Jenu V. Chacko

A novel nanoscopic tool by combining AFM with STED microscopy

We present a new instrument for nanoscopic investigations by coupling an atomic force microscope (AFM) with a super resolution stimulated emission depletion (STED) microscope. This nanoscopic tool allows high resolution fluorescence imaging, topographical imaging and nano-mechanical imaging, such as, stiffness. Results obtained from technical and biological samples are shown illustrating different functions and the versatility of the presented tool. We assert that, this highly precise tractable tool paves the way to a new set of comprehensive studies in medicine, biology and materials science.

Probing cytoskeletal structures by coupling optical superresolution and AFM techniques for a correlative approach

In this article, we describe and show the application of some of the most advanced fluorescence superresolution techniques, STED AFM and STORM AFM microscopy towards imaging of cytoskeletal structures, such as microtubule filaments. Mechanical and structural properties can play a relevant role in the investigation of cytoskeletal structures of interest, such as microtubules, that provide support to the cell structure. In fact, the mechanical properties, such as the local stiffness and the elasticity, can be investigated by AFM force spectroscopy with tens of nanometers resolution. Force curves can be analyzed in order to obtain the local elasticity (and the Youngs modulus calculation by fitting the force curves from every pixel of interest), and the combination with STED/STORM microscopy integrates the measurement with high specificity and yields superresolution structural information. This hybrid modality of superresolution‐AFM working is a clear example of correlative multimodal microscopy.

Elucidation of Exosome Migration across the Blood-Brain Barrier Model In Vitro.

The delivery of therapeutics to the central nervous system remains a major challenge in part due to the presence of the blood–brain barrier (BBB). Recently, cell-derived vesicles, particularly exosomes, have emerged as an attractive vehicle for targeting drugs to the brain, but whether or how they cross the BBB remains unclear. Here, we investigated the interactions between exosomes and brain microvascular endothelial cells (BMECs) in vitro under conditions that mimic the healthy and inflamed BBB in vivo. Transwell assays revealed that luciferase-carrying exosomes can cross a BMEC monolayer under stroke-like, inflamed conditions (TNF-α activated) but not under normal conditions. Confocal microscopy showed that exosomes are internalized by BMECs through endocytosis, co-localize with endosomes, in effect primarily utilizing the transcellular route of crossing. Together, these results indicate that cell-derived exosomes can cross the BBB model under stroke-like conditions in vitro. This study encourages further development of engineered exosomes as drug delivery vehicles or tracking tools for treating or monitoring neurological diseases.

Sub-diffraction nano manipulation using STED AFM.

In the last two decades, nano manipulation has been recognized as a potential tool of scientific interest especially in nanotechnology and nano-robotics. Contemporary optical microscopy (super resolution) techniques have also reached the nanometer scale resolution to visualize this and hence a combination of super resolution aided nano manipulation ineluctably gives a new perspective to the scenario. Here we demonstrate how specificity and rapid determination of structures provided by stimulated emission depletion (STED) microscope can aid another microscopic tool with capability of mechanical manoeuvring, like an atomic force microscope (AFM) to get topological information or to target nano scaled materials. We also give proof of principle on how high-resolution real time visualization can improve nano manipulation capability within a dense sample, and how STED-AFM is an optimal combination for this job. With these evidences, this article points to future precise nano dissections and maybe even to a nano-snooker game with an AFM tip and fluorospheres.

Cellular level nanomanipulation using atomic force microscope aided with superresolution imaging

Abstract. Atomic force microscopes (AFM) provide topographical and mechanical information of the sample with very good axial resolution, but are limited in terms of chemical specificity and operation time-scale. An optical microscope coupled to an AFM can recognize and target an area of interest using specific identification markers like fluorescence tags. A high resolution fluorescence microscope can visualize fluorescence structures or molecules below the classical optical diffraction limit and reach nanometer scale resolution. A stimulated emission depletion (STED) microscopy superresolution (SR) microscope coupled to an AFM is an example in which the AFM tip gains nanoscale manipulation capabilities. The SR targeting and visualization ability help in fast and specific identification of subdiffraction-sized cellular structures and manoeuvring the AFM tip onto the target. We demonstrate how to build a STED AFM and use it for biological nanomanipulation aided with fast visualization. The STED AFM based bionanomanipulation is presented for the first time in this article. This study points to future nanosurgeries performable at single-cell level and a physical targeted manipulation of cellular features as it is currently used in research domains like nanomedicine and nanorobotics.

Mechanoresponsive stem cells to target cancer metastases through biophysical cues

Targeting the mechano-niche of the tumor microenvironment is a strategy to interrogate, detect, and treat cancer. A stiff punishment for tumors In patients, tumor cells do not grow in isolation, and their behavior is regulated not only by their own biology but also by interactions with their microenvironment. A key part of the microenvironment is the extracellular matrix, which typically has a greater stiffness in tumors than in surrounding normal tissues. To take advantage of this, Liu et al. engineered mechanoresponsive mesenchymal stem cells to act as vehicles for cancer drug delivery. These engineered stem cells accumulated in tumors, delivering the first half of a two-part cancer therapy: the enzyme cytosine deaminase. A drug called 5-fluorocytosine was then delivered systemically, and cytosine deaminase in the tumors activated the drug, providing local anticancer activity with no off-target damage in mice. Despite decades of effort, little progress has been made to improve the treatment of cancer metastases. To leverage the central role of the mechanoenvironment in cancer metastasis, we present a mechanoresponsive cell system (MRCS) to selectively identify and treat cancer metastases by targeting the specific biophysical cues in the tumor niche in vivo. Our MRCS uses mechanosensitive promoter–driven mesenchymal stem cell (MSC)–based vectors, which selectively home to and target cancer metastases in response to specific mechanical cues to deliver therapeutics to effectively kill cancer cells, as demonstrated in a metastatic breast cancer mouse model. Our data suggest a strong correlation between collagen cross-linking and increased tissue stiffness at the metastatic sites, where our MRCS is specifically activated by the specific cancer–associated mechano-cues. MRCS has markedly reduced deleterious effects compared to MSCs constitutively expressing therapeutics. MRCS indicates that biophysical cues, specifically matrix stiffness, are appealing targets for cancer treatment due to their long persistence in the body (measured in years), making them refractory to the development of resistance to treatment. Our MRCS can serve as a platform for future diagnostics and therapies targeting aberrant tissue stiffness in conditions such as cancer and fibrotic diseases, and it should help to elucidate mechanobiology and reveal what cells “feel” in the microenvironment in vivo.

Ultracompact alignment-free single molecule fluorescence device with a foldable light path

Instruments with single-molecule level detection capabilities can potentially benefit a wide variety of fields, including medical diagnostics. However, the size, cost, and complexity of such devices have prevented their widespread use outside sophisticated research laboratories. Fiber-only devices have recently been suggested as smaller and simpler alternatives, but thus far, they have lacked the resolution and sensitivity of a full-fledged system, and accurate alignment remains a critical requirement. Here we show that through-space reciprocal optical coupling between a fiber and a microscope objective, combined with wavelength division multiplexing in optical fibers, allows a drastic reduction of the size and complexity of such an instrument while retaining its resolution. We demonstrate a 4 × 4 × 18 cm(3) sized fluorescence correlation spectrometer, which requires no alignment, can analyze kinetics at the single-molecule level, and has an optical resolution similar to that of much larger microscope based devices. The sensitivity can also be similar in principle, though in practice it is limited by the large background fluorescence of the commonly available optical fibers. We propose this as a portable and field deployable single molecule device with practical diagnostic applications.

Correlative nanoscopy: super resolved fluorescence and atomic force microscopy towards nanoscale manipulation and multimodal investigations

Super resolved fluorescence microscopy combined with Atomic Force Microscopy allows to access different data sets and functionalities of investigated samples [1]. We named this hybrid approach coupling not specific force probing and fluorescence biochemical targeting correlative nanoscopy. It opens opens new windows for approaching original questions to study behaviour of biological and materials science samples.

Taking Two-Photon Excitation (2PE) further: 2PE coupling to Far-field Optical Nanoscopy and Super Resolution Microscopy towards Three-dimensional (3D) Imaging of Thick Scattering Specimens

Two-photon excitation (2PE) microscopy implementation in biological sciences and medicine [1] was immediately identified as a powerful fluorescence imaging method for studying living cell aggregates or tissues up to a significative depth, today close to one millimeter, not allowed by single photon approaches. Further benefits of two-photon excitation include: localized volume of excitation and emission, background rejection especially in 3D imaging, intrinsic optical sectioning, 3D localized photobleaching, photoswitching and uncaging, long term experiments [2]. These features have made possible experiments and innovations beyond the capability of traditional confocal microscopy. Unfortunately, one of the practical drawbacks in 2PE fluorescence microscopy is given by the worsening of spatial resolution due to red-shift of the excitation wavelength, only partially compensated by the improved signal-to-noise ratio. So far, image restoration [3] or aperture engineering [4] approaches have been used to alleviate such a condition.

Development of Optics Kit for Schools in Developing Countries — International School of Photonics Model

In India, the pedagogy of science education is “believe what text book says”. Providing schools with appropriate teaching materials to enhance teaching has always been a challenge in a developing country like India. Generally it is not possible for a normal school in India to afford the expensive teaching materials to teach through demonstrations and experiments. Thus students are forced to believe what text book says rather than learning concepts through experiments. The International School of Photonics SPIE (International Society for Optical Engineering) student chapter came up with ‘Optics kit’ to supplement the teaching of optics in school level. ‘Optics kit’, developed with indigenously procured components, could be sold at an affordable prize for an average Indian School. The chapter is currently selling the kit for less than