
==== Front
Proc Natl Acad Sci U S A
Proc Natl Acad Sci U S A
PNAS
Proceedings of the National Academy of Sciences of the United States of America
0027-8424
1091-6490
National Academy of Sciences

38498716
202321825
10.1073/pnas.2321825121
videoVideoresearch-articleResearch ArticlechemChemistry410
Physical Sciences
Chemistry
Cascaded momentum-space polarization filters enabled label-free black-field microscopy for single nanoparticles analysis
Liu Yang a https://orcid.org/0000-0001-8711-4593

Chen Qiankun a
Zhang Hongli b
Feng Zeyu b
Zou Gang b
Zhang Douguo dgzhang@ustc.edu.cn
a c d 1 https://orcid.org/0000-0003-1230-8742

aAdvanced Laser Technology Laboratory of Anhui Province, Department of Optics and Optical Engineering, University of Science and Technology of China, Hefei, Anhui 230026, China
bChinese Academy of Sciences Key Laboratory of Soft Matter Chemistry, Department of Polymer Science and Engineering, University of Science and Technology of China, Hefei, Anhui 230026, China
cHefei National Research Center for Physical Sciences at the Microscale, University of Science and Technology of China, Hefei, Anhui 230026, China
dHefei National Laboratory, University of Science and Technology of China, Hefei 230088, China
1To whom correspondence may be addressed. Email: dgzhang@ustc.edu.cn.
Edited by David Weitz, Harvard University, Cambridge, MA; received December 12, 2023; accepted February 25, 2024

18 3 2024
26 3 2024
18 9 2024
121 13 e232182512112 12 2023
25 2 2024
Copyright © 2024 the Author(s). Published by PNAS.
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This article is distributed under Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND).

Significance

One central challenge in optically detecting single nanoparticles is that the background intensity is much stronger than the intensity of the scattering light from single nano-objects. In this work, we invent cascaded momentum-space polarization filters that can be installed in various optical microscopies to realize a black-field imaging. It can perform vector field modulation to block most of the background field and result in an almost black background; in contrast, only a small proportion of the scattering field is blocked, leading to obvious imaging contrast enhancement for single nanoparticles. The proposed black-field microscopy provides a powerful platform for single nanoparticle analysis and will find wide applications in the biological, physical, environmental, and materials sciences.

Label-free optical imaging of single-nanometer-scale matter is extremely important for a variety of biomedical, physical, and chemical investigations. One central challenge is that the background intensity is much stronger than the intensity of the scattering light from single nano-objects. Here, we propose an optical module comprising cascaded momentum-space polarization filters that can perform vector field modulation to block most of the background field and result in an almost black background; in contrast, only a small proportion of the scattering field is blocked, leading to obvious imaging contrast enhancement. This module can be installed in various optical microscopies to realize a black-field microscopy. Various single nano-objects with dimensions smaller than 20 nm appear distinctly in the black-field images. The chemical reactions occurring on single nanocrystals with edge lengths of approximately 10 nm are in situ real-time monitored by using the black-field microscopy. This label-free black-field microscopy is highly promising for a wide range of future multidisciplinary science applications.

single-nanoparticle analysis
momentum-space polarization filters
label-free optical microscopy
MOST | National Key Research and Development Program of China (NKPs) 501100012166 2021YFA1400700 Douguo Zhang MOST | National Natural Science Foundation of China (NSFC) 501100001809 12134013 62127818 Douguo Zhang
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pmcThe ability to detect, image, and quantify the properties of single nanoscopic objects, including their structures, masses, and dynamics, on nanoscopic length scales is essential to understand and ultimately control the properties and functions of nanoscale matter; this is desirable for both fundamental research and clinical and industrial applications (1–5). Light-based microscopy has been a popular tool in the work toward this goal because of its distinct advantages, including its noninvasive nature, dynamic operation, potential for high throughput, real-time study capability, and compatibility for integration with other technologies (6). In recent decades, various forms of fluorescence microscopy, including super-resolution methods, have been developed rapidly to characterize physical or chemical phenomena, including morphology, motion, dynamic reactions, and mechanical properties, on the nanometer scale (7–12). The specific advantage of fluorescence microscopy is the spectral separation between its illumination and emission profiles, which enables highly efficient background suppression. However, these fluorescence methods are limited by the brightness and photostability of the label dyes or fluorescent proteins, which restrict the duration, speed, and resolution of optical measurements (6). This has led to intensive research into label-free optical microscopy techniques. One example is scattering-based optical microscopy, which includes dark-field microscopy, interferometric detection microscopy, and surface wave illumination microscopy methods (13–24). In these techniques, the imaging contrast originates from scattering of the incident light by the single nanoscale objects. However, because of the rapid decrease of the scattering intensity with the size of the dielectric object, it is difficult to detect scattering signals from single-nanometer-scale objects because the signal is much smaller than the imaging background intensity (6, 25). This challenge is being addressed by using various approaches to reduce the background intensity or amplify the signal of interest. Interferometric scattering microscopy (iSCAT) and surface plasmon resonance microscopy (SPRM) are the most rapidly developing interferometric approaches, with sensitivity boundaries that have been pushed significantly from detection of single metal nanoparticles to detection of single proteins (21, 22, 26–32). The reference light used in these interferometric approaches is to amplify the signal of interest. The intensity of the reference light is larger than that of the scattering light from single nano-objects. Therefore, software development steps involving the invention of innovative image processing and data analysis techniques are necessary for extracting the signals of the single nanoparticles or proteins from the unique interference field.

In this work, we address this challenge in another way, which is through the invention of a hardware component comprising cascaded momentum-space polarization filters (MSPFs) to perform vector field modulation in momentum space and reduce the background intensity. These adjustable filter modules can be installed in a variety of label-free microscopy devices to perform extremely efficient background rejection without the use of image postprocessing and thus greatly enhance the imaging contrast and the signal-to-noise ratio (SNR) for single nano-objects. This results in a unique type of microscopy, the black-field microscopy, whose images’ background is almost black. Due to the adoption of homogeneous polarization filtering in the momentum space, the label-free black-field microscopy will have a wide field of view (FoV). The MSPFs can also be installed on a fluorescence microscope to combine the strengths of both label-based and label-free detection technologies in a single setup (17, 21), and their capability for in-suit real-time monitoring of chemical reactions in single nanocrystals is demonstrated successfully.

Results

Principle of the Momentum-Space Polarization Filters (MSPFs).

As shown in Fig. 1A, when an initial image that was captured via a label-free optical microscopy technique, e.g., surface plasmon resonance microscopy or total internal reflection microscopy (TIRM), is processed by the proposed MSPFs, its contrast can be enhanced greatly. The cascaded MSPFs are formed from several combinations of a polarizer (P0), zero-order vortex half-wave retarders (Vn), and an analyzer (Pn). The polarizer is a common optical component for linearly polarized light generation and is used as a polarization analyzer. The vortex retarder uses a liquid crystal polymer with aligned molecules, like aligned nematic liquid crystals, and thus, its fast axis rotates continuously over the area of the optic. The point of rotation for the fast axis is nominally located at the center of the plate. This type of vortex retarder is designed to generate radially or azimuthally polarized vector light field from a linearly polarized source (33, 34). Here, we use the retarder in an unusual manner to spatially modulate the light beam’s polarization state in the momentum space.

Fig. 1. Principle and functions of the cascaded MSPFs. (A) Schematic diagram of the cascaded MSPFs, which are composed of polarizers (Pn) and vortex half-wave retarders (Vn). (B) Schematic diagram of the first type of MSPF (filter 1), which is made from two polarizers and one vortex retarder. The Right image shows the calculated momentum-space intensity distribution of a beam when transmitted through filter 1. (C) Schematic diagram of the second type of MSPF (filter 2), which is made from two polarizers and two vortex retarders. The two retarders at the center are shifted vertically from the optical axis by a distance d. The Right image shows the calculated momentum-space intensity distribution of a beam when transmitted through filter 2. The colored one-way arrows inserted into right-side images in (B) and (C) indicate the polarization directions of the transmitted light. (D) Schematic diagram of a captured momentum-space image that contains both the light scattered from extremely small nanoparticles under illumination by p-polarized light (represented by the yellow ring) and the reflected illumination light that was not scattered by the nanoparticles (represented by the green spots). (E and F) The two green spots, which contain no information about the nanoparticles, can be erased by the first type of MSPF with a different orientation. The MSPFs are placed on the BFP of the imaging system. The gray fan-shaped area corresponds to the black region in the Right image of part (B). (G) The second type of MSPF is used to remove the background noise around the yellow ring. (H) The cascade MSPFs remove most of the background light and only the scattering light that contains the information about the single nanoparticles remains on the BFP image.

The function of this combination of polarizers and vortex retarders (filter 1 and filter 2) is generating vector light fields as illustrated in Fig. 1 B and C. As Fig. 1B shows, when a plane wave passes through the aligned polarizer (P0) and the vortex retarder (V1), the light polarization orientations will be spatially tuned as indicated by the colored one-way arrows in the image on the right of Fig. 1B, which represents the typical features of a radially polarized beam. Then, after passing through the analyzer (second polarizer P1), the intensity distribution of the vector light field is partially suppressed and shows a pair of fan-shaped dark patterns orientated along the vertical direction, indicating that this portion of the light has been filtered out. The orientation of this pair of fan-shaped dark patterns can be tuned by tuning the orientations of the polarizers and retarders, as illustrated in Fig. 1F (filter 1’). The second group of polarizers and vortex retarders (filter 2) is shown in Fig. 1C, where two retarders with vertically shifted centers (by a distance d) are used. In this case, a pair of dark cambered patterns is generated by the light field modulation in the momentum space (Right image, Fig. 1C). The generation of these filters’ patterns on the back focal plane (BFP) is described theoretically in SI Appendix, Supplementary section 1 and Fig. S1. The functions of filter 1 and filter 2 are verified using experimental momentum-space images of the light transmission through either filter 1 or filter 2, as shown in SI Appendix, Fig. S1 B and C, which are consistent with the images on the right of Fig. 1 B and C, respectively.

The dark regions in the images on the right of Fig. 1 B and C demonstrate the important role of the MSPFs, which will be used to filter out background light, as illustrated in Fig. 1 D–H. When the nano-objects are illuminated using a collimated beam, the reflected illumination beam from the sample substrate that has not interacted with the nano-objects will be represented by one spot on the BFP image (the Left green spot in Fig. 1 D–H). The appearance of another green spot (the Right green spot in Fig. 1 D–H) is caused by reflection from the surfaces of the beam splitter used in the microscopy setup. The diffraction intensity of the two focusing spots is considerable and will generate a little big pattern around the spots (represented by the green ring in Fig. 1 D–H) on the BFP. In contrast, under the illumination of p-polarized light, the scattered light from the single nano-objects (such as a nanoparticle) will be displayed as a circular pattern on the BFP image (represented by the yellow ring in Fig. 1 D–H), which can be explained by using a point-dipole approximation model when the illumination light has p-polarization. The details of generation of the scattering and background patterns on the BFP are described theoretically in SI Appendix, Supplementary section 2 and Fig. S2. On the other hand, the inevitable tiny dirt and defects in the optics will scatter the illumination light to large angles that locate around the yellow ring, which are another source of the background noise. The different polarization-states and noncommon path characteristics between the background noise and the scattering signal, provide an opportunity to separate the signals from the background noise in the momentum space using the proposed MSPFs.

The first type of MSPF (filter 1 and filter 1’) can be used to block the two green dots and their associated patterns (Fig. 1 E and F) because these two spots are located inside the dark regions in the Right image in Fig. 1B. The background noise that are located outside of the yellow ring can be erased by using the dark pattern generated by filter 2 (Fig. 1G). Finally, only the scattered light (yellow ring) from the single nano-objects remains on the BFP image and the background noise is strongly suppressed, leaving a contrast-enhanced image with a high SNR.

It should be noted that beam blockers were always used to remove the background noise in optical microscopy. However, for different illumination angle, nano-objects of interest, substrates of samples, objectives, tube lens, and microscopy configurations, the spatial distributions of the background noise and scattering signals on the BFP are different. It means that the blockers should be redesigned and refabricated and realigned if only one factor of the microscopy was changed. It is inconvenient for practical applications. On the other hand, the MSPFs are composed of several commercial optical elements, which do not need to be redesigned or refabricated, and can be used in various microscopy configurations. The direction of the fan-shaped pattern introduced by filter 1 can be rotated by rotating the vortex retarder plate; The diameter and shape of the cambered pattern introduced by filter 2 can be changed by tuning the vertical distance between the pair of vortex retarders, as shown in SI Appendix, Fig. S3. Therefore, the background noise can be efficiently blocked by using the MSPFs, resulting in a microscopic image of high contrast and SNR.

Experimental Demonstrations of the MSPFs Enabled Black-Field Microscopy for Single Nanoparticles Imaging.

To verify the principle and function of the proposed cascaded MSPFs, the module was first installed on the SPRM, which is a powerful biomedical imaging platform because of its wide-field, label-free, and high-surface-sensitivity imaging capabilities (35–38). As shown in Fig. 2, the laser beam at a wavelength of 635 nm was focused on the BFP of an oil-immersed objective (100×; numerical aperture (NA): 1.49). By tuning the position of this focal point on the BFP appropriately, an expanded and collimated beam (parallel beam) will exit the objective and strike the substrate at a given angle (surface plasmon resonance (SPR) angle) to excite surface plasmons (SPs) on the gold film. The nano-objects of interest will be illuminated by these SPs. The scattered light from these single nano-objects, the reflected laser beam, and all the background noise induced by reflections, refractions, or diffractions from all the optical elements, are collected by the same objective lens. All these collected light beams will pass through the proposed cascaded MSPFs, which are composed of three combinations of polarizers and vortex retarders (filter 1, filter 1’, and filter 2, which are all located at the BFP position of the objective to perform polarization filtering in the momentum space). To demonstrate the functions of the MSPF fully, both front focal plane (FFP) and BFP images of the objective lens will be captured. The camera used in this work has 1,200 × 1,200 pixels with a single pixel area of 11 × 11 μm2; the imaging system’s field of view can thus reach approximately 100 × 100 μm2 based on the 133× system magnification (0.0825 μm/pixel). Details of the experimental setup are presented in Materials and Methods.

Fig. 2. Schematic diagram of the experimental setup containing the MSPFs. Single nanoparticles can be placed on the thin Au film-coated glass substrate (for SPRM) or the bare glass substrate (for TIRM). The laser beam at 532 nm can be used to perform fluorescence imaging and the beam at 635 nm is used to perform label-free imaging. L1 to L12: lenses. BS1 to BS5: beam splitters. R1 to R6: reflectors. The sCMOS camera was used to perform either fluorescence imaging or label-free FFP/BFP imaging. Three MSPFs were cascaded and installed in this setup.

Single gold (Au) nanoparticles (ANPs) with a diameter of approximately 20 nm were imaged using the black-field microscopy, as shown in Fig. 2 and SI Appendix, Fig. S4A. For comparison, a conventional SPRM image of single nanoparticles without the cascade MSPFs is shown in Fig. 3 A, i, which shows a wave-like pattern with parabolic tails and is consistent with previously reported works (19, 35, 39). The tails of the curved fringes are of several micrometers length in the plasmon propagation direction. This pattern can be regarded as the point spread function (PSF) of conventional SPRM, and thus, the spatial resolution of SPRM along the plasmon propagation direction is quite poor. Apart from the bright spot, many background noise signals appear on the image, indicating the bad imaging contrast in the SPRM image without image postprocessing.

Fig. 3. Experimental demonstration of the functions of the MSPFs installed in the SPRM. (A) Captured FFP images in the cases of imaging with no MSPFs (A, i), with filter 1 (A, ii), with F1 + F1’ (A, iii), and with F1+F1’+F2 (A, iv). (B) BFP images corresponding to those in (A), showing the procedure used to erase the background light and allow only the scattering light from the nanoparticles to remain. The two green rings indicate the positions at which the numerical aperture is slightly larger than 1.0 and equal to 1.49. B1 and B1 are the point-like background noise that are induced by the reflected beams from the substrate and beam-splitter. S0 represents the background noise located inside the two green rings, which are induced by diffraction and scattering from tiny dirt and defects in the optics. The intensities of B1, B2, and S0 were highly attenuated by the MSPFs. (C) Values of the image contrast and SNR derived from the four different situations shown in (A), illustrating the enhanced contrast and SNR induced by the MSPFs. The yellow bars (Isca¯), the green bars (Ibac¯), and the blue bars (σ¯) represent the normalized mean intensities of the signal and the noise, and the rms noise, respectively. (D) Intensities of the first spot (IB1¯), the second spot (IB2¯), and the hollow ring (IS0¯) derived from the four BFP images in (B). (E and F) SPRM images of individual 20 nm PS nanoparticles acquired without (E) and with (F) the cascaded MSPFs. The inserted boxes demonstrate the size of the PSF without and with the cascaded MSPFs. (G and H) SPRM images of individual 5 nm ANPs acquired without (G) and with (H) the cascaded MSPFs.

In contrast, when the cascade MSPFs were used in the experimental setup (Fig. 2 and SI Appendix, Fig. S4A), the captured FFP images demonstrated obviously enhanced imaging contrast for the nanoparticles, as shown in Fig. 3 A, ii–iv, in which one, two, and three MSPFs are used, respectively. It is noted that the wings-shaped scattering spot appeared after the use of the MSPFs and the generation of this wings-shaped spot is described in SI Appendix, Supplementary section 3. In addition to the progressive suppression and smoothing of the background noise, the parabolic tails around the nanoparticles were also removed, resulting in enhancement of the spatial resolution that was discussed in SI Appendix, Supplementary section 4, Fig. 3 E and F. To demonstrate the functions of the three MSPFs directly, four corresponding BFP images were also recorded, as shown in Fig. 3 B, i–iv. Fig. 3 B, i shows two bright spots (corresponding to the two spots in Fig. 1 D–H), which can be attributed to the reflected laser beam from the two surfaces of beam splitter BS3. As shown in Fig. 2 and SI Appendix, Fig. S4A, the incident laser beam at 635 nm was focused on the BFP of the objective; the sample was placed on the FFP of the objective and the reflected beam from the sample would then be focused on the BFP. Because of the finite thickness of beam splitter BS3, the reflected beam from the upper and bottom surfaces of this beam splitter BS3 will generate two spots on the BFP image (B1 and B2), which will increase the background intensity for the entire FFP (Figs. 1 D–H and 3 B, i). The intensity of two reflected beams is evidently much stronger than that of the scattered beam from the single gold nanoparticles, and thus, the scattered light signal does not appear in Fig. 3 B, i.

When the first and second MSPFs are used, the intensity of the two distinct spots on the BFP image will be greatly weakened, as shown in Fig. 3 B, ii and iii. The two bright spots are linearly polarized after passing through polarizer P0, but their polarization orientations will rotate by approximately 90° after passing through the vortex retarder. To tune the orientation of the analyzer located behind the retarder properly, the intensity of these two bright spots will be weakened to almost zero.

Therefore, as shown in Fig. 3 B, ii and iii, a bright ring composed of scattered light from the single ANPs appears. The ring also can be regarded as the leaky radiation of the excited SPs (40, 41). The ring diameter is corresponding to the SPR angle (42, 43). The ring does not appear in Fig. 3 B, i because its intensity is much weaker than that of the two bright spots. Unlike the two bright spots located at two small points on the momentum space, the bright ring is spread on the BFP and thus will be tuned to a radially or azimuthally polarized beam after passing through the polarizer and the vortex retarder. Then, after passing through the analyzer, the ring’s intensity can be much greater than that of the two spots (Fig. 3 B, iii). The scattering and diffraction related background noise that is located outside of this ring will generate background roughness on the FFP. This background roughness can be removed by using the third MSPF (filter 2, Fig. 3 B, iv). Finally, only the scattered light from the single ANPs (represented by the bright ring) will reach the camera, and the imaging contrast and SNR of the nanoparticles will be greatly enhanced.

To describe the imaging enhancement effect induced by the MSPF quantitatively, the intensities of the background light and the scattered light from single 20-nm Au nanoparticles were derived from the captured FFP and BFP images, as shown in Fig. 3 C and D. Here, the intensity of the bright spot pixels (from Fig. 3 A, i–iv) is summed and then divided by the number of pixels, and the result is used as the scattered light intensity from single nanoparticles. There are several single nanoparticles inside the FoV, and thus, a mean value of the scattered light intensity of particle n can be obtained (Iscan). The mean background light intensity of particle n (Ibacn) is obtained from the intensity of the pixels that are in areas without the nanoparticles. The rms of the fluctuations in the background of the captured images is called σn. Details of the data processing based on captured images with several nanoparticles are presented in SI Appendix, Supplementary section 5 and Fig. S5. The mean value of the scattered light intensity, background and RMS of all particles are calculated as Isca¯, Ibac¯, and σ¯. To remove the influence of the incident power density, the sum of the three values of Isca¯, Ibac¯, and σ¯ is normalized to 1 (normalized Isca¯+Ibac¯+σ¯=1) for the four images shown in Fig. 3A.

As shown in the bar graph in Fig. 3C, the intensity ratio of the background light is reduced and the ratio of the signals (scattered light from single nanoparticles) is increased using three MSPFs. The image contrast is defined as C=1N∑n=1NIscan-Ibacn/Iscan+Ibacn. The SNR of the imaged nanoparticles is defined as SNR=1N∑n=1NIscan/σn. The measured imaging contrast and the SNR of the four images are presented in Fig. 3C (dots on the lines), which directly demonstrate the enhanced imaging contrast (with a value of nearly 0.9) and SNR (with the highest value approaching 22) caused using three MSPFs. The enhanced imaging contrast and SNR can still be preserved in the case of high incident power density (e.g., from 10 to 30 W/cm2, where the intensities of both the background and the scattering light are increased), as illustrated in SI Appendix, Supplementary section 6 and Fig. S6, because of the efficient background noise suppression.

This enhancement effect can be explained quantitatively based on Fig. 3D, where the data were derived from the four BFP images (Fig. 3B). Both the diffraction and scattering-related noise pattern (IS0¯; mean intensity of the area between the two larger green-circles in Fig. 3B, from NA ≈1 to NA = 1.49) and the point-like noise patterns (IB1¯ and IB2¯; mean intensities of the two bright spots in Fig. 3B) are weakened by the MSPFs. For example, the normalized intensity of the two bright spots in Fig. 3 B, i of approximately 8.94 × 104 was reduced to 5.56 × 101 (Fig. 3 B, iv) through use of the three MSPFs. In contrast, the diffraction and scattering-related noise intensity changes from 2.89 × 102 to 1.02 × 101. The point-like noise patterns are successively suppressed after installing filter 1 and filter 1’, and the diffraction and scattering-related noise is attenuated by filter 2 subsequently. Therefore, the imaging contrast and the SNR of the nanoparticles can be greatly enhanced. In other words, because of the use of the MSPFs, most of the background light that is not associated with the imaged nano-objects is erased, and only the light scattered from the single nano-objects can be detected by the camera, as illustrated in Fig. 1 D–H.

As a result of this enhanced imaging contrast and SNR, very small single nanoparticles, e.g., polystyrene (PS) nanoparticles with diameters of approximately 20 nm and ANPs with diameters of approximately 5 nm, can be detected clearly in a wide-field imaging manner and without the need for image postprocessing techniques, as shown in Fig. 3 F and H. In contrast, if the MSPFs were removed from the experimental setup, these two types of tiny single nanoparticles then could not be found clearly in the captured images, as shown in Fig. 3 E and G, where the background light overwhelms the images because the light scattered from these single tiny nanoparticles is much weaker than the background light. Transmission electron microscopy (TEM) images of these PS (20 nm) and Au (5 nm, 20 nm) nanoparticles are shown in SI Appendix, Fig. S7.

MSPFs Convert Traditional Label-Free Microscopes into Black-Field Microscopes.

In addition to SPRM, the proposed MSPFs are also compatible with other microscopy techniques, including TIRM. TIRM has the same configuration as SPRM except that the gold film-coated glass substrate is replaced with a bare glass substrate. The illumination source is then the evanescent waves generated at the glass/air interface through the total internal reflection (TIR), as shown in SI Appendix, Fig. S4A. SI Appendix, Fig. S8 A and B show corresponding images of single 20 nm PS nanoparticles and 5 nm ANPs, respectively, that were placed on the bare glass substrate, with both demonstrating high imaging contrast. Single proteins with mass of 66 kDa and 150 kDa can also be detected in this configuration (shown in SI Appendix, Figs. S9 and S10), which provides a method to achieve the quantitative mass imaging of single biological macromolecules (3, 4). There are three aspects that will influence the limit of detection: shot noise and thermal noise of detector, background suppression efficiency, and scattering signal collection efficiency, which are described in SI Appendix, Supplementary section 7 in details.

Furthermore, the proposed MSPFs can also be used to detect nano-defects on nontransparent substrates, e.g., silicon wafers, where both PS nanoparticles and 5 nm ANPs were placed on the wafer to act as defects. Here, an upright microscope (modified from an inverted microscope) was used and operates in reflection mode, as shown in Fig. 2 and SI Appendix, Fig. S11A. A dry objective was used to collect the scattering signals. Comparisons between SI Appendix, Fig. S11 B and C and between SI Appendix, Fig. S11 D and E verify that inclusion of the cascaded MSPFs can greatly enhance the imaging contrast, thus allowing the nanoparticles to be observed clearly. We call all these microscopies combined with the MSPFs as the label-free black-field microscopy; the common features of these microscopies are the nearly black background that is enabled by the MSPFs.

Sensing the Anion-Exchange Reactions in Single Perovskite Nanocrystals.

Finally, we demonstrate the compatibility of the proposed cascaded MSPF with other imaging instruments, e.g., the fluorescence microscope. The main advantages of fluorescence imaging include the wide range of available genetically encodable fluorescent proteins and fluorescent materials with various emission/excitation wavelengths and its superior background suppression performance, and this method has thus been used widely in biological and materials science (44, 45). As shown in Fig. 2 and SI Appendix, Fig. S4B, the cascaded MSPFs were installed in one channel of a total internal reflection fluorescence microscopy (TIRFM) instrument, which makes it possible to combine the strengths of label-based and label-free detection technologies within a single setup (21, 46). To test the feasibility of this approach, standard fluorescent PS nanoparticles with diameters of approximately 20 nm were monodispersed on a cover glass substrate. Fig. 4 A and B show recorded fluorescence and scattering images of the same single nanoparticles, with both demonstrating high imaging contrast.

Fig. 4. Simultaneous labeled and label-free imaging of single nanoparticles. (A and B) TIRFM image (A) and label-free TIRM image (B) of individual 20 nm fluorescent PS nanoparticles. (C) TEM image of CsPbI3 NCs with an edge length of approximately 10 nm. (D) TIRFM image of individual CsPbI3 NCs. (E–G) Label-free TIRM images of CsPbI3 NCs before reaction with the HCl vapor (E), after reaction with HCl (F), and after reaction with HI (G). (H) Curves of the time-varying intensities of individual CsPbX3 NCs inside boxes 1 to 7 during the anion-exchange reaction process. The cascaded MSPFs were installed on the label-free TIRM.

To demonstrate the power of this setup fully, fast anion-exchange reactions in single highly luminescent nanocrystals composed of cesium lead halide perovskites (CsPbX3, X = Cl, Br, I) were monitored in real time. Fig. 4C shows a TEM image of the monodisperse colloidal CsPbI3 nanocubes (NCs), which have an edge length of approximately 10 nm. Fig. 4 D and E show the traditional TIRFM image (excitation wavelength of 325 nm and a long-pass filter at 500 nm that was used to reject the excitation laser beam) and the MSPF image (incident wavelength of 635 nm) of single CsPbI3 NCs in the same FoV. Here, MSPF images means the optical images captured in the detection channel installed with the cascaded MSPFs, as shown in SI Appendix, Fig. S4B.

The anion-exchange reactions in the cesium lead halide perovskites provide an avenue toward tuning of the bright photoluminescence over the entire visible spectral region while also maintaining high quantum yields and narrow emission linewidths. For example, during the anion-exchange reaction between CsPbI3 and Cl−, the excitation spectrum and the optical absorption spectrum are both blue-shifted simultaneously (SI Appendix, Fig. S12 A and B). In addition, the emission spectrum peak is changed from 675 nm to 405 nm, which is shorter than the incident wavelength used in the MSPF channel (635 nm; see SI Appendix, Fig. S12C). The spectral measurements are described in SI Appendix, Supplementary section 8. These optical spectra demonstrate that, under illumination by a laser beam at 635 nm, the CsPbI3 NCs emit fluorescent light, while in contrast, the CsPbCl3 NCs do not emit fluorescent light. These phenomena are consistent with the MSPF images shown in Fig. 4 E and F. After HCl vapor was introduced onto the cover glass on which the CsPbI3 NCs were spin-coated, the intensity of the single NCs in the MSPF image (Fig. 4F) decreased gradually because of the anion-exchange reaction in which the single NCs changed from CsPbI3 to CsPbCl3. A TEM image of the CsPbCl3 NCs is shown in SI Appendix, Fig. S7D, where the diameters of the NCs did not apparently change after the anion-exchange reaction. This anion-exchange reaction is very fast and is complete in only a few seconds.

In the next step, HI vapor was added and another anion-exchange reaction then occurred, in which the NCs were recovered from CsPbCl3 to CsPbI3; the intensity of the single NCs on the MSPF image thus increased again, as shown in Fig. 4G. This anion-exchange reaction between CsPbCl3 and HI is long and lasts for approximately 40 min (Movie S1). All these reactions involving the single NCs were monitored in real time through the intensity changes in the MSPF images.

To demonstrate the advantages of this in situ real-time measurement of single NCs in comparison to macroscopic statistical results, seven areas (marked using boxes 1 to 7) were selected as shown in Fig. 4 E–G. The sum of the pixel intensities inside the seven boxes was derived from the captured MSPF images and the time variations in the pixel intensities were plotted as shown in Fig. 4H. The intensities of the particles inside yellow boxes 1 to 3 decreased with addition of the HCl vapor and increased with addition of the HI vapor; this can be attributed to the normal anion-exchange reactions (Fig. 4H and Movie S2).

No particles were inside boxes 4 to 6, according to Fig. 4 D–F; however, particles appeared in the same positions in Fig. 4G. This is interpreted as the pixel intensities in boxes 4 to 6 remaining unchanged with addition of the HCl vapor and increasing with addition of the HI vapor, as shown in Fig. 4H and Movie S2, and can be attributed to the oxidation and deposition processes of HI vapor acting on the cover glass. In addition, the background of the MSPF image in Fig. 4G became rougher when compared with the image in Fig. 4E because of new roughness being formed on the substrate by the HI vapor. A particle was present inside box 7 in the MSPF image, but this particle did not appear in the TIRFM image, indicating that this particle was not a perovskite NC. Therefore, the MSPF intensity of this particle remained unchanged (Fig. 4H and Movie S2) during addition of both HCl and HI vapors. All the various features of the single NCs are clearly shown in the captured images because of the high imaging contrast and high SNR performance of the proposed experimental setup for single nanoparticle analysis. This setup combines the advantages of label imaging, which can identify objects specifically, with the advantage of label-free imaging, which can be used to perform long-term in situ observations.

Furthermore, the experimental results above show that the intensity of the NCs in the MSPF images comes from both scattering light and fluorescent emissions under laser beam illumination at 635 nm. To find further information about the fast anion-exchange reaction (from CsPbCl3 to CsPbI3) that occurred on the single fluorescent NCs, a 50:50 beam splitter (BS5) was placed behind the cascade MSPFs, as shown in SI Appendix, Fig. S4C. Two identical cameras were used to capture the signals that were transmitted and reflected by this beam splitter. A 650 nm long-pass filter was placed before one camera, thus ensuring that only the fluorescent emission could reach this camera, and the captured fluorescence images are shown in Fig. 5A. The images captured by the other camera that contain both the fluorescence and scattering signals (named the total signals) from the single NCs are shown in Fig. 5B. The whole reaction process was recorded as Movie S3. The images show that the fluorescent intensity decayed very rapidly and then disappeared (Fig. 5A), whereas the total signals in Fig. 5B decayed slowly throughout the entire reaction stage.

Fig. 5. In situ real-time monitoring of the CsPbX3 NC anion-exchange reactions. (A and B) Captured images of individual CsPbX3 NCs after addition of HCl vapor, which were imaged using the MSPF-installed TIRM system with (A) and without (B) a 650 nm long-pass filter placed before the sCMOS camera. (C) Curves of the time-varying intensities of the individual NCs derived from the images in SI Appendix, Fig. S13 A and B. (D) Captured images of the illuminated and nonilluminated areas acquired with and without the long-pass filter. (E) Curves of the time-varying intensities of the individual NCs derived from the images in SI Appendix, Fig. S13 C and D that are both inside the illuminated area acquired with (blue curve) and without (orange curve) a long-pass filter placed before the sCMOS camera. No HCl vapor was present during the imaging process.

To describe this phenomenon quantitatively, the intensities of the fluorescence and total signals from 113 single NCs were derived from the two wide-field images (SI Appendix, Fig. S13 A and B) and averaged, and their time variations were plotted as shown in Fig. 5C. In this case, the 113 single NCs were imaged or detected simultaneously because of the wide-field optical imaging ability of the proposed method. The effective imaging area was approximately 50 × 50 μm2. As shown in Fig. 5C, the intensities of both the fluorescence and total signals remained unchanged before the reaction started but shook when the container of the HCl vapor was placed on the sample holder. The duration of the fluorescence emission was approximately 6 s before it disappeared; however, the reaction was ongoing because the total signals still changed with time, which further demonstrates the advantages of label-free imaging for long-term observation. This phenomenon, as shown in Fig. 5C, shows that the anion-exchange reaction influenced the intensities of both the fluorescence emission and scattering signals from the single NCs. The intensity change in the scattering signals (5.8 × 102) was 65 times greater than that of the fluorescent signals (8.99), which indicates that the scattering signals are more sensitive to the reactions.

Another interesting phenomenon was observed when we translated the glass substrate. As shown in Fig. 5D and Movie S3, the single NCs outside the original illuminated area demonstrated higher fluorescence and scattering intensities than those inside the original illuminated area. In the experiments, the HCl vapor spread over the entire glass substrate, and thus, all NCs on the substrate experienced the anion-exchange reaction; the difference was that some of the NCs were illuminated by the laser beam and some were not. After the laser was turned off for 20 min, the perovskite NCs in the HCl vapor environment were reacted completely (Movie S4). This phenomenon indicated that laser illumination may accelerate the anion-exchange reaction, apart from the photobleaching effect. To verify this point, a control experiment was performed, and the results are shown in Fig. 5E and SI Appendix, Fig. S13 C and D, where no HCl vapor was added to the NCs, and thus, no anion-exchange reaction occurred. The two curves in Fig. 5E were derived from the fluorescence and total signals from the 116 NCs shown in SI Appendix, Fig. S13 C and D. The fluorescence was shown to decay gradually under the laser illumination because of the photobleaching effect. However, the decay amplitude and speed are much smaller than in the two curves in Fig. 5C. Therefore, from the results shown in Fig. 5 C–E, we can conclude that the anion-exchange reaction of single NCs can be accelerated under laser beam illumination, which may generate additional heat around the NCs.

Discussion

We have invented a cascaded MSPF module that can be installed as an add-on component in conventional optical microscopy equipment, including SPRM, TIRM, TIRFM, and upright microscopy systems; benefitting from the modulation of vector field, this MSPF module can eliminate most of the background light and thus enhance the imaging contrast and SNR greatly for wide-field label-free imaging of single nanoparticles. A unique type of microscopy, the black-field microscopy, was then proposed, which have an almost black background. The background intensity of the black-field microscopy is much weaker than that of the conventional dark-field microscopy (6, 47, 48), so it has a better imaging contrast, higher SNR, and sensitivity for single-nanoparticle analysis. Different from the imaging techniques that are based on the two-beam interference, the black-field microscopy is independent of interference; thus, it has a lower requirement on the stability of the setup. It only records the scattering light from the single nano-objects; thus, there is no need for complicated image postprocessing and data analysis techniques. In addition, the functional elements of the MSPFs are all made of thin films; thus, it is possible to integrate the three elements into one and form an optical element. This element is more compact and can be used as an add-in to convert commercial microscopy into black-field microscopy conveniently.

Single gold nanoparticles with diameters of approximately 5 nm and 20 nm, and PS nanoparticles with diameters of approximately 20 nm can all be imaged or detected in real time with high contrast and without using image postprocessing techniques, thus providing an opportunity to perform real-time experimental operations through black-field microscopy on the nanoscale. The cascaded MSPFs can also be installed in fluorescence microscopy systems to combine the strengths of label-based and label-free detection technologies within a single setup. Because of the wide-field, high-contrast, and direct imaging capabilities of the black-field microscopy, the anion-exchange reactions of single perovskite NCs with edge lengths of approximately 10 nm were monitored and manipulated in situ in real time. The results showed some phenomena that can only be observed from single nanoparticle analysis, including the different reaction behaviors among single NCs, and the effects of laser illumination on the anion-exchange reaction. Note that the black-field microscopy is not suitable for super-resolution imaging because of the poor shape of the PSF, which does not break the diffraction limit (49, 50). The main benefit of black-field microscopy is for wide-field imaging of single point-like scatterers with diameters of less than 20 nm that cannot be detected clearly and monitored in real time using conventional label-free imaging methods. The black-field microscopy provides a powerful platform for single nanoparticle analysis and will find wide applications in the biological, physical, environmental, and materials sciences (3, 4, 51–54).

Materials and Methods

Experimental Setup.

All optical measurements were performed using a modified optical microscope (Ti2-U, Nikon, Japan) equipped with a high-numerical-aperture oil immersion objective (100×; NA = 1.49; CFI Apochromat TIRF 100XC Oil, Nikon, Japan). As shown in Fig. 2, the collimated incident laser beam (635 nm) was focused on the BFP of the objective, where the position corresponds to the illumination angle (SI Appendix, Fig. S1A), and then used to illuminate the substrate in the form of a parallel beam. The reflected light generates a point-like pattern and the scattered light generates a ring-like pattern on the BFP. The MSPFs composed of a combination of polarizers and m = 1 vortex retarders, which were installed on BFP 1, BFP 2, and BFP 3, are used to suppress the background noise and extract the scattering signals. The FFP image was separated into two channels and captured using two identical scientific complementary–metal–oxide semiconductor (sCMOS) cameras (Prime 95B, Photometrics). A 650 nm long-pass filter was inserted into one of the channels to filter out the fluorescence signals. In the BFP imaging section, a setup containing a 50:50 beam splitter (BS4) followed by a lens (L12) was used to image the BFP on another sCMOS camera (Neo, Andor Oxford Instruments). In the TIRF system section, a collimated laser beam (532 nm) was used to excite the fluorescence of the fluorescent PS nanoparticles (Fig. 4A), and the 600 nm long-pass filter was used to filter out the fluorescence images. The substrates can be set to be gold films, cover glass, and silicon slices, corresponding to SPRM, TIRM, and upright microscopy systems, respectively. When a silicon slice is inserted into the configuration, the dry objective (NA = 0.60; CFI S Plan Fluor ELWD 40XC, Nikon, Japan) must be used.

Data Acquisition.

In the section where the MSPFs are installed in the system, images were acquired using sCMOS cameras (Photometrics Prime 95B) operating at a frame rate of 40 fps with an exposure time of 10 ms. The incident laser power density was controlled at 12.2 W/cm2 during acquisition of the data shown in Figs. 3–5, except for the data shown in Fig. 3 A, i (acquired at 0.1 W/cm2). The reason for adopting different incident laser power densities for images in Fig. 3 A, i is explained in SI Appendix, Supplementary section 9 and Fig. S14. The total number of pixels in this type of camera is 1,200 × 1,200, and the pixel size is 11 µm. The final magnification of the MSPF-installed microscope system used in this work is 133×, resulting in a size of 82.5 nm/ pixel for the camera used. Benefiting from the homogeneous modulation of the output field by the MSPFs, the full FoV in this work reached 99×99 μm2.

Surface Treatment.

The coverslips (22 × 22 mm2, Fisherfinest, Fisher Scientific) and the silicon slices (25 × 25 mm2, Center for Micro and Nanoscale Research and Fabrication of USTC) were cleaned sequentially in acetone and anhydrous ethanol followed by an ultrapure deionized water rinse and were then dried using a N2 stream. A gold film with a thickness of 45 nm was then deposited on the clean cover glass using a magnetron sputtering system (PRO Line PVD75, Kurt J. Lesker). Prior to deposition, a thin titanium film with a thickness of approximately 3 nm was deposited on the bare glass substrate to enhance the stability of the subsequent thin gold film.

Preparation of the Single Nanoparticles.

Twenty nanometer PS nanoparticles were purchased from Thermo Fisher Scientific. Five nanometer Au nanoparticles and 20 nm fluorescent PS nanoparticles were purchased from XFNANO Materials Tech. Co., Ltd. Human immunoglobulin G (IgG), anti-human IgG, bovine serum albumin (BSA), Cs2CO3, PbI2, and oleyl amine were purchased from Aladdin. Octadecene was purchased from Sigma-Aldrich. Oleic acid and tert-butanol were purchased from Sinopharm Chemical Reagent Co., Ltd. Deionized water with a resistivity of 18.2 MΩ cm−1 from a water purifier (YTUP45S, Shanghai Yetuo Technology Co., Ltd.) was used in all experiments. The Au particles and PS particles were diluted with anhydrous ethanol and dropped on the substrate to dry up. All the biological samples were prepared with deionized water (18.2 MΩ cm−1). The solution was dropped on the cover glass, and then, it was dried under nitrogen. This process was repeated for multiple times so that enough single particles can be left on the cover glass.

Preparation of Cs-oleate.

Cs2CO3 (0.814 g, 99.9%) was loaded into a 100 mL three-neck flask along with octadecene (40 mL, ODE, 90%) and oleic acid (2.5 mL, OA), dried for 1 h at 120 °C, and then heated under a N2 atmosphere to 150 °C until all the Cs2CO3 reacted with the OA. Since Cs-oleate precipitates out of ODE at room temperature, it must be preheated to 100 °C before injection.

Synthesis of CsPbI3 NCs.

ODE (50 mL) and PbI2 (0.87 g, 99.99%) were loaded into a 150 mL three-neck flask and dried under a vacuum for 1 h at 120 °C. Dried oleyl amine (5 mL, OLA, 80 to 90%) and dried OA (5 mL) were injected at 120 °C under a N2 atmosphere. After complete solubilization of a PbI2 salt, the temperature was increased to 170 °C, and the Cs-oleate solution (4 mL, 0.125 M in ODE, prepared as described above) was quickly injected; 5 s later, the reaction mixture was cooled using an ice-water bath.

Isolation and Purification of CsPbI3 NCs.

The crude solution was cooled with the ice-water bath, and the aggregated NCs were separated by centrifuging. Addition of tert-butanol (tBuOH) to the crude solution (ODE:tBuOH = 1:1 by volume) was found to be helpful in obtaining complete precipitation. After centrifugation, the supernatant was discarded and the particles were redispersed in hexane to form long-term colloidally stable solutions.

Solid-State Chlorination Process.

Following the preparation process above, the CsPbI3 NCs solution was spin-coated onto the substrate, which exhibited red fluorescence. The solid-state samples were then exposed to a HCl gas environment for various time intervals, and spectroscopic analysis suggested that the fluorescence emission of the sample was transferred to around 405 nm. This behavior could be attributed to the substitution reaction of CsPbI3 NCs with HCl to produce CsPbCl3, which was accompanied by the variation in the fluorescence emission.

Supplementary Material

Appendix 01 (PDF)

Movie S1. Anion-exchange reactions process of CsPbI3 NCs recorded by the MSPFs installed TIRM system during reacting with HCl (left) and HI (right) in sequence.

Movie S2. Anion-exchange reactions process of 7 areas in Fig. 4 recorded by the MSPFs installed TIRM system during reacting with HCl (left, time scale: seconds) and HI (right, time scale: minutes) in sequence.

Movie S3. Reaction rates of CsPbI3 NCs in different regions after adding HCl vapor, which are imaged by the MSPFs installed TIRM system with (left) and without (right) a 650 nm long pass filter placed before the sCMOS.

Movie S4. Reaction results of CsPbI3 NCs in different regions after adding HCl vapor for 20 mins, which are imaged by the MSPFs installed TIRM system with (left) and without (right) a 650 nm long pass filter placed before the sCMOS.

This work was supported by the National Nature Science Foundation of China (grant nos. 12134013 and 62127818, 52373122, 22071233), the National Key Research and Development Program of China (2021YFA1400700), the Key Research & Development Program of Anhui Province (202104a05020010), and the Fundamental Research Funds for the Central Universities (WK2340000109), Innovation Program for Quantum Science and Technology (no. 2021ZD0303301). D.Z. is supported by a USTC Tang Scholarship. The work was partially performed at the University of Science and Technology of China’s Center for Micro and Nanoscale Research and Fabrication.

Author contributions

Y.L. and D.Z. designed research; Y.L., Q.C., H.Z., Z.F., G.Z., and D.Z. performed research; Y.L. and D.Z. contributed new reagents/analytic tools; Y.L. and D.Z. analyzed data; D.Z. supervised the work; and Y.L. and D.Z. wrote the paper.

Competing interests

The authors declare no competing interest.

Data, Materials, and Software Availability

All study data are included in the article and/or supporting information.

Supporting Information

This article is a PNAS Direct Submission.
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