarization beam splitter are arranged to transmit the return iSCAT signal through the rotating disk to the polarization beam splitter. The plurality of pinholes are arranged to spatially filter the return iSCAT signal for confocal-based detection. The polarization beam splitter is arranged to direct the return iSCAT signal into an optical path for image observation. A scanning device is provided, characterized by the above features.

[0005] In one embodiment, the polarization beam splitter is arranged to reflect the return iSCAT signal into the optical path for image observation, and the return iSCAT signal is for causing an image capture device to form an iSCAT confocal image. In one embodiment, the incident light is linearly polarized before reaching the polarization beam splitter, and the quarter-wave plate is arranged to convert the incident light from linear polarization to circular polarization and to convert the return iSCAT signal from circular polarization to linear polarization, and the polarization beam splitter has high transmissivity for the linear polarization of the incident light and high reflectivity for the linear polarization of the return iSCAT signal. In one embodiment, the incident light is p-polarized before reaching the polarization beam splitter, and the quarter-wave plate is arranged to convert the incident light from p-polarization to circular polarization and to convert the return iSCAT signal from circular polarization to s-polarization, and the polarization beam splitter has high transmissivity for p-polarization and high reflectivity for s-polarization. In one embodiment, the quarter-wave plate is inserted into a filter cube turret of a microscope. In one embodiment, the optical path for image observation further includes a linear polarizing element. In one embodiment, the incident light is a laser, the rotating disk is a rotatable Nipkow disk, and the plurality of pinholes are arranged to be irradiated with the incident light and optically project the incident light through a microscope onto a sample.

[0006] In one aspect of the present invention, a microscope and a scanning device are included. The scanning device includes a lens that receives incident light, a rotating disk having a plurality of pinholes, and a polarization beam splitter positioned between the lens and the rotating disk. The lens, the polarization beam splitter, the rotating disk, and the microscope are arranged such that the incident light passes through the lens, the polarization beam splitter, the rotating disk, and the microscope. The microscope, the rotating disk, and the polarization beam splitter are arranged such that the return iSCAT signal passes through the microscope and the rotating disk and is transmitted to the polarization beam splitter. The plurality of pinholes are arranged to spatially filter the return iSCAT signal for confocal-based detection. The polarization beam splitter is arranged to direct the return iSCAT signal into an optical path for image observation. An iSCAT confocal microscope system is provided, which is characterized by the above features.

[0007] In one embodiment, the polarization beam splitter is arranged to reflect the return iSCAT signal into the optical path for image observation, and the return iSCAT signal is for causing an image capture device to form an iSCAT confocal image. In one embodiment, the incident light is linearly polarized before reaching the polarization beam splitter, and the quarter-wave plate is arranged to convert the incident light from linearly polarized light to circularly polarized light and to convert the return iSCAT signal from circularly polarized light to linearly polarized light, and the polarization beam splitter has high transmissivity with respect to the linearly polarized light of the incident light and high reflectivity with respect to the linearly polarized light of the return iSCAT signal. In one embodiment, the incident light is p-polarized before reaching the polarization beam splitter, and the quarter-wave plate is arranged to convert the incident light from p-polarized light to circularly polarized light and to convert the return iSCAT signal from circularly polarized light to s-polarized light, and the polarization beam splitter has high transmissivity with respect to p-polarized light and high reflectivity with respect to s-polarized light. In one embodiment, the quarter-wave plate is located in the filter cube turret of the microscope. In one embodiment, the scanning device further includes a linear polarization element in the optical path for image observation. In one embodiment, the incident light is a laser, the rotating disk is a rotatable Nipkow disk, and the plurality of pinholes are arranged to be irradiated with the incident light and optically project the incident light through the microscope onto the sample. In one embodiment, the microscope is an inverted optical microscope or a transmission confocal microscope.

[0008] In one aspect of the present invention, there is provided an observation method used for observation by an iSCAT confocal microscope, the method including receiving, by a lens, laser incident light that is linearly polarized; irradiating, with the laser incident light, a plurality of pinholes of a rotating disk to optically project the laser incident light through an optical microscope onto a sample; converting the laser incident light from linearly polarized light to circularly polarized light before the laser incident light reaches the sample; converting the return iSCAT signal from circularly polarized light to linearly polarized light; spatially filtering the return iSCAT signal for confocal-based detection by the plurality of pinholes; and guiding the return iSCAT signal into the optical path for image observation.

[0009] In certain embodiments, the rotating disk is a rotatable Nipkow disk, and the return iSCAT signal is linearly polarized in the optical path for image observation and is for forming an iSCAT confocal image. In certain embodiments, the background in the iSCAT confocal image is eliminated based on the spatial heterogeneity of the background. In certain embodiments, the microscope is an inverted optical microscope or a transmission confocal microscope. In certain embodiments, the return iSCAT signal is guided to the optical path for image observation by a polarization beam splitter. In certain embodiments, the laser incident light is p-polarized. In certain embodiments, the laser incident light and the return iSCAT signal are different linearly polarized lights.

Advantages of the Invention

[0010] According to the present invention, it can be used for the observation of an iSCAT confocal microscope.

Brief Description of the Drawings

[0011]

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Mode for Carrying Out the Invention

[0012] In order to fully understand the essence, advantages, and preferred embodiments of the present invention, by referring to the accompanying drawings, the following detailed description can be more clearly understood.

[0013] The following description is a preferred embodiment of the present invention. Hereinafter, the present invention will be described in detail with reference to the embodiments and the drawings. Also, the present invention is not intended to be limited to the following embodiments, but follows the principles disclosed herein. Furthermore, those having ordinary knowledge in the technical field to which the present invention pertains can make various improvements or modifications according to the disclosure herein and include them within the gist and scope of the present invention and the appended claims.

[0014] Regarding the disclosure of "spinning disk interferometric scattering confocal microscopy captures millisecond timescale dynamics of living cells" (Optics Express 45233, December 2022 issue, Volume 30, Number 25 / 5) and its supplementary documents published on November 29, 2022, all of its contents are incorporated herein by reference.

[0015] Label-free optical interference microscopy is a tool for biological research. Cell biologists have been monitoring the morphology of living cells using phase contrast and differential interference contrast (DIC) microscopy. For a long time, non-invasive live cell imaging has enabled the study of complex cell dynamics over large spatial and temporal scales, within which photo-bleaching and phototoxic effects have also limited fluorescence methods.

[0016] Due to the essential three-dimensional (3D) nature of biological cell structures, cell imaging requires high resolution at all 3D scales. Despite the use of interference microscopy in label-free cell imaging, nanoscale cell dynamics remain a challenge for most conventional techniques due to weak signals.

[0017] To capture nanoscale cell dynamics, there are several requirements for visualization. First is the detection sensitivity. Since small organisms have little light scattering, a sensitive microscope is required to detect them. Photon scattering noise determines the weakest signal detectable above the photon scattering noise, which in turn determines the sensitivity of the interference microscope. For this reason, the microscope should be able to adapt to sufficient illumination intensity without saturating the detector. Low homology light sources (LEDs and incandescent lamps commonly used in QPI) are difficult to provide high brightness to the sample and ultimately limit the detection sensitivity. The second requirement is that the dynamic properties of cells complicate the achievable detection sensitivity during live cell imaging applications. Small biological entities (e.g., vesicles and filamentous structures smaller than 100 nm) are driven by thermal fluctuations and cell activities to continuously move in 3D space. The microscope should have a fast enough data acquisition speed to reduce blurring caused by the rapid movement of the object. Therefore, interference microscopy techniques face challenges in capturing the rapid dynamics of nano-sized objects as they require multiple frames for reconstruction. The requirement for rapid image acquisition precludes the use of the widely used method of frame averaging, which is a common way to actually improve the signal-to-noise ratio (SNR). The third requirement for visualizing nanoscale cell dynamics is the scattering background that includes the non-specific scattering background generated by optical components and the out-of-focus signal of the sample. In principle, if the background can be accurately measured or estimated, the scattering background can be effectively removed by post-processing the image. At the same time, the out-of-focus scattering background of the sample is removed by an optical section (e.g., confocal-based detection).

[0018] A common-path wide-field interferometric scattering (iSCAT) microscope can be utilized. The iSCAT microscope detects backscattered light from a sample by interference and uses the reflection from a supported coverslip as a reference beam. When using laser illumination, light can be efficiently passed through the field of view (FOV). A high-speed camera is used to capture interference images at a high frame rate. The iSCAT microscope is similar to the reflection interference contrast microscopy (RICM) described using the low-homology light source. The iSCAT microscope can capture the nanoscale movement of small nanoparticles with a time resolution of microseconds. In the iSCAT microscope, a single label-free biopolymer can be directly visualized on a clean coverslip through appropriate signal alignment and background correction.

[0019] The wide-field iSCAT microscope is excellent in sensitivity and speed and can analyze the dynamics of the plasma membrane and intracellular vesicles. However, wide-field iSCAT still requires further optimization for label-free cell imaging. Cells have a complex 3D structure that generates numerous out-of-focus backgrounds. In particular, iSCAT well detects cell membrane reflections, generating interference fringes that make it difficult to visualize intracellular organisms. The iSCAT confocal microscope is essentially a reflection confocal microscope equipped with a pinhole mask that defines the detection volume. When using a reflection confocal microscope to acquire an image near the coverslip of a cell (within 1 - 2 μm), the reflection of the coverslip acts as a reference field, which interferes with the backscattered signal of the cell. Interference detection enhances the contrast of the signal so that weak scattering objects can be detected. Furthermore, the out-of-focus cell background is significantly reduced, and it has been possible to visualize nanoscale cell dynamics in fine detail. However, the point-scanning method inherently limits the image capture speed. If the typical pixel dwell time is 1 μs, hundreds of milliseconds are required to capture a high-resolution image (e.g., at a frame rate of several hertz, the resolution is 512×512).

[0020] In one embodiment, the iSCAT confocal microscope system uses a high-speed wide-field iSCAT microscope combined with confocal optical sections. By multi-beam scanning of a rotating disk confocal microscope, the iSCAT confocal microscope system can acquire images at a speed of 100 or more, for example, about 1000 frames per second (fps). The iSCAT confocal microscope system is very sensitive and can, for example, successfully detect each of 10 nm gold nanoparticles. Using high-speed iSCAT confocal imaging, the rapid movement of a single nanoparticle on a model membrane and a single endosome in a living cell can be captured. The iSCAT confocal microscope system can be applied to label-free iSCAT confocal imaging, thereby enabling detailed visualization of nanoscale cell dynamics in the most native form.

[0021] In one embodiment, the iSCAT confocal microscope system is fast and can capture hundreds of frames per second (fps), that is, about two orders of magnitude faster than those compatible with the point scanning method. By reducing the FOV, an image capture speed of 1000 fps can be achieved. The iSCAT confocal microscope system can be modified from a rotating disk confocal fluorescence microscope. By adopting a polarization optical element, the signal collection efficiency can be increased and the surrounding background noise can be reduced. The detection sensitivity and speed of the iSCAT confocal microscope system can be evaluated by using a nanoparticle sample. For example, a single very small gold nanoparticle (AuNP) such as 10 nm in diameter can be successfully detected. Furthermore, in an iSCAT confocal microscope system with high-speed 3D cell imaging, the nanoscale diffusion of endosomes can be analyzed.

[0022] FIG. 1 is a schematic diagram showing an iSCAT confocal microscope system 100 according to an embodiment. In FIG. 1, the iSCAT confocal microscope system 100 includes a microscope 3 and a scanning device 2. The scanning device 2 includes a lens 21 that receives an incident light IL1, a rotating disk 23 having a plurality of pinholes, and a polarization beam splitter 22 positioned between the lens 21 and the rotating disk 23. The polarization beam splitter 22, the rotating disk 23, and the microscope 3 can polarize, guide, or direct light so that the incident light IL1 passes through the lens 21, the polarization beam splitter 22, the rotating disk 23, and the microscope 3 to irradiate the sample 4. After the incident light reaches the sample 4, a return iSCAT signal RL1 is generated from the sample 4. The microscope 3, the rotating disk 23, and the polarization beam splitter 22 pass the return iSCAT signal RL1 through the microscope 3 and the rotating disk 23 and transmit it to the beam splitter 22. The pinhole spatially filters the return iSCAT signal RL2 for confocal-based detection. The return iSCAT signal RL2 is for the optical path (through which the return iSCAT signal RL3 passes) for image observation. For example, the polarization beam splitter 22 guides the return iSCAT signal RL2 to the optical path for image observation. Image observation can be performed by an image capture device 6 such as a camera. The camera may be a high-speed camera capable of capturing a still image, a moving image, or a video.

[0023] In one embodiment, the incident light IL1 may be a laser generated by the light source 1. The light source 1 may be a laser diode module having a predetermined wavelength, for example, a wavelength of 561 nm. The lens 21 can include a plurality of microlenses or a microlens array. The rotating disk 23 may be a rotatable Nipkow disk. The pinhole is formed by a pinhole array and irradiated with the incident light IL1, and the incident light IL1 can be optically projected onto the sample 4 through the microscope 3. The microscope 3 may be an inverted optical microscope or a transmission confocal microscopy. Further, the quarter-wave plate 5 can convert the incident light IL2 from linearly polarized light to circularly polarized light and convert the return iSCAT signal RL1 from circularly polarized light to linearly polarized light (return iSCAT signal RL2). For example, the quarter-wave plate 5 can convert the incident light IL2 from p-polarized light to circularly polarized light and convert the return iSCAT signal RL1 from circularly polarized light to s-polarized light (return iSCAT signal RL2). In one embodiment, the quarter-wave plate 5 can be mounted within the scanning device 2, within the microscope 3, or between the scanning device 2 and the microscope 3.

[0024] In one embodiment, the polarization beam splitter 22 reflects the return iSCAT signal RL2 into the optical path for image observation (the one through which the return iSCAT signal RL3 passes), and the return iSCAT signal RL3 is for causing the image capture device 6 to form an iSCAT confocal image. The polarization beam splitter 22 has high transmissivity for the linear polarization of the incident light IL1 and high reflectivity for the linear polarization of the return iSCAT signal RL2. The incident light IL1 is linearly polarized before reaching the polarization beam splitter 22. The polarization beam splitter 22 can be manufactured by sputtering on a fused silica substrate. For example, the incident light IL1 may be p-polarized before reaching the polarization beam splitter 22, and the polarization beam splitter 22 has high transmissivity for p-polarization and high reflectivity for s-polarization. Alternatively, the incident light IL1 may be s-polarized before reaching the polarization beam splitter 22, so the optical path or optical device can be correspondingly changed. In one embodiment, the polarization beam splitter 22 may have an extinction ratio of Tp:Ts>250:1 and / or Rs:Rp>10:1 at an incident angle of 45° at a wavelength of, for example, 561 nm or other wavelengths. In one embodiment, the polarization beam splitter 22 may have an extinction ratio of Tp:Ts>500:1 and / or Rs:Rp>20:1 at an incident angle of 45° at a wavelength of, for example, 561 nm or other wavelengths. In one embodiment, the polarization beam splitter 22 may have an extinction ratio of Tp:Ts>1000:1 and / or Rs:Rp>40:1 at an incident angle of 45° at a wavelength of, for example, 561 nm or other wavelengths. Tp:Ts is the ratio of transmission of p-polarization to s-polarization. Rs:Rp is the ratio of reflection of s-polarization to p-polarization. The polarization beam splitter 22 can be manufactured by sputtering on a substrate such as a fused silica substrate, for example.

[0025] In one embodiment, the incident light IL1 is received by the lens 21. The incident light IL1 is a linearly polarized laser such as p-polarized light. Then, the incident light IL1 passes through the pinhole of the polarization beam splitter 22 and the rotating disk 23 to become the incident light IL2. The incident light IL2 is converted from linearly polarized light to circularly polarized light to become the incident light IL3. Before the incident light IL3 reaches the sample 4, the pinhole is irradiated with the incident light IL2 / IL3 and optically projected onto the sample 4. After the incident light IL3 reaches the sample 4, the return iSCAT signal RL1 is generated from the sample 4. The return iSCAT signal RL1 is converted from circularly polarized light to linearly polarized light to become the return iSCAT signal RL2. The incident light IL1 and the return iSCAT signal RL2 are linearly polarized lights different from each other. For example, when the incident light IL1 is p-polarized light, the return iSCAT signal RL2 is s-polarized light, the incident light IL2 is converted from p-polarized light to circularly polarized light to become the incident light IL3, and the return iSCAT signal RL1 is converted from circularly polarized light to s-polarized light to become the return iSCAT signal RL2. The return iSCAT signal RL2 is spatially filtered by the pinhole for confocal detection, and the return iSCAT signal RL2 is reflected by the polarization beam splitter 22 into the optical path for image observation to become the return iSCAT signal RL3. Then, the return iSCAT signal RL3 forms an iSCAT confocal image on the image capture device 6. The iSCAT confocal image can be generated by the image capture device 6. The iSCAT confocal image may be a still image, a moving image, or a part of a video. The combination of the polarization beam splitter 22 and the quarter-wave plate 5 can increase the collection efficiency of the return iSCAT signal of the sample 4.

[0026] In one embodiment, the scanning device 2 further includes a lens 25 and a linear polarizing element 27 in the optical path for image observation. The linear polarizing element 27 is attached in front of the image capture device 6 in the optical path to remove an unspecified reflective background of the equipment (mainly caused by the reflection of a rotating disk having pinholes). The reason why a very weak reflective background of the rotating disk having pinholes remains is considered to be due to the depolarization effect of the backscattering of the pinholes. The scanning device 2 or the microscope 3 can include other members such as a lens or a turret (not shown in FIG. 1), for example.

[0027] FIG. 2a is a schematic diagram showing an iSCAT confocal microscope system 200a according to an embodiment. In FIG. 2a, the microscope 3a of the iSCAT confocal microscope system 200a includes a mirror 31, a tube lens 32, an objective lens 33 for the microscope, a filter cube turret 34, and an objective lens turret 35. The quarter-wave plate 5 may be attached inside the filter cube turret 34. The iSCAT confocal microscope system 200a and the microscope 3a operate in a reflection mode. Also, the scanning device 2a may include a mirror 24 and a lens 26 further arranged in the optical path for image observation. The scanning device 2a or the microscope 3a can include other members such as a lens or a turret (not shown in FIG. 2a), for example.

[0028] FIG. 2b is a schematic diagram showing an iSCAT confocal microscope system 200b according to an embodiment. In FIG. 2b, the polarization-maintaining fiber 11 is connected between the light source 1 and the scanning device 2b such that the incident light IL1 reaches the mirror 21 from the light source 1. The sample 4 is placed on the sample stage 41. FIG. 2b also shows the iSCAT detection of the backscattering interference of cells due to the reflection from the water - cover glass interface as a reference field. The iSCAT confocal microscope system 200b and the microscope 3b operate in a reflection mode. The scanning device 2b or the microscope 3b can include other members such as a lens or a turret (not shown in FIG. 2b), for example.

[0029] FIG. 3 is a schematic diagram showing an iSCAT confocal microscope system 300 according to an embodiment. In FIG. 3, the iSCAT confocal microscope system 300 and the microscope 7 operate in a transmission mode. Although the incident optical path to the sample and the path of the return iSCAT signal from the sample are different, they have the same path between the microscope 7 and the scanning device 2. The microscope 7 includes a polarization beam splitter 71, a mirror 72, an objective lens 73 for the microscope, a mirror 74, and a mirror 75. The quarter-wave plate 51 is attached to the incident optical path to the sample, for example, between the mirror 72 and the objective lens 73 for the microscope. The quarter-wave plate 51 converts the incident light IL2 from linearly polarized light (p-polarized light) to circularly polarized light to obtain incident light IL3. The incident light is guided by an optical element and irradiated onto the sample 4. The quarter-wave plate 52 is attached to the path of the return iSCAT signal from the sample, for example, between the mirror 74 and the mirror 75. The quarter-wave plate 52 converts the return iSCAT signal RL1 from circularly polarized light to linearly polarized light (s-polarized light) to obtain the return iSCAT signal RL2. The return iSCAT signal is returned by being guided by an optical element. The scanning device 2 or the microscope 7 can include other members such as a lens or a turret (not shown in FIG. 3), for example.

[0030] In other embodiments, the quarter-wave plates 51 and 52 can be attached to the incident optical path to the sample. For example, after the quarter-wave plate 51 converts the incident light IL2 from linearly polarized light (p-polarized light) to circularly polarized light, the quarter-wave plate 52 converts the incident light from circularly polarized light to linearly polarized light (s-polarized light). The linearly polarized light (s-polarized light) incident light is guided by an optical element and irradiated onto the sample 4. The return iSCAT signal has no polarization conversion. In other embodiments, the quarter-wave plates 51 and 52 may be attached to the path of the return iSCAT signal from the sample. For example, the linearly polarized light (p-polarized light) incident light is guided by an optical element and irradiated onto the sample 4. After the quarter-wave plate 51 converts the return iSCAT signal RL1 from linearly polarized light (p-polarized light) to circularly polarized light, the quarter-wave plate 52 converts the return iSCAT signal RL1 from circularly polarized light to linearly polarized light (s-polarized light).

[0031] Transmission geometries have several advantages. In transmission, it is easy to use an unscattered transmitted beam as a stable reference beam on a common path. Therefore, the imaging depth is not limited as in the case of reflection. Furthermore, unlike reflection geometries that detect many film reflections, transmission imaging is more effective for detecting intracellular signals. A transmission wide-field iSCAT microscope called a coherent bright (COBRI) microscope has already been proven to be capable of analyzing intracellular nanoscale cellular dynamics, including vesicle trafficking, chromatin remodeling, and viral membrane interactions. Transmission confocal microscopes are an underdeveloped technology compared to reflection confocal microscopes because it is difficult to synchronize and reverse-scan the transmitted beam with the incident beam. The reverse scanning of the transmitted beam can be achieved by returning it to its original optical path, or theoretically, by adding a synchronous reverse scanning unit in transmission, the transmitted beam can be reverse-scanned.

[0032] In the above embodiments, polarization optics are used to improve the signal collection efficiency and suppress the unspecified scattered background. The remaining background can be further removed by image data processing to achieve high detection sensitivity. An iSCAT confocal microscope system with a rotating disk enables rapid and sensitive imaging of nano-sized objects based on scattering by optical sections and is widely applied to the detection of organic substances and dynamics of biological cells. Therefore, the polarization optical element will improve the image quality and detection sensitivity. The unspecified background can be removed by image data processing.

[0033] An iSCAT confocal microscope system with a rotating disk can be used to visualize small biological nanoparticles, intracellular organelles, and their dynamics without exogenous labels. The iSCAT confocal microscope system can be easily integrated into a fluorescence rotating disk confocal microscope for providing iSCAT and fluorescence confocal images of a sample. Furthermore, the iSCAT confocal microscope system is fast and sensitive and can be configured based on commercially available components with minimal optical alignment.

[0034] An iSCAT confocal microscope with a rotating disk can detect backscattered light from nano-objects in combination with high-speed iSCAT confocal imaging technology. Compared with a reflectance confocal microscope (RCM) that uses a near-infrared laser to optimize deep tissue imaging at the spatial resolution of sub-cells, iSCAT confocal measures interference signals, while RCM imaging usually lacks a reference field for well-defined interference detection. Therefore, RCM measures the intensity of backscattering, and its image becomes like a dark field (i.e., the backscattered light of the object appears bright in an image with an overall dark background). By using a high numerical aperture (NA) objective lens and a visible laser for illumination, RCM generates an image of backscattered cells similar to a dark field. The maximum detection sensitivity of dark field detection is usually lower than that of interference detection because of its high sensitivity to a given background and reading noise.

[0035] An iSCAT confocal microscope system can operate near the interface of a cover glass and can use the reflection from the interface of the cover glass as a reference field for interference detection. When the focus is not on the interface, the reflection from the cover glass disappears, so the imaging depth for iSCAT detection is substantially limited to several micrometers. This imaging depth is usually sufficient to analyze thin adherent cells. Within this detection volume, iSCAT detection significantly improves the imaging sensitivity and reveals weak scattering signals from nano-objects. Recent laser-scanning iSCAT confocal microscopes have been demonstrated to be capable of successfully analyzing a variety of intracellular organelles and nano-scale cell structures, including endoplasmic reticulum and microtubules. With a confocal pinhole, an iSCAT confocal microscope system with a rotating disk can also analyze these intracellular organelles, as well as mitochondria and lipid droplets that are clearly visualized in other phase-sensitive interference phase microscopes. The difficulty in label-free visualization of intracellular organelles often results from the lack of signal-to-background ratio (SBR) rather than the lack of detection sensitivity. This is because all cell structures scatter light and it is difficult to distinguish them solely based on iSCAT images when they are densely packed. Analyzing different intracellular organelles at the cell periphery is relatively easy, where most cell structures are spatially separated in two dimensions.

[0036] By using a rotating disk confocal microscope and a high-speed camera, iSCAT confocal imaging achieved a speed of 1000 fps. With faster rotating disks and cameras, higher frame rates can be realized. The iSCAT confocal microscope system with a rotating disk has the potential advantage of achieving a large field of view (FOV) at high speed. For example, in the operation of a Yokogawa rotating disk confocal microscope, approximately 1000 laser foci scan the entire sample at a maximum speed of about 1000 Hz. In contrast, in a wide-field iSCAT microscope, typically a sample is irradiated by scanning a single beam with a pair of acousto-optic deflectors (AODs), and its scanning speed is up to about 100 kHz. As a result, the multi-beam scanning of the rotating disk confocal is thought to be able to cover a wider field of view in a shorter frame time. This is because by using Michelson and Mirau objective lenses, a stable reference beam can be generated to increase the imaging depth of thick 3D samples. By having a stable reference field, the axial range of iSCAT confocal imaging is determined by the operating distance of the objective lens.

[0037] Figures 4a - 4d show iSCAT confocal images of 30-nm gold nanoparticles deposited on a glass substrate. Figure 4a shows the original image without a linear polarizer or a quarter-wave plate attached. Due to the intense heterogeneous background, no particles could be observed at all. Figure 4b shows the iSCAT confocal image of the particle sample. After attaching a quarter-wave plate, each of the nanoparticles appears as a small black dot. Figure 4c shows the image after digitally removing the heterogeneous background or the illumination background by image processing. Each of the particles can be clearly observed. Figure 4d plots the optimized image of the sample after a linear polarizer is attached. It was found that the signal-to-noise ratio was slightly improved.

[0038] Figures 4e to 4i show the background removal of the iSCAT confocal images. Figures 4e to 4f show the iSCAT confocal images after background removal. In certain embodiments, background removal by post-processing of the image data improves the image quality for visualization of small cellular structures. In the original rotating disk iSCAT confocal images, spatially non-uniform intensity variations due to unwanted reflections and interferences of the rotating disk scanning unit are shown. These variations are called "background" and inhibit the direct visualization of weak scattering signals from small objects. The background was spatially heterogeneous but temporally stable. Therefore, it can be measured in advance and digitally erased in the post-processing of the image. To achieve this, a reference background was obtained by imaging a blank sample consisting of a clean cover glass. Since confocal detection relates to an optical section, the background image varies depending on the axial position of the sample. Therefore, it is beneficial to acquire a library of background images at different axial positions of the cover glass. Then, the cover glass was replaced with the sample of interest, and its iSCAT confocal image was captured. To remove the background from the original image, the intensity of the original image is divided by the intensity of the corresponding background image on a pixel-by-pixel basis. The visibility of small cellular structures can be further enhanced by image flattening as shown in FIG. 4g.

[0039] In FIG. 4h, for a static sample, since both the signal and the background are stationary, the sample is intentionally moved laterally, during which the signal changes spatially and temporally while the background remains stationary. Specifically, when the sample position is moved laterally at (0 μm, 0 μm), (3 μm, 0 μm), (6 μm, 0 μm), (9 μm, 0 μm), (0 μm, 3 μm), (3 μm, 3 μm), (6 μm, 3 μm), (9 μm, 3 μm), eight original images are acquired. As the displacement increases, the background changes significantly. Then, the background image is estimated by calculating the median image of the eight images for each pixel. The median background image is removed from the original image by division. Then, spatial registration and averaging are performed on the eight background-removed images to generate the final background-removed image.

[0040] In FIG. 4i, the background estimation of the sample of the moving target of interest can be easily performed by recording the original video and calculating the image of the time median of the video. Thereafter, the background of the time median is removed from the original image for each pixel. FIG. 4i shows an example of the original image (left) of the nanoparticles diffused on the model film, the corresponding background image (middle), and the background-removed image (right).

[0041] (Example) The confocal-based iSCAT microscope system as shown in Fig. 2b was developed using a commercially available rotating disk scanning unit. A laser diode module (OBIS561, Coherent) with a wavelength of 561 nm is used as the light source. The Yokogawa confocal scanning unit (CSU-X1) is attached to an inverted microscope (Eclipse Ti2, Nikon). Polarization optical technology is utilized to convert the original fluorescence detection to scattering detection. Specifically, a polarization beam splitter (PBS) (Yuqun Optoelectronic Technology Co., Ltd., Control Optics Taiwan Inc.) is prepared. The PBS is manufactured by sputtering on a fused silica substrate and has extinction ratios of Tp:Ts > 1000:1 and Rs:Rp > 40:1 at an incident angle of 45° at a wavelength of 561 nm. Also, a quarter-wave plate (QWP) (WPQ10M-561, Thorlabs) is inserted into the filter cube turret. The combination of the PBS and the QWP significantly improves the collection efficiency of the iSCAT signal of the sample. Furthermore, a linear polarizer (LPVISC100, Thorlabs) is attached in front of the camera to remove the unspecified reflection background of the equipment (mainly caused by the reflection of the pinhole disk). The reason why a very weak reflection background of the pinhole disk remains is considered to be due to the depolarization effect of the backscattering of the pinhole. An oil immersion objective lens with a high numerical aperture (NA) (CFI Plan Apochromat Lambda 100X Oil, Nikon) is used. As the detector, a scientific CMOS camera (Zyla, Andor) enables the recording of high-speed iSCAT videos. The iSCAT confocal microscope system can be converted from iSCAT to fluorescence imaging by simply replacing the PBS and the linear polarizer with a dichroic spectroscopic element and a fluorescence emission filter, respectively.

[0042] The rotating disk operates at a maximum rotational speed of 10,000 rpm. The Yokogawa scanning unit employs a Nipkow disk and scans the sample every 30° rotation. Therefore, the scanning duration for a single sample is 0.5 ms, and the sample is scanned 12 times per rotation. To stabilize the illumination intensity, the camera was set to the global shutter mode with the frame time and exposure time being multiples of 0.5 ms. For example, when the exposure time is 0.5 ms, the frame time is set to 1 ms (1000 fps), when the exposure time is 1.5 ms, the frame time is set to 2 ms (500 fps), and when the exposure time is 5.5 ms, the frame time is set to 6 ms (166.67 fps). Using the above settings, it is possible to avoid spatial moiré patterns in the video caused by the frequency mismatch between the rotation of the disk and the image capture. The measured intensity showed a 10% peak-to-peak periodic change every 1 ms. Such periodic changes can be interpreted as a result of imperfect manufacturing of the rotating disk that produces an obvious but reproducible illumination pattern every 30° rotation. To minimize the intensity variation between each scan, each image of the video and its average spatial intensity are normalized. After normalization, the intensity fluctuated by approximately 1%.

[0043] The imaging sensitivity of this iSCAT confocal microscope is characterized by gold nanoparticles (AuNP). A single gold nanoparticle with a diameter of 30 nm (BBI Solutions) was fixed onto a clean coverslip by spin coating and immersed in water for imaging. When the particle of the sample is located in the focal plane of the microscope objective lens, the backscattered light of the particle and the reflection at the water-glass interface are collected and projected onto the camera. The measured intensity is represented by the following equation (1).

Equation

[0044] Er is the reflected reference field, Es is the backscattered signal field, and θ is the phase difference between the two fields. For weakly scattering objects, |Es| 2can be ignored compared to the other two terms in Equation (1). The iSCAT contrast is defined as the visibility of the interference of the signals. iSCAT contrast

Number

[0045] Note that the interference contrast is determined by the ratio of the backscattered signal field to the reference field and the phase difference between the two fields. Although both optical fields are spatially filtered by the confocal pinhole, since these two optical fields still have different spatial patterns (because the pinhole is not infinitesimal), they will undergo different phase evolutions along the z-direction (i.e., θ is a function of z).

[0046] Figures 5a - 5f show the iSCAT confocal imaging and tracking of a single 30-nm gold nanoparticle. In Figure 5a, a single 30-nm gold nanoparticle is fixed on a coverslip. Shown in the figure is a close-up of the iSCAT image of the 30-nm gold nanoparticle and the contrast line profile of its Gaussian fitting. Figure 5a shows the iSCAT confocal contrast image of the 30-nm gold nanoparticle, indicating the cancellation interference between the signal and the reference (cosθ is approximately equal to -1, and the particle is displayed as a black dot in the image). Figure 5b is a histogram of the iSCAT contrast of a single 30-nm gold nanoparticle. The normalized iSCAT contrast of the 30-nm gold nanoparticle is approximately 0.19 (Figure 5b), which is roughly the same as the contrast obtained using a wide-field iSCAT microscope. The high contrast of the gold nanoparticle indicates that the polarization optical elements effectively removed the unspecified background reflections of the scanning unit. Conversely, when these polarization optical elements are removed and replaced with an unpolarized beam splitter, the unspecified background will significantly reduce the sensitivity.

[0047] Reference intensity |Er| 2Accurate measurement is essential for detecting weak scattering objects with low iSCAT contrast. The reference intensity (also called the "background intensity") is typically spatially heterogeneous due to non-uniform illumination and inevitable reflections from optical elements. When the object of interest moves, the background intensity is estimated by calculating the temporal median image of the iSCAT video. A rotating disk confocal microscope produces a reproducible illumination background (with an intensity variation of about 1%) that is different between each scan. Also, if the frame time is shorter than the time required for the rotating disk to make one revolution (i.e., 6 ms), multiple temporal median background images for correction are calculated to further reduce background fluctuations. For example, three background images are calculated for a 2 ms frame time, and six background images are calculated for a 1 ms frame time. For static samples, the sample position was set horizontally, and the static background intensity was extracted from the movement signal. For details of the background correction procedure and its performance, see FIGS. 4g-4i and the associated description.

[0048] An iSCAT confocal microscope system with a rotating disk can obtain high-speed videos by optical sectioning. Here, we showed high-speed imaging of a single nanoparticle diffusing on a supported lipid bilayer membrane. This model membrane system has been used as a platform to study the dynamics of monolayers. An artificial lipid bilayer membrane was prepared on a coverslip, biotinylated phospholipids (1 mol%) were added to the membrane, and gold nanoparticles were labeled with streptavidin (see Fig. 5c). Fig. 5c is a schematic diagram of gold nanoparticles attached to lipid molecules in a lipid bilayer membrane supported on a coverslip. After attachment, the gold nanoparticles diffused laterally on the membrane, and their movement was recorded at 166.67 fps using an iSCAT confocal microscope system. The positions of the gold nanoparticles were located in each image using custom MATLAB (registered trademark) code, and consecutive localizations were linked to reconstruct the diffusion trajectories (Fig. 5d). The SNR of the 30-nm gold nanoparticles was approximately 18, and the positioning accuracy was approximately 6.5 nm. Using a narrow field of view, images can be captured at a higher speed. The SNR decreased slightly with an increase in the imaging speed (the SNR at 500 fps was approximately 11, and the SNR at 1000 fps was approximately 9). The decrease in the signal-to-noise ratio is due to a two-fold increase in the noise floor caused by a low optical quantum flux. Diffusion trajectories recorded at 500 fps and 1000 fps are depicted in Fig. 5e and Fig. 5f, respectively. A higher image capture speed enables the analysis of detailed diffusion motion at the nanoscale.

[0049] As shown in Equation (2), the iSCAT contrast of the particles is determined by the ratio of the signal field to the reference field. Therefore, to detect very small nanoparticles, the iSCAT contrast of the particles can be enhanced by reducing the intensity of the reference beam. This is achieved by replacing the immersion water of the sample with a high refractive index liquid, which can reduce the reflectivity of the sample - cover glass interface and thereby reduce the intensity of the reference beam. Water was replaced with glycerol (G2025, Sigma - Aldrich), and the refractive index became 1.474. This reduced the reflectivity by 21.5 times (from 0.43% to 0.02% with the refractive index of water being 1.33 and that of the cover glass being 1.517). At the same time, changing the medium from water to glycerol also slightly changed the scattering cross - section of the gold nanoparticles (increased by about 33%. Since gold has a large complex refractive index, the change is small). Overall, the medium exchange increased the iSCAT contrast by a factor of (1.33×21.5) 1 / 2 (almost equal to 5.3) times. Due to the enhanced contrast, a single 10 - nm gold nanoparticle fixed on the cover glass could be detected (Figure 6). The enhanced iSCAT contrast of the 10 - nm gold nanoparticle was about 0.04, resulting in an SNR of about 4. Figure 6 shows the iSCAT confocal image of a single 10 - nm gold nanoparticle. This figure shows the contour of the iSCAT contrast line and the Gaussian fitting. A spatial Gaussian filter with a standard deviation of 0.7 pixels was applied.

[0050] Finally, since the measurement of iSCAT confocal imaging is limited by the noise of the scattered particles, it was verified that the detection sensitivity can be improved by increasing the number of detected photons by the averaging method. The iSCAT image of a clean cover glass was captured at a frame rate of up to 1000 fps, and the fluctuations of the iSCAT contrast could be analyzed. The noise was defined as the standard deviation of the contrast fluctuations. The dependence of the noise on the detected number of photons N followed the power law of 1 / (N 1 / 2 )), indicating that the main noise in iSCAT imaging is the noise of the scattered photons at the highest image capture speed.

[0051] The 3D imaging ability of the iSCAT confocal microscope system can be confirmed by using biological cells. Human osteosarcoma epithelial (U2OS) cells were cultured on a cover glass bottom dish (WillCo Wells). The optical sectioning ability of the iSCAT confocal was confirmed by z-stack images of the captured cells. When the water-cover glass interface is out of focus, the intensity of the reference beam decreases due to the confocal section. However, within an axial distance of about 1 μm, the intensity of the reference beam was still sufficient for iSCAT imaging. The strongly attenuated reference beam is challenging for capturing iSCAT images of deeper parts of the sample.

[0052] Figures 7a to 7d show a comparison between confocal images and wide-field iSCAT images of biological cells. Figures 7a and 7b show iSCAT confocal images of U2OS cells at z = 0 μm (Figure 7a) and z = 0.5 μm (Figure 7b). Figures 7c and 7d show wide-field iSCAT images of the same cells shown in Figures 7a and 7b at the corresponding axial positions, i.e., z = 0 μm (Figure 7c) and z = 0.5 μm (Figure 7d). Since the two confocal images in Figures 7a and 7b show different features of the optical section, the two wide-field images in Figures 7c and 7d show similar features due to the low axial resolution. Note that Figures 7c and 7d are tiled images of the original 4×4 images because the field of view of the wide-field iSCAT microscope is small.

[0053] Figure 7a shows an iSCAT confocal image of a cell where the focal plane is located at the basement membrane of the cell (i.e., the cell-cover glass interface). At this z-position, due to the morphology of the basement membrane, a distinct change in iSCAT contrast was observed, reminiscent of the adhesion factors previously observed with the wide-field iSCAT microscope. When the focal plane was moved 0.5 μm into the cell, distinct features became visible, and the cell nucleus surrounded by the nuclear membrane was observed (Figure 7b). The large difference in the two axial positions of the two iSCAT confocal images indicates that the microscope has excellent optical sectioning ability due to the removal of the out-of-focus scattering signal.

[0054] To explain the influence of the optical section on 3D cell imaging, z-stack iSCAT images of the same cells were captured as a comparison using a normalized wide-field iSCAT microscope. The wide-field iSCAT images at two corresponding heights (z = 0 μm and z = 0.5 μm) are shown in FIGS. 7c and 7d, respectively. Different from the two iSCAT confocal images, the two wide-field iSCAT images show very similar features because of the low axial resolution of the wide-field iSCAT microscope. Note that in the wide-field iSCAT image, concentric interference fringes appear on the cell periphery within a long axial range due to the reflection of the plasma membrane. The optical section of the iSCAT confocal microscope system largely removed this interference ring.

[0055] The iSCAT confocal microscope system is compatible with confocal fluorescence imaging. As a demonstration, DNA in the cell nucleus was labeled with the fluorescent dye DRAQ5 (ab108410, Abcam), and the cell membrane was stained with a lipophilic dye (D3898, FASTDiO, Invitrogen). The iSCAT confocal images of the cells and the corresponding fluorescence images of the two dyes are shown in FIGS. 8a - 8c. FIGS. 8a - 8c show the iSCAT and fluorescence confocal images of U2OS cells. FIG. 8a shows the iSCAT confocal image of the cells. FIG. 8b shows the two-color fluorescence image of the cells. The fluorescence signals of the lipophilic dye and the DNA dye are shown in green and red, respectively. FIG. 8c shows the overlap of the iSCAT image in FIG. 8a and the fluorescence image in FIG. 8b. The figure shows a close-up of the cell vesicles. The magenta arrow indicates the vesicles present in both the iSCAT and the fluorescence channels, and the blue arrow indicates the particles that are clearly detected by iSCAT but show low fluorescence intensity. Both the plasma membrane and the nuclear membrane generate strong iSCAT signals. Furthermore, in the iSCAT channel, many small particles were observed to be positioned together with DNA and the lipophilic dye (FIGS. 8a - 8c), suggesting their involvement in the uptake of DNA dyes and lipid-rich vesicles.

[0056] Through the iSCAT channel, it was observed that many nanoparticles continued to move inside living cells. The diffusion motion of these rapidly moving vesicles can be measured by using single-particle tracking and high-speed image capture with an iSCAT confocal microscope system. Figures 9a - 9e show the diffusion motion of biological nanoparticles observed using a high-speed iSCAT confocal microscope. Figure 9a shows a snapshot of an iSCAT confocal video of multiple cells. The region of the cell nucleus is shown in red and corresponds to the fluorescence image of DNA staining. Figures 9b - 9e show close-ups of four ROIs shown in Figure 9a. The diffusion trajectories of the vesicles are drawn in red. Particles undergoing orientational diffusion are indicated by magenta arrows. In Figure 9b, ROI1 shows the region around the cell where particles move along the cell boundary. In Figure 9c, ROI2 shows the region of the nuclear boundary where the orientational motion of the particles was observed. In Figure 9d, in ROI3, the association between the particle and a large cellular structure (indicated by a yellow arrow) is being monitored. In Figure 9e, ROI4 shows the random diffusion of particles in the cytoplasm. The snapshot of the iSCAT images of multiple cells was plotted in Figure 9a. Four regions of interest (ROIs) are indicated by rectangular frames, and their close-up views are shown in Figures 9b - 9e. Inside these ROIs, nano-sized biological particles can be detected and their diffusion motion can be measured. To improve the positioning accuracy, the static cell background was measured by using temporal median filtering, and then the background was removed from the original image by division. The trajectories of the biological nanoparticles are shown in Figures 9b - 9e. Most of the particles spread locally, and the maximum displacement was less than 450 nm per second. These particles are restricted within the cytoskeletal network. Occasionally, it was observed that some particles displaced over a longer distance of more than about 1.5 μm through orientational motion, which was interpreted as the result of active transport in the cell. When the particles diffuse from the focal plane, the tracking of consecutive single particles is usually interrupted. Indeed, the iSCAT confocal microscope system has a fairly thin detection volume due to its optical sectioning ability. Although the tracking of the particles is shown in 2D, appropriate modeling of the point spread function and calibration should enable 3D particle tracking.

[0057] In one embodiment, the iSCAT confocal microscope system may be a high-speed rotating disk iSCAT confocal microscope for wide-field iSCAT imaging using optical sections. The operating conditions are the synchronization of the polarization optical element and the rotation of the disk with the image capture. The iSCAT confocal microscope enables visualization of small single nanoparticles up to 10 nm gold nanoparticles. Furthermore, high-speed tracking (up to 1000 fps) of the nanoscale motion of single gold nanoparticles was demonstrated. Also, iSCAT confocal imaging of live cells was demonstrated. The plasma membrane and nuclear membrane could be clearly visualized and analyzed using optical sections. Furthermore, multiple nanoscale vesicles diffusely distributed in three dimensions in the cytoplasm could also be observed. iSCAT confocal imaging can provide information on the rich structure and dynamics of cell samples by combining with multi-color fluorescence confocal imaging. Therefore, the iSCAT confocal microscope system is suitable as a confocal microscope for examining cell samples.

[0058] Even without further detailed description, those skilled in the art are considered to be able to utilize the present invention to the maximum extent based on the above description. Therefore, specific embodiments should be construed as merely illustrative without limiting the present disclosure in any way.

[0059] All references described in this specification are hereby incorporated by reference in their entirety for all purposes to the extent that each individual reference (e.g., published gazette or patent gazette or patent application) is specifically and individually indicated. Other embodiments are included in the claims of the claims.

Explanation of Signs

[0060] 100, 200a, 200b, 300... iSCAT confocal microscope system 1... Light source 11... Polarization-maintaining fiber 2, 2a, 2b... Scanning device 21... Lens 22... Polarizing beam splitter 23…Rotating disk 24…Mirror 25, 26…Lenses 27…Linear polarizing element 3, 3a, 3b…Microscopes 31…Mirror 32…Tube lens 33…Objective lens for microscope 34…Filter cube turret 35…Objective lens turret 4…Sample 41…Sample stage 5, 51, 52…Quarter-wave plate 6…Image capture device 7…Microscope 71…Polarizing beam splitter 72…Mirror 73…Objective lens for microscope 74, 75…Mirrors IL1, IL2, IL3…Incident light RL1, RL2, RL3…Return iSCAT signal

Claims

1. A scanning device used for observation with an iSCAT confocal microscope, comprising: a lens for receiving incident light; a rotating disk having a plurality of pinholes; a polarization beam splitter positioned between the lens and the rotating disk, wherein the lens, the polarization beam splitter, and the rotating disk are arranged to illuminate a sample by passing the incident light through the lens, the polarization beam splitter, and the rotating disk; the rotating disk and the polarization beam splitter are arranged to transmit a return iSCAT signal through the rotating disk and transmit it to the polarization beam splitter; the plurality of pinholes are arranged to spatially filter the return iSCAT signal for confocal-based detection; the polarization beam splitter is arranged to direct the return iSCAT signal into an optical path for image observation, characterized by the scanning device.

2. The polarization beam splitter is arranged to reflect the return iSCAT signal into the optical path for the image observation, wherein the return iSCAT signal is for forming an iSCAT confocal image in an image capture device, characterized by the scanning device according to Claim 1.

3. The incident light is linearly polarized before reaching the polarization beam splitter, a quarter-wave plate is arranged to convert the incident light from linearly polarized light to circularly polarized light and convert the return iSCAT signal from circularly polarized light to linearly polarized light, the polarization beam splitter has high transmissivity with respect to the linearly polarized light of the incident light and high reflectivity with respect to the linearly polarized light of the return iSCAT signal, characterized by the scanning device according to Claim 1.

4. The quarter-wave plate is inserted into a filter cube turret of a microscope, characterized by the scanning device according to Claim 3.

5. The scanning device according to Claim 1, further comprising a linear polarization element in the optical path for the image observation.

6. The incident light is a laser, the rotating disk is a rotatable Nipkow disk, the plurality of pinholes are arranged to be irradiated by the incident light and optically project the incident light through a microscope onto a sample, characterized by the scanning device according to Claim 1.

7. a microscope; a scanning device, wherein the scanning device is A lens that receives incident light, A rotating disk having a plurality of pinholes, A polarization beam splitter positioned between the lens and the rotating disk, and The lens, the polarization beam splitter, the rotating disk, and the microscope are arranged so that the incident light passes through the lens, the polarization beam splitter, the rotating disk, and the microscope. The microscope, the rotating disk, and the polarization beam splitter are arranged so that a return iSCAT signal passes through the microscope and the rotating disk and is transmitted to the polarization beam splitter. The plurality of pinholes are arranged to spatially filter the return iSCAT signal for confocal-based detection. An iSCAT confocal microscope system, wherein the polarization beam splitter is arranged to direct the return iSCAT signal into an optical path for image observation.

8. The polarization beam splitter is arranged to reflect the return iSCAT signal into the optical path for the image observation. The return iSCAT signal is for causing an image capture device to form an iSCAT confocal image. The iSCAT confocal microscope system according to claim 7, characterized in that.

9. The incident light is linearly polarized before reaching the polarization beam splitter. A quarter-wave plate is arranged to convert the incident light from linearly polarized light to circularly polarized light and to convert the return iSCAT signal from circularly polarized light to linearly polarized light. The polarization beam splitter has high transmissivity with respect to the linearly polarized light of the incident light and high reflectivity with respect to the linearly polarized light of the return iSCAT signal. The iSCAT confocal microscope system according to claim 7, characterized in that.

10. The quarter-wave plate is located in a filter cube turret of the microscope. The iSCAT confocal microscope system according to claim 9, characterized in that.

11. The scanning device further includes a linear polarizing element in the optical path for the image observation. The iSCAT confocal microscope system according to claim 7, characterized in that.

12. The incident light is a laser. The rotating disk is a rotatable Nipkow disk. The iSCAT confocal microscope system according to claim 7, wherein the plurality of pinholes are arranged to be irradiated with the incident light and pass the incident light through a microscope and optically project it onto a sample.

13. The iSCAT confocal microscope system according to claim 7, wherein the microscope is an inverted optical microscope or a transmission confocal microscope.

14. An observation method used for observing an iSCAT confocal microscope, receiving laser incident light that is linearly polarized by a lens, irradiating a plurality of pinholes of a rotating disk with the laser incident light to pass the laser incident light through an optical microscope and optically project it onto a sample, converting the laser incident light from linearly polarized light to circularly polarized light before the laser incident light reaches the sample, converting a return iSCAT signal from circularly polarized light to linearly polarized light, spatially filtering the return iSCAT signal for detection based on confocal by the plurality of pinholes, guiding the return iSCAT signal to an optical path for image observation, and characterized by including the above.

15. The rotating disk is a rotatable Nipkow disk, The observation method according to claim 14, wherein the return iSCAT signal is linearly polarized in the optical path for the image observation and is for forming an iSCAT confocal image.

16. The observation method according to claim 15, characterized by eliminating the background in the iSCAT confocal image based on the spatial heterogeneity of the background.

17. The observation method according to claim 14, wherein the microscope is an inverted optical microscope or a transmission confocal microscope.

18. The observation method according to claim 14, wherein the return iSCAT signal is guided to the optical path for the image observation by a polarization beam splitter.

19. The observation method according to claim 14, wherein the laser incident light is p-polarized.

20. The observation method according to claim 14, wherein the laser incident light and the return iSCAT signal are different linearly polarized lights.

Images