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Author: Heintzmann, Rainer × Has files: Yes × Type: Text × WGL Institute: IPHT ×

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    cellSTORM - Cost-effective Super-Resolution on a Cellphone using dSTORM
    (San Francisco : Public Library of Science, 2019) Diederich, Benedict; Then, Patrick; Jügler, Alexander; Förster, Ronny; Heintzmann, Rainer
    High optical resolution in microscopy usually goes along with costly hardware components, such as lenses, mechanical setups and cameras. Several studies proved that Single Molecular Localization Microscopy can be made affordable, relying on off-the-shelf optical components and industry grade CMOS cameras. Recent technological advantages have yielded consumer-grade camera devices with surprisingly good performance. The camera sensors of smartphones have benefited of this development. Combined with computing power smartphones provide a fantastic opportunity for “imaging on a budget”. Here we show that a consumer cellphone is capable of optical super-resolution imaging by (direct) Stochastic Optical Reconstruction Microscopy (dSTORM), achieving optical resolution better than 80 nm. In addition to the use of standard reconstruction algorithms, we used a trained image-to-image generative adversarial network (GAN) to reconstruct video sequences under conditions where traditional algorithms provide sub-optimal localization performance directly on the smartphone. We believe that “cellSTORM” paves the way to make super-resolution microscopy not only affordable but available due to the ubiquity of cellphone cameras.High optical resolution in microscopy usually goes along with costly hardware components, such as lenses, mechanical setups and cameras. Several studies proved that Single Molecular Localization Microscopy can be made affordable, relying on off-the-shelf optical components and industry grade CMOS cameras. Recent technological advantages have yielded consumer-grade camera devices with surprisingly good performance. The camera sensors of smartphones have benefited of this development. Combined with computing power smartphones provide a fantastic opportunity for “imaging on a budget”. Here we show that a consumer cellphone is capable of optical super-resolution imaging by (direct) Stochastic Optical Reconstruction Microscopy (dSTORM), achieving optical resolution better than 80 nm. In addition to the use of standard reconstruction algorithms, we used a trained image-to-image generative adversarial network (GAN) to reconstruct video sequences under conditions where traditional algorithms provide sub-optimal localization performance directly on the smartphone. We believe that “cellSTORM” paves the way to make super-resolution microscopy not only affordable but available due to the ubiquity of cellphone cameras.
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    Using machine-learning to optimize phase contrast in a low-cost cellphone microscope
    (San Francisco, CA : Public Library of Science, 2018) Diederich, Benedict; Wartmann, Rolf; Schadwinkel, Harald; Heintzmann, Rainer
    Cellphones equipped with high-quality cameras and powerful CPUs as well as GPUs are widespread. This opens new prospects to use such existing computational and imaging resources to perform medical diagnosis in developing countries at a very low cost. Many relevant samples, like biological cells or waterborn parasites, are almost fully transparent. As they do not exhibit absorption, but alter the light’s phase only, they are almost invisible in brightfield microscopy. Expensive equipment and procedures for microscopic contrasting or sample staining often are not available. Dedicated illumination approaches, tailored to the sample under investigation help to boost the contrast. This is achieved by a programmable illumination source, which also allows to measure the phase gradient using the differential phase contrast (DPC) [1, 2] or even the quantitative phase using the derived qDPC approach [3]. By applying machine-learning techniques, such as a convolutional neural network (CNN), it is possible to learn a relationship between samples to be examined and its optimal light source shapes, in order to increase e.g. phase contrast, from a given dataset to enable real-time applications. For the experimental setup, we developed a 3D-printed smartphone microscope for less than 100 $ using off-the-shelf components only such as a low-cost video projector. The fully automated system assures true Koehler illumination with an LCD as the condenser aperture and a reversed smartphone lens as the microscope objective. We show that the effect of a varied light source shape, using the pre-trained CNN, does not only improve the phase contrast, but also the impression of an improvement in optical resolution without adding any special optics, as demonstrated by measurements.
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    Patterned illumination single molecule localization microscopy (piSMLM): user defined blinking regions of interest cellSTORM - Cost-effective Super-Resolution on a Cellphone using dSTORM
    (Washington D.C. : Optical Society of America, 2018) Chen, S.-Y.; Bestvater, F.; Heintzmann, Rainer; Cremer, Christoph
    Single molecule localization microscopy (SMLM) has been established as an important super-resolution technique for studying subcellular structures with a resolution down to a lateral scale of 10 nm. Usually samples are illuminated with a Gaussian shaped beam and consequently insufficient irradiance on the periphery of the illuminated region leads to artifacts in the reconstructed image which degrades image quality. We present a newly developed patterned illumination SMLM (piSMLM) to overcome the problem of uneven illumination by computer-generated holography. By utilizing a phase-only spatial light modulator (SLM) in combination with a modified Gerchberg-Saxton algorithm, a user-defined pattern with homogeneous and nearly speckle-free illumination is obtained. Our experimental results show that irradiance 1 to 5 kW/cm2 was achieved by using a laser with an output power of 200 mW in a region of 2000 µm2 to 500 µm2, respectively. Higher irradiance of up to 20 kW/cm2 can be reached by simply reducing the size of the region of interest (ROI). To demonstrate the application of the piSMLM, nuclear structures were imaged based on fluctuation binding-activated localization microscopy (fBALM). The super-resolution fBALM images revealed nuclear structures at a nanometer scale.Single molecule localization microscopy (SMLM) has been established as an important super-resolution technique for studying subcellular structures with a resolution down to a lateral scale of 10 nm. Usually samples are illuminated with a Gaussian shaped beam and consequently insufficient irradiance on the periphery of the illuminated region leads to artifacts in the reconstructed image which degrades image quality. We present a newly developed patterned illumination SMLM (piSMLM) to overcome the problem of uneven illumination by computer-generated holography. By utilizing a phase-only spatial light modulator (SLM) in combination with a modified Gerchberg-Saxton algorithm, a user-defined pattern with homogeneous and nearly speckle-free illumination is obtained. Our experimental results show that irradiance 1 to 5 kW/cm2 was achieved by using a laser with an output power of 200 mW in a region of 2000 µm2 to 500 µm2, respectively. Higher irradiance of up to 20 kW/cm2 can be reached by simply reducing the size of the region of interest (ROI). To demonstrate the application of the piSMLM, nuclear structures were imaged based on fluctuation binding-activated localization microscopy (fBALM). The super-resolution fBALM images revealed nuclear structures at a nanometer scale.
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    IMAGE-IN: Interactive web-based multidimensional 3D visualizer for multi-modal microscopy images
    (San Francisco, California, US : PLOS, 2022) Gupta, Yubraj; Costa, Carlos; Pinho, Eduardo; A. Bastião Silva, Luís; Heintzmann, Rainer
    Advances in microscopy hardware and storage capabilities lead to increasingly larger multidimensional datasets. The multiple dimensions are commonly associated with space, time, and color channels. Since “seeing is believing”, it is important to have easy access to user-friendly visualization software. Here we present IMAGE-IN, an interactive web-based multidimensional (N-D) viewer designed specifically for confocal laser scanning microscopy (CLSM) and focused ion beam scanning electron microscopy (FIB-SEM) data, with the goal of assisting biologists in their visualization and analysis tasks and promoting digital work-flows. This new visualization platform includes intuitive multidimensional opacity fine-tuning, shading on/off, multiple blending modes for volume viewers, and the ability to handle multichannel volumetric data in volume and surface views. The software accepts a sequence of image files or stacked 3D images as input and offers a variety of viewing options ranging from 3D volume/surface rendering to multiplanar reconstruction approaches. We evaluate the performance by comparing the loading and rendering timings of a heterogeneous dataset of multichannel CLSM and FIB-SEM images on two devices with installed graphic cards, as well as comparing rendered image quality between ClearVolume (the ImageJ open-source desktop viewer), Napari (the Python desktop viewer), Imaris (the closed-source desktop viewer), and our proposed IMAGE-IN web viewer.
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    Video-rate multi-color structured illumination microscopy with simultaneous real-time reconstruction
    (Berlin : Nature Publishing, 2019) Markwirth, A; Lachetta, Mario; Mönkemöller, V.; Heintzmann, Rainer; Hübner, Wolfgang; Huser, Thomas; Müller, Marcel
    Super-resolved structured illumination microscopy (SR-SIM) is among the fastest fluorescence microscopy techniques capable of surpassing the optical diffraction limit. Current custom-build instruments are able to deliver two-fold resolution enhancement with high acquisition speed. SR-SIM is usually a two-step process, with raw-data acquisition and subsequent, time-consuming post-processing for image reconstruction. In contrast, wide-field and (multi-spot) confocal techniques produce high-resolution images instantly. Such immediacy is also possible with SR-SIM, by tight integration of a video-rate capable SIM with fast reconstruction software. Here we present instant SR-SIM by VIGOR (Video-rate Immediate GPU-accelerated Open-Source Reconstruction). We demonstrate multi-color SR-SIM at video frame-rates, with less than 250 ms delay between measurement and reconstructed image display. This is achieved by modifying and extending high-speed SR-SIM image acquisition with a new, GPU-enhanced, network-enabled image-reconstruction software. We demonstrate high-speed surveying of biological samples in multiple colors and live imaging of moving mitochondria as an example of intracellular dynamics.
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    Linear and non-linear optical imaging of cancer cells with silicon nanoparticles
    (Basel : Molecular Diversity Preservation International (MDPI), 2016) Tolstik, Elen; Osminkina, Liubov A.; Akimov, Denis; Gongalsky, Maksim B.; Kudryavtsev, Andrew A.; Timoshenko, Victor Yu.; Heintzmann, Rainer; Sivakov, Vladimir; Popp, Jürgen
    New approaches for visualisation of silicon nanoparticles (SiNPs) in cancer cells are realised by means of the linear and nonlinear optics in vitro. Aqueous colloidal solutions of SiNPs with sizes of about 10–40 nm obtained by ultrasound grinding of silicon nanowires were introduced into breast cancer cells (MCF-7 cell line). Further, the time-varying nanoparticles enclosed in cell structures were visualised by high-resolution structured illumination microscopy (HR-SIM) and micro-Raman spectroscopy. Additionally, the nonlinear optical methods of two-photon excited fluorescence (TPEF) and coherent anti-Stokes Raman scattering (CARS) with infrared laser excitation were applied to study the localisation of SiNPs in cells. Advantages of the nonlinear methods, such as rapid imaging, which prevents cells from overheating and larger penetration depth compared to the single-photon excited HR-SIM, are discussed. The obtained results reveal new perspectives of the multimodal visualisation and precise detection of the uptake of biodegradable non-toxic SiNPs by cancer cells and they are discussed in view of future applications for the optical diagnostics of cancer tumours.
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    Bessel beam CARS of axially structured samples
    ([London] : Macmillan Publishers Limited, part of Springer Nature, 2015) Heuke, Sandro; Zheng, Juanjuan; Akimov, Denis; Heintzmann, Rainer; Schmitt, Michael; Popp, Jürgen
    We report about a Bessel beam CARS approach for axial profiling of multi-layer structures. This study presents an experimental implementation for the generation of CARS by Bessel beam excitation using only passive optical elements. Furthermore, an analytical expression is provided describing the generated anti-Stokes field by a homogeneous sample. Based on the concept of coherent transfer functions, the underling resolving power of axially structured geometries is investigated. It is found that through the non-linearity of the CARS process in combination with the folded illumination geometry continuous phase-matching is achieved starting from homogeneous samples up to spatial sample frequencies at twice of the pumping electric field wave. The experimental and analytical findings are modeled by the implementation of the Debye Integral and scalar Green function approach. Finally, the goal of reconstructing an axially layered sample is demonstrated on the basis of the numerically simulated modulus and phase of the anti-Stokes far-field radiation pattern.
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    Engineering an achromatic Bessel beam using a phase-only spatial light modulator and an iterative Fourier transformation algorithm
    (Amsterdam [u.a.] : Elsevier, 2016) Walde, Marie; Jost, Aurélie; Wicker, Kai; Heintzmann, Rainer
    Bessel illumination is an established method in optical imaging and manipulation to achieve an extended depth of field without compromising the lateral resolution. When broadband or multicolour imaging is required, wavelength-dependent changes in the radial profile of the Bessel illumination can complicate further image processing and analysis. We present a solution for engineering a multicolour Bessel beam that is easy to implement and promises to be particularly useful for broadband imaging applications. A phase-only spatial light modulator (SLM) in the image plane and an iterative Fourier Transformation algorithm (IFTA) are used to create an annular light distribution in the back focal plane of a lens. The 2D Fourier transformation of such a light ring yields a Bessel beam with a constant radial profile for different wavelength.
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    PEGylation of Guanidinium and Indole Bearing Poly(methacrylamide)s - Biocompatible Terpolymers for pDNA Delivery
    (Weinheim : Wiley-VCH, 2021) Cokca, Ceren; Hack, Franz J.; Costabel, Daniel; Herwig, Kira; Hülsmann, Juliana; Then, Patrick; Heintzmann, Rainer; Fischer, Dagmar; Peneva, Kalina
    This study describes the first example for shielding of a high performing terpolymer that consists of N-(2-hydroxypropyl)methacrylamide (HPMA), N-(3-guanidinopropyl)methacrylamide (GPMA), and N-(2-indolethyl)methacrylamide monomers (IEMA) by block copolymerization of a polyethylene glycol derivative – poly(nona(ethylene glycol)methyl ether methacrylate) (P(MEO9MA)) via reversible addition–fragmentation chain transfer (RAFT) polymerization. The molecular weight of P(MEO9MA) is varied from 3 to 40 kg mol–1 while the comonomer content of HPMA, GPMA, and IEMA is kept comparable. The influence of P(MEO9MA) block with various molecular weights is investigated over cytotoxicity, plasmid DNA (pDNA) binding, and transfection efficiency of the resulting polyplexes. Overall, the increase in molecular weight of P(MEO9MA) block demonstrates excellent biocompatibility with higher cell viability in L-929 cells and an efficient binding to pDNA at N/P ratio of 2. The significant transfection efficiency in CHO-K1 cells at N/P ratio 20 is obtained for block copolymers with molecular weight of P(MEO9MA) up to 10 kg mol–1. Moreover, a fluorescently labeled analogue of P(MEO9MA), bearing perylene monoimide methacrylamide (PMIM), is introduced as a comonomer in RAFT polymerization. Polyplexes consisting of labeled block copolymer with 20 kg mol–1 of P(MEO9MA) and pDNA are incubated in Hela cells and investigated through structured illumination microscopy (SIM).
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    Successful optimization of reconstruction parameters in structured illumination microscopy
    (Amsterdam [u.a.] : Elsevier, 2019) Karras, Christian; Smedh, Maria; Förster, Ronny; Deschout, Hendrik; Fernandez-Rodriguez, Julia; Heintzmann, Rainer
    The impact of the different reconstruction parameters in super-resolution structured illumination microscopy (SIM) on image artifacts is carefully analyzed. These parameters comprise the Wiener filter parameter, an apodization function, zero-frequency suppression and modifications of the optical transfer function. A detailed investigation of the reconstructed image spectrum is concluded to be suitable for identifying artifacts. For this purpose, two samples, an artificial test slide and a more realistic biological system, were used to characterize the artifact classes and their correlation with the image spectra as well as the reconstruction parameters. In addition, a guideline for efficient parameter optimization is suggested and the implementation of the parameters in selected up-to-date processing packages (proprietary and open-source) is depicted. © 2018 The Authors