Figure - available from: Communications Biology
This content is subject to copyright. Terms and conditions apply.
Operating modes of IR nanoscopy in an asymmetric Michelson interferometer scheme a IR radiation, either broadband fs laser for nanoFTIR or QCL for sSNOM, is guided through a beamsplitter (BS) and focused on an oscillating AFM tip via a parabolic mirror (PM). For nanoFTIR (1), the reference arm mirror is mounted on a movable stage. In sSNOM mode (2), the reference arm is equipped with piezo-driven mirror vibrating with frequency M. The scattered light from the tip is recombined with the reflected light from the reference arm at the BS, focused on a mercury-cadmium-telluride (MCT) detector, and fed to a lock-in amplifier. b Simultaneously to AFM imaging, the demodulated sSNOM scattering amplitude and phase are recorded. sSNOM tomography is performed by imaging of serial sections. c The detected interferogram is demodulated at sidebands nΩ ± mM for harmonics n and m of the tip’s resonance frequency Ω and the mirror’s vibration frequency M, respectively. Fast Fourier transformation (FFT) of the interferogram is used to obtain nanoFTIR spectra.
Source publication
Although techniques such as fluorescence-based super-resolution imaging or confocal microscopy simultaneously gather both morphological and chemical data, these techniques often rely on the use of localized and chemically specific markers. To eliminate this flaw, we have developed a method of examining cellular cross sections using the imaging powe...
Similar publications
The folding of newly synthesized polypeptides requires the coordinated action of molecular chaperones. Prokaryotic cells and the chloroplasts of plant cells possess the ribosome-associated chaperone trigger factor, which binds nascent polypeptides at their exit stage from the ribosomal tunnel. The structure of bacterial trigger factor has been well...
Citations
... (a) Infrared spectroscopy, nearfield imaging, and structural model of phospholipid bilayers [65] ; (b) distribution of nuclear components in lymphocytes [66] ; (c) nearfield images of influenza X31 virus in an environment with a pH of 7 [67] ; (d) comparison of nearfield imaging effects of tobacco mosaic virus before and after spectral signal processing [68] 特邀综述 第 44 卷 第 10 期/2024 年 5 月/光学学报 Fig. 7 Applications of nearfield detection technology in cells and microorganisms. (a) SNOM morphology, reflection, and transmission images of sperm [69] ; (b) nano -FTIR spectra detection of single sEV [70] ; (c) tomography reconstruction of Chlamydomonas reinhardtii [71] 特邀综述 第 44 卷 第 10 期/2024 年 5 月/光学学报 Fig. 8 Detecting various types of polaritons by SNOM imaging. (a) Nearfield imaging of graphene plasmons [81] ; (b) phonon polariton images detected at different frequencies [84] ; (c) nanooptical imaging of a MoSe 2 planar waveguide [98] ; (d) timedomain interferometry nanoimaging of hyperbolic polariton pulse propagation [100] 特邀综述 第 44 卷 第 10 期/2024 年 5 月/光学学报 Fig. 9 Nearfield optics and nonlinear effects of lowdimensional materials. ...
... However, the disadvantage is a small penetration depth (about 100 μm) in the visible wavelength range, but it can reach 1-2 cm when using excitation wavelengths in the NIR range. Thus, using nanoscopy in the IR range, it was possible to obtain a tomogram of intracellular structures of eukaryotic (Chlamydomonas reinhardtii) and prokaryotic (Escherichia coli) species with less interference due to the low energy of infrared radiation, though not of the highest resolution (about 20 nm) [22]. ...
This study focuses on the molecular design and synthesis of salt spiropyrans with near-IR fluorescence. The structure of the obtained compounds was confirmed by NMR, IR and mass spectroscopy. In the course of studying the spectral and photoluminescent characteristics, it was possible to reveal the effect of some substituents in various positions on the properties of spiropyran dyes. Due to the structural similarity of one of the isomers to cyanine dyes, the obtained compounds are of interest as potential fluorescent probes for bioimagimg, in particular, for DNA studies. To reveal their ability of binding to DNA molecules molecular docking was carried out. Toxic effects of compounds demonstrating NIR fluorescence were studied on biofilms, as well as using bacterial lux-biosensors.
... Recording of the probe-scattered near fields yields information on the dielectric properties of the sample with nanoscale spatial resolution far beyond the diffraction limit. As a result, s-SNOM has found use in a wide range of applications, including the label-free identification of biological nanoobjects [4][5][6][7][8][9], the compositional mapping of polymer films [10][11][12][13][14][15], the discovery of exotic optical properties of two-dimensional materials [16][17][18] and free-carrier mapping in semiconductors [19][20][21]. ...
... (5) or Eq. (9) to obtain the near-field signals, s n,l and φ n,l . This behavior sets a limit on the useful range of phase modulation depth, γ. ...
We report the development and characterization of a detection technique for scattering-type scanning near-field optical microscopy (s-SNOM) that enables near-field amplitude and phase imaging at two or more wavelengths simultaneously. To this end, we introduce multispectral pseudoheterodyne (PSH) interferometry, where infrared lasers are combined to form a beam with a discrete spectrum of laser lines and a time-multiplexing scheme is employed to allow for the use of a single infrared detector. We first describe and validate the implementation of multispectral PSH into a commercial s-SNOM instrument. We then demonstrate its application for the real-time correction of the negative phase contrast (NPC), which provides reliable imaging of weak IR absorption at the nanoscale. We anticipate that multispectral PSH could improve data throughput, reduce effects of sample and interferometer drift, and help to establish multicolor s-SNOM imaging as a regular imaging modality, which could be particularly interesting as new infrared light sources become available.
... s-SNOM has previously been employed for imaging isolated biological specimens such as protein complexes 6-8 , viruses 9 , and amyloid fibrils 10 , as well as surface imaging of whole cells [11][12][13] . Intracellular structures in cells and tissue have also been imaged in single-celled organisms 14 and zebrafish retinal tissue 15 , respectively. Here we build on this by chemically mapping intracellular structures in human multiple myeloma cells, including, to the authors' knowledge, the first directly optical, sub-diffraction images of endoplasmic reticulum (ER), mitochondria (Mt) and fibrillar components in nucleoli. ...
... The amide I and II absorption bands are aggregates of many vibrational modes exhibited by amide moieties and are routinely used in IR spectroscopy for analysing the protein content of samples 34 , including in nanoscale studies with s-SNOM 10,14 . The phosphodiester (PO 2 ) band corresponds to the PO 2 moieties found in the backbones of nucleic acids and phospholipids. ...
The ability to image cell chemistry at the nanoscale is key for understanding cell biology, but many optical microscopies are restricted by the ~(200–250)nm diffraction limit. Electron microscopy and super-resolution fluorescence techniques beat this limit, but rely on staining and specialised labelling to generate image contrast. It is challenging, therefore, to obtain information about the functional chemistry of intracellular components. Here we demonstrate a technique for intracellular label-free chemical mapping with nanoscale (~30 nm) resolution. We use a probe-based optical microscope illuminated with a mid-infrared laser whose wavelengths excite vibrational modes of functional groups occurring within biological molecules. As a demonstration, we chemically map intracellular structures in human multiple myeloma cells and compare the morphologies with electron micrographs of the same cell line. We also demonstrate label-free mapping at wavelengths chosen to target the chemical signatures of proteins and nucleic acids, in a way that can be used to identify biochemical markers in the study of disease and pharmacology.
... To address this issue, a streamlined sliceand-view method could be used to obtain volumetric images of biological systems. 129 Another limitation is the imaging speed. Compared to laser scans in CRS microscopy, AFM stage scan has a much slower frame time from minutes to hours, leaving many dynamic biological processes 130 untraceable. ...
Chemical imaging based on vibrational contrasts can extract molecular information entangled in complex biological systems. To this end, nonlinear Raman scattering microscopy, mid-infrared photothermal (MIP) microscopy, and atomic force microscopy (AFM)-based force-detected photothermal microscopies are emerging with better chemical sensitivity, molecular specificity, and spatial resolution than conventional vibrational methods. Their utilization in bioimaging applications has provided biological knowledge in unprecedented detail. This Perspective outlines key methodological developments, bioimaging applications, and recent technical innovations of the three techniques. Representative biological demonstrations are also highlighted to exemplify the unique advantages of obtaining vibrational contrasts. With years of effort, these three methods compose an expanding vibrational bioimaging toolbox to tackle specific bioimaging needs, benefiting many biological investigations with rich information in both label-free and labeling manners. Each technique will be discussed and compared in the outlook, leading to possible future directions to accommodate growing needs in vibrational bioimaging.
... The contrast mechanisms of s-SNOM enable mapping the dielectric properties [28][29][30][31] and chemical composition 14 of samples, which has so far facilitated a wide range of discoveries in condensed phase and two-dimensional materials. [32][33][34][35][36][37][38][39][40] Additionally, s-SNOM applications in biology enable precise mapping of specific cellular and subcellular structures of interest, [41][42][43][44] and even mapping of the exact values of important optical parameters such as the refractive index. 45 Although s-SNOM is generally acknowledged as a surface characterization technique, it nonetheless exhibits valuable capabilities for subsurface and 3D imaging. ...
... 107,108 This pump beam would complement the probe beam, which in many s-SNOM applications is tuned to the absorption band of the sample to promote phase contrast. 14,43,81,109 An additional layer of (faster) modulation can eventually provide higher SNR. Furthermore, this alternative modulation scheme based on onoff switching of the pump beam could even entirely replace the traditional modulation schemes based on the tapping movement of the probe, which could enable s-SNOM imaging in contact mode, which would be faster than tapping-mode s-SNOM (which is the current default way of performing s-SNOM investigations), and more suited for some applications, depending on the sample type and scanning environment. ...
A thorough understanding of biological species and emerging nanomaterials requires, among other efforts, their in-depth characterization through optical techniques capable of nano-resolution. Nanoscopy techniques based on tip-enhanced optical effects have gained tremendous interest over the past years, given their potential to obtain optical information with resolutions limited only by the size of a sharp probe interacting with focused light, irrespective of the illumination wavelength. Although their popularity and number of applications is rising, tip-enhanced nanoscopy (TEN) techniques still largely rely on probes that are not specifically developed for such applications, but for atomic force microscopy. This limits their potential in many regards, e.g., in terms of signal-to-noise ratio, attainable image quality, or extent of applications. We take the first steps toward next-generation TEN by demonstrating the fabrication and modeling of specialized TEN probes with known optical properties. The proposed framework is highly flexible and can be easily adjusted to be used with diverse TEN techniques, building on various concepts and phenomena, significantly augmenting their function. Probes with known optical properties could potentially enable faster and more accurate imaging via different routes, such as direct signal enhancement or facile, and ultra-fast, optical signal modulation. We consider that the reported development can pave the way for a vast number of novel TEN imaging protocols and applications, given the many advantages that it offers.
... The contrast mechanisms of s-SNOM enable mapping the dielectric properties 21-24 of samples and their chemical composition 11 , which have facilitated so far a wide range of discoveries in the condensed phase materials, and two-dimensional materials [25][26][27][28][29][30][31][32][33] . Additionally, s-SNOM applications in biology enable precise mapping of specific cellular and sub-cellular structures of interest [34][35][36][37] , and even mapping the exact values of important optical parameters such as the refractive index 38 . TERS, combining the advantages of surface-enhanced Raman spectroscopy and scanning probe microscopy technologies [39][40][41] , found its utilization in the investigation of a wide variety of nanostructured materials ranging from organic to inorganic chemical substances. ...
... We speculate that this type of modulation, achieved by mechanical means, can be replaced, or augmented with an alternative/additional modulation scheme, by means of a pump beam tuned to the plasmon band of the tip, which can intermittently plasmonically activate/deactivate the tip, at ultra-fast rates, and depending on the properties of the sample, it can modify the interaction of the two mirror dipoles (in probe and sample) accounting for s-SNOM contrast 97,98 . This pump beam, would complement the probe beam, which in many s-SNOM applications is tuned to the absorption band of the sample to promote phase contrast 36,72,96,99 . An additional layer of (faster) modulation can eventually provide higher SNR. ...
A thorough understanding of biological species and of emerging nanomaterials requires, among others, their in-depth characterization with optical techniques capable of nano-resolution. Nanoscopy techniques based on tip-enhanced optical effects have gained over the past years tremendous interest given their potential to probe various optical properties with resolutions depending on the size of a sharp probe interacting with focused light, irrespective of the illumination wavelength. Although their popularity and number of applications is rising, tip-enhanced nanoscopy techniques (TEN) still largely rely on probes that are not specifically developed for such applications, but for Atomic Force Microscopy. This cages their potential in many regards, e.g. in terms of signal-to-noise ratio, attainable image quality, or extent of applications. In this article we place first steps towards next-gen TEN, demonstrating the fabrication and modelling of specialized TEN probes with known optical properties. The proposed framework is highly flexible and can be easily adjusted to be of o benefit to various types of TEN techniques, for which probes with known optical properties could potentially enable faster and more accurate imaging via different routes, such as direct signal enhancement or novel signal modulation strategies. We consider that the reported development can pave the way for a vast number of novel TEN imaging protocols and applications, given the many advantages that it offers.
... We consider these observations to be important in light of s-SNOM's capabilities for nanoscale spatial resolution, which represent an important advantage when studying Au NPrelated aspects, such as their distribution in doped materials or inside cells. Such latter studies are greatly facilitated by recent efforts that have been focused on s-SNOM imaging of living 51 and fixed cells, 52 which have resulted in valuable investigation platforms that can characterize various types of cell structures and processes. We argue that these currently introduced methodologies can be straightforwardly used to assess as well subtle aspects related to the interactions occurring between cells and biomedically functionalized (or hazardous) nanomaterials, to resolve important pending issues such as nanoparticle trafficking by cells. ...
Scattering-type scanning near-field optical microscopy (s-SNOM) has emerged over the past years as a powerful characterization tool that can probe important properties of advanced materials and biological samples in a label-free manner, with spatial resolutions lying in the nanoscale realm. In this work, we explore such usefulness in relationship with an interesting class of materials: polymer-coated gold nanoparticles (NPs). As thoroughly discussed in recent works, the interplay between the Au core and the polymeric shell has been found to be important in many applications devoted to biomedicine. We investigate bare Au NPs next to polystyrenesulfonate (PSS) and poly(diallyldimethylammonium chloride) (PDDA) coated ones under 532 nm laser excitation, an wavelength matching the surface plasmon band of the custom-synthesized nanoparticles. We observe consistent s-SNOM phase signals in the case of bare and shallow-coated Au NPs, whereas for thicker shell instances, these signals fade. For all investigated samples, the s-SNOM amplitude signals were found to be very weak, which may be related to reduced scattering efficiency due to absorption of the incident beam. We consider these observations important, as they may facilitate studies and applications in nanomedicine and nanotechnology where the precise positioning of polymer-coated Au NPs with nanoscale resolution is needed besides their dielectric function and related intrinsic optical properties, which are also quantitatively available with s-SNOM.
... In fact, several s-SNOM studies have already investigated slices microtomed from the bulk sample, such as hair [93], silk [108], and cellulose materials [125]. Recently, Kanevche et al. has reported s-SNOM imaging of 100 nm-thick cellular crosssections of eukaryotic and prokaryotic cells, and a 3D tomogram with 20-nm IR resolution was composed [126]. The smooth combination of AFM-based nano-IR techniques with advanced microtome methodology will streamline the discovery of spatialtemporal correlations in life-relevant cells and tissues. ...
Small biomolecules at the subcellular level are building blocks for the manifestation of complex biological activities. However, non-intrusive in situ investigation of biological systems has been long daunted by the low spatial resolution and poor sensitivity of conventional light microscopies. Traditional infrared (IR) spectro-microscopy can enable label-free visualization of chemical bonds without extrinsic labeling but is still bound by Abbe’s diffraction limit. This review article introduces a way to bypass the optical diffraction limit and improve the sensitivity for mid-IR methods – using tip-enhanced light nearfield in atomic force microscopy (AFM) operated in tapping and peak force tapping modes. Working principles of well-established scattering-type scanning near-field optical microscopy (s-SNOM) and two relatively new techniques, namely, photo-induced force microscopy (PiFM) and peak force infrared (PFIR) microscopy, will be briefly presented. With ∼10-20 nm spatial resolution and monolayer sensitivity, their recent applications in revealing nanoscale chemical heterogeneities in a wide range of biological systems, including biomolecules, cells, tissues, and biomaterials, will be reviewed and discussed. We also envision several future improvements of AFM-based tapping and peak force tapping mode nano-IR methods that permit them to better serve as a versatile platform for uncovering biological mechanisms at the fundamental level.
This study utilizes nanoscale Fourier transform infrared spectroscopy (nanoFTIR) to perform stable isotope probing (SIP) on individual bacteria cells cultured in the presence of 13C-labelled glucose. SIP-nanoFTIR simultaneously quantifies single-cell metabolism through infrared spectroscopy and acquires cellular morphological information via atomic force microscopy. The redshift of the amide I peak corresponds to the isotopic enrichment of newly synthesized proteins. These observations of single-cell translational activity are comparable to those of conventional methods, examining bulk cell numbers. Observing cells cultured under conditions of limited carbon, SIP-nanoFTIR is used to identify environmentally-induced changes in metabolic heterogeneity and cellular morphology. Individuals outcompeting their neighboring cells will likely play a disproportionately large role in shaping population dynamics during adverse conditions or environmental fluctuations. Additionally, SIP-nanoFTIR enables the spectroscopic differentiation of specific cellular growth phases. During cellular replication, subcellular isotope distribution becomes more homogenous, which is reflected in the spectroscopic features dependent on the extent of 13C-13C mode coupling or to specific isotopic symmetries within protein secondary structures. As SIP-nanoFTIR captures single-cell metabolism, environmentally-induced cellular processes, and subcellular isotope localization, this technique offers widespread applications across a variety of disciplines including microbial ecology, biophysics, biopharmaceuticals, medicinal science, and cancer research.
