Label-free Microscopy Techniques Research Articles

Intraoperative microscopic autofluorescence detection and characterization in brain tumors using stimulated Raman histology and two-photon fluorescence.

The intrinsic autofluorescence of biological tissues interferes with the detection of fluorophores administered for fluorescence guidance, an emerging auxiliary technique in oncological surgery. Yet, autofluorescence of the human brain and its neoplasia is sparsely examined. This study aims to assess autofluorescence of the brain and its neoplasia on a microscopic level by stimulated Raman histology (SRH) combined with two-photon fluorescence. With this experimentally established label-free microscopy technique unprocessed tissue can be imaged and analyzed within minutes and the process is easily incorporated in the surgical workflow. In a prospective observational study, we analyzed 397 SRH and corresponding autofluorescence images of 162 samples from 81 consecutive patients that underwent brain tumor surgery. Small tissue samples were squashed on a slide for imaging. SRH and fluorescence images were acquired with a dual wavelength laser (790 nm and 1020 nm) for excitation. In these images tumor and non-tumor regions were identified by a convolutional neural network that reliably differentiates between tumor, healthy brain tissue and low quality SRH images. The identified areas were used to define regions.of- interests (ROIs) and the mean fluorescence intensity was measured. In healthy brain tissue, we found an increased mean autofluorescence signal in the gray (11.86, SD 2.61, n=29) compared to the white matter (5.99, SD 5.14, n=11, p<0.01) and in the cerebrum (11.83, SD 3.29, n=33) versus the cerebellum (2.82, SD 0.93, n=7, p<0.001), respectively. The signal of carcinoma metastases, meningiomas, gliomas and pituitary adenomas was significantly lower (each p<0.05) compared to the autofluorescence in the cerebrum and dura, and significantly higher (each p<0.05) compared to the cerebellum. Melanoma metastases were found to have a higher fluorescent signal (p<0.01) compared to cerebrum and cerebellum. In conclusion we found that autofluorescence in the brain varies depending on the tissue type and localization and differs significantly among various brain tumors. This needs to be considered for interpreting photon signal during fluorescence-guided brain tumor surgery.

Label-free microscopy for virus infections.

Microscopy has been essential to elucidate micro- and nano-scale processes in space and time and has provided insights into cell and organismic functions. It is widely employed in cell biology, microbiology, physiology, clinical sciences and virology. While label-dependent microscopy, such as fluorescence microscopy, provides molecular specificity, it has remained difficult to multiplex in live samples. In contrast, label-free microscopy reports on overall features of the specimen at minimal perturbation. Here, we discuss modalities of label-free imaging at the molecular, cellular and tissue levels, including transmitted light microscopy, quantitative phase imaging, cryogenic electron microscopy or tomography and atomic force microscopy. We highlight how label-free microscopy is used to probe the structural organization and mechanical properties of viruses, including virus particles and infected cells across a wide range of spatial scales. We discuss the working principles of imaging procedures and analyses and showcase how they open new avenues in virology. Finally, we discuss orthogonal approaches that enhance and complement label-free microscopy techniques.

End-to-end reconstruction of multi-scale holograms based on CUE-NET

Digital holography is one of the most widely used label free microscopy techniques in biomedical imaging. It is an important step in hologram reconstruction to recover the missing phase information. A phase recovery method based on deep learning is presented here, which enables the phase and amplitude information of multiple holograms with different out-of-focus distances to be reconstructed quickly, and the multi-scale and large field of view can be reconstructed through the generated network model. We demonstrate the success of this deep learning multi-scale hologram self-focusing reconstruction method by self-focusing the holographic microscopic images of human red blood cells and chicken blood cells. Compared with the existing methods, this method improves the quality of the reconstructed image and the reconstruction speed. Compared with U-NET, the peak signal-to-noise ratio (PSNR) and structural similarity of the reconstructed network output are increased from 18.5 dB and 0.89 to 23.98 dB and 0.97, respectively, which can suppress the influence of speckle noise well.

Holographic Image Reconstruction with Phase Recovery and Autofocusing Using Recurrent Neural Networks

Digital holography is one of the most widely used label-free microscopy techniques in biomedical imaging. Recovery of the missing phase information of a hologram is an important step in holographic image reconstruction. Here we demonstrate a convolutional recurrent neural network (RNN) based phase recovery approach that uses multiple holograms, captured at different sample-to-sensor distances to rapidly reconstruct the phase and amplitude information of a sample, while also performing autofocusing through the same network. We demonstrated the success of this deep learning-enabled holography method by imaging microscopic features of human tissue samples and Papanicolaou (Pap) smears. These results constitute the first demonstration of the use of recurrent neural networks for holographic imaging and phase recovery, and compared with existing methods, the presented approach improves the reconstructed image quality, while also increasing the depth-of-field and inference speed.

Combined approach using circular intensity differential scattering microscopy under phasor map data analysis.

Circular intensity differential scattering (CIDS) is based on the analysis of circular polarized light scattering and has been proven to be an interesting label-free microscopy technique sensitive to the chiral organization at the submicroscopic level. However, this approach averages the localized contrasts related to the sample polarimetric properties in the illumination volume. Additionally, the detection sensitivity suffers from the confinement of the mixture of structures, and it becomes an arduous task to discriminate the source of the signal. In this work, we show that a phasor map approach combined with CIDS microscopy has provided an intuitive view of the sample organization to recognize the presence of different molecular species in the illumination volume. The data represented in terms of polarization response mapped to a single point called a phasor also have the potential to pave the way for the analysis of large data sets. We validated this method by numerical simulations and compared the results with that of experimental data of optical devices of reference.

Nanoelectromechanical photothermal polarization microscopy with 3 Å localization precision

Single-molecule microscopy has become an indispensable tool for biochemical analysis. The capability of characterizing distinct properties of individual molecules without averaging has provided us with a different perspective for the existing scientific issues and phenomena. Recently, super-resolution fluorescence microscopy techniques have overcome the optical diffraction limit by the localization of molecule positions. However, the labeling process can potentially modify the intermolecular dynamics. Based on the highly sensitive nanomechanical photothermal microscopy reported previously, we propose optimizations on this label-free microscopy technique toward localization microscopy. A localization precision of 3 Å is achieved with gold nanoparticles, and the detection of polarization-dependent absorption is demonstrated, which opens the door for further improvement with polarization modulation imaging.

Three-dimensional imaging with reflection synthetic confocal microscopy.

Biomedical imaging lacks label-free microscopy techniques able to reconstruct the contour of biological cells in solution, in 3D and with high resolution, as required for the fast diagnosis of numerous diseases. Inspired by computational optical coherence tomography techniques, we present a tomographic diffractive microscope in reflection geometry used as a synthetic confocal microscope, compatible with this goal and validated with the 3D reconstruction of a human effector T lymphocyte.

Phase imaging and synthetic aperture super-resolution via total internal reflection microscopy.

Total internal reflection microscopy is mainly used in its fluorescence mode and is the reference technique to image fluorescent proteins in the vicinity of cell membranes. Here, we show that this technique can easily become a phase microscope by simply detecting the coherent signal resulting from the interference between the field scattered by the probed sample and the total internal reflection. Moreover, combining several illumination angles permits generating synthetic aperture reconstructions with improved resolutions compared to standard label-free microscopy techniques.

Surface plasmon microscopy by spatial light switching for label-free imaging with enhanced resolution.

In this Letter, we describe spatially switched surface plasmon microscopy (ssSPM) based on two-channel momentum sampling. The performance evaluated with periodic nanowires in comparison with conventional SPM and bright-field microscopy shows that the resolution of ssSPM is enhanced by almost 15 times over conventional SPM. ssSPM provides an extremely simple way to attain diffraction limit in SPM and to go beyond for super-resolution in label-free microscopy techniques.

Label-free Imaging and Bending Analysis of Microtubules by ROCS Microscopy and Optical Trapping

Mechanical manipulation of single cytoskeleton filaments and their monitoring over long times is difficult because of fluorescence bleaching or phototoxic protein degradation. The integration of label-free microscopy techniques, capable of imaging freely diffusing, weak scatterers such as microtubules (MTs) in real-time, and independent of their orientation, with optical trapping and tracking systems, would allow many new applications. Here, we show that rotating-coherent-scattering microscopy (ROCS) in dark-field mode can also provide strong contrast for structures far from the coverslip such as arrangements of isolated MTs and networks. We could acquire thousands of images over up to 30 min without loss in image contrast or visible photodamage. We further demonstrate the combination of ROCS imaging with fast and nanometer-precise 3D interferometric back-focal-plane tracking of multiple beads in time-shared optical traps using acoustooptic deflectors to specifically construct and microrheologically probe small microtubule networks with well-defined geometries. Thereby, we explore the frequency-dependent elastic response of single microtubule filaments between 0.5 Hz and 5 kHz, which allows for investigating their viscoelastic response up to the fourth-order bending mode. Our spectral analysis reveals constant filament stiffness at low frequencies and frequency-dependent stiffening following a power law ∼ωp with a length-dependent exponent p(L). We find further evidence for the dependence of the MT persistence length on the contour length L, which is still controversially debated. We could also demonstrate slower stiffening at high frequencies for longer filaments, which we believe is determined by the molecular architecture of the MT. Our results shed new light on the nanomechanics of this essential, multifunctional cytoskeletal element and pose new questions about the adaptability of the cytoskeleton.

Fast label-free microscopy technique for 3D dynamic quantitative imaging of living cells.

The refractive index (RI) is an important optical characteristic that is often exploited in label-free microscopy for analysis of biological objects. A technique for 3D RI reconstruction of living cells has to be fast enough to capture the cell dynamics and preferably needs to be compatible with standard wide-field microscopes. To solve this challenging problem, we present a technique that provides fast measurement and processing of data required for real-time 3D visualization of the object RI. Specifically, the 3D RI is reconstructed from the measurement of bright-field intensity images, axially scanned by a high-speed focus tunable lens mounted in front of a sCMOS camera, by using a direct deconvolution approach designed for partially coherent light microscopy in the non-paraxial regime. Both the measurement system and the partially coherent illumination, that provides optical sectioning and speckle-noise suppression, enable compatibility with wide-field microscopes resulting in a competitive and affordable alternative to the current holographic laser microscopes. Our experimental demonstrations show video-rate 3D RI visualization of living bacteria both freely swimming and optically manipulated by using freestyle laser traps allowing for their trapping and transport along 3D trajectories. These results prove that is possible to conduct simultaneous 4D label-free quantitative imaging and optical manipulation of living cells, which is promising for the study of the cell biophysics and biology.

Phase Measurement Using DIC Microscopy

The development of fluorescent probes and proteins has helped make light microscopy more popular by allowing the visualization of specific subcellular components, location and dynamics of biomolecules. However, it is not always feasible to label the cells as it may be phototoxic or perturb their functionalities. Label-free microscopy techniques allow us to work with live cells without perturbation and to evaluate morphological differences, which in turn can provide useful information for high-throughput assays. In this study, we use one of the most popular label-free techniques called differential interference contrast (DIC) microscopy to estimate the phase of cells and other nearly transparent objects and instantly estimate their height. DIC images provide detailed information about the optical path length (OPL) differences in the sample and they are visually similar to a gradient image. Our previous DIC construction algorithm outputs an image where the values are proportional to the OPL (or implicitly the phase) of the sample. Although the reconstructed images are capable of describing cellular morphology and to a certain extent turn DIC into a quantitative technique, the actual OPL has to be computed from the input DIC image and the microscope calibration settings. Here we propose a computational method to measure the phase and approximate height of cells after microscope calibration, assuming a linear formation model. After a calibration step the phase of further samples can be determined when the refractive indices of the sample and the surrounding medium is known. The precision of the method is demonstrated on reconstructing the thickness of known objects and real cellular samples.

Fast, label-free super-resolution live-cell imaging using rotating coherent scattering (ROCS) microscopy.

Living cells are highly dynamic systems with cellular structures being often below the optical resolution limit. Super-resolution microscopes, usually based on fluorescence cell labelling, are usually too slow to resolve small, dynamic structures. We present a label-free microscopy technique, which can generate thousands of super-resolved, high contrast images at a frame rate of 100 Hertz and without any post-processing. The technique is based on oblique sample illumination with coherent light, an approach believed to be not applicable in life sciences because of too many interference artefacts. However, by circulating an incident laser beam by 360° during one image acquisition, relevant image information is amplified. By combining total internal reflection illumination with dark-field detection, structures as small as 150 nm become separable through local destructive interferences. The technique images local changes in refractive index through scattered laser light and is applied to living mouse macrophages and helical bacteria revealing unexpected dynamic processes.

Noncontact three-dimensional mapping of intracellular hydromechanical properties by Brillouin microscopy.

Current measurements of the biomechanical properties of cells require physical contact with cells or lack sub-cellular resolution. Here, we developed a label-free optical microscopy technique based on Brillouin light scattering capable of measuring intracellular longitudinal modulus with optical resolution. We obtained 3D Brillouin maps of cells in 2D and 3D microenvironments, which reveal mechanical changes due to cytoskeletal modulation and cell volume regulation.

Digital Holographic Microscopy: A Quantitative Label-Free Microscopy Technique for Phenotypic Screening

Digital Holographic Microscopy (DHM) is a label-free imaging technique allowing visualization of transparent cells with classical imaging cell culture plates. The quantitative DHM phase contrast image provided is related both to the intracellular refractive index and to cell thickness. DHM is able to distinguish cellular morphological changes on two representative cell lines (HeLa and H9c2) when treated with doxorubicin and chloroquine, two cytotoxic compounds yielding distinct phenotypes. We analyzed parameters linked to cell morphology and to the intracellular content in endpoint measurements and further investigated them with timelapse recording. The results obtained by DHM were compared with other optical label-free microscopy techniques, namely Phase Contrast, Differential Interference Contrast and Transport of Intensity Equation (reconstructed from three bright-field images). For comparative purposes, images were acquired in a common 96-well plate format on the different motorized microscopes. In contrast to the other microscopies assayed, images generated with DHM can be easily quantified using a simple automatized on-the-fly analysis method for discriminating the different phenotypes generated in each cell line. The DHM technology is suitable for the development of robust and unbiased image-based assays.

Quantifying Biomass Changes of Single CD8+ T Cells during Antigen Specific Cytotoxicity

Existing approaches that quantify cytotoxic T cell responses rely on bulk or surrogate measurements which impede the direct identification of single activated T cells of interest. Single cell microscopy or flow cytometry methodologies typically rely on fluorescent labeling, which limits applicability to primary cells such as human derived T lymphocytes. Here, we introduce a quantitative method to track single T lymphocyte mediated cytotoxic events within a mixed population of cells using live cell interferometry (LCI), a label-free microscopy technique that maintains cell viability. LCI quantifies the mass distribution within individual cells by measuring the phase shift caused by the interaction of light with intracellular biomass. Using LCI, we imaged cytotoxic T cells killing cognate target cells. In addition to a characteristic target cell mass decrease of 20–60% over 1–4 h following attack by a T cell, there was a significant 4-fold increase in T cell mass accumulation rate at the start of the cytotoxic event and a 2–3 fold increase in T cell mass relative to the mass of unresponsive T cells. Direct, label-free measurement of CD8+ T and target cell mass changes provides a kinetic, quantitative assessment of T cell activation and a relatively rapid approach to identify specific, activated patient-derived T cells for applications in cancer immunotherapy.

Imaging impedance

A label-free microscopy technique based on electrochemical impedance offers a new way of studying electrochemical processes in single cells.