Conference Agenda

Surpassing the diffraction limit has become daily practice in the field of microscopy. This is made possible by methodological breakthroughs in which specific modifications to the optical hardware and labeling biochemistry are combined with computational advances in an integral approach. In the presentation, I will give an overview of current developments in our lab at Delft University of Technology. Topics that will be presented range within limitations imposed by noise, advances for large field or volume of view, and efforts toward cryogenic microscopy. Specifically I will highlight:

(1) Recent results on the classical Richardson-Lucy deconvolution method, in which we use a Cramer-Rao Lower Bound analysis to show that amplification of noise and ill-convergence are a necessary consequence of the method.

(2) The impact of field dependent aberrations, where we use so-called Nodal Aberration Theory to model and fit field-dependent aberrations from single-molecule data, and show how this improves the accuracy of localization.

(3) Cryogenic 4pi-microscopy, where we use the self-interference of fluorescent light collected trough two opposing objective lenses, in order to overcome the poor axial localization precision in low NA localization inherent to cryogenic setups.

Structured illumination microscopy (SIM) is a powerful super-resolution technique that relies on patterned light to surpass the diffraction limit. However, conventional SIM setups require complex optical configurations that limit their adaptability and usability. In this work, we present an integrated optical chip fabricated using femtosecond laser micromachining, designed to simplify the generation of structured light. This chip, coupled with an optical fiber, enables direct generation of structured illumination on its surface, where the biological samples can be positioned for imaging. Our approach eliminates bulky optics, enhances system stability, and offers a compact and scalable solution for super-resolution microscopy. We present the design, the fabrication process, and the first validations of this system for advanced biological imaging.

Live-cell imaging enables observation of dynamic cellular processes, yet many subcellular structures remain inaccessible due to the diffraction limit. Fluctuation-based super-resolution techniques overcome this barrier by exploiting intensity variations caused by fluorescence blinking. Among these methods, Super-resolution Optical Fluctuation Imaging (SOFI) enhances spatial resolution by leveraging the statistical properties of blinking fluorophores, achieving an n-fold resolution gain through nth-order calculations. However, SOFI requires the acquisition of hundreds of frames and substantial post-processing, making it unsuitable for real-time visualization of rapid cellular dynamics.

To address these limitations, we utilize a recurrent neural network (RNN) model, MISRGRU, inspired by SOFI. MISRGRU processes sequences of low-resolution frames to extract correlated temporal signals, effectively enhancing temporal resolution while achieving a twofold increase in spatial resolution. We also compare this approach with a widely adopted fully convolutional architecture, the U-Net, evaluating both in terms of resolution enhancement and computational latency. While both models achieve comparable resolution improvements using significantly fewer frames than SOFI, the U-Net’s requirement for frame interpolation prior to inference limits its suitability for real-time applications. In contrast, MISRGRU offers a 250-fold reduction in reconstruction latency, demonstrating strong potential for real-time super-resolution imaging in live-cell experiments.

Super-resolution microscopy based on the localization of single molecules facilitates the visualization of cellular structures at a resolution approaching the molecular level. However, their low-throughput nature hampers their applicability in biomolecular research and screening. Here, we propose an efficient workflow, starting with the scanning of large areas using fast fluctuation-based imaging, followed by single-molecule localization microscopy of selected cells. We exploit the versatility of DNA oligo hybridization kinetics with DNA-PAINT probes to tailor the fluorescent blinking toward high-throughput and high-resolution imaging. Additionally, we employ super-resolution optical fluctuation imaging (SOFI) to analyze statistical fluctuations in the DNA-PAINT binding kinetics, thereby tolerating much denser blinking and facilitating accelerated imaging speeds. We demonstrate 30–300-fold faster imaging of different cellular structures compared to conventional DNA-PAINT imaging, albeit at a lower resolution. Notably, by tuning the image medium and data processing, we can flexibly switch between high-throughput SOFI (scanning an FOV of 0.65 mm × 0.52 mm within 4 min of total acquisition time) and super-resolution DNA-PAINT microscopy and thereby demonstrate that combining DNA-PAINT and SOFI enables one to adapt image resolution and acquisition time based on the imaging needs. We envision this approach to be especially powerful when combined with multiplexing and 3D imaging.

We present a method to generate vector beams using a single spatial light modulator (SLM) for depletion-based super-resolution microscopy techniques such as STED, RESOLFT, and subtraction microscopy. We demonstrate the generation of circularly polarized Laguerre-Gaussian LG01 beams and vector beams with spatially varying polarization, such as azimuthal beams. Their intensity profiles, polarization distributions, and robustness to optical aberrations are experimentally analyzed under low- and high-numerical aperture conditions to assess their suitability for super-resolution applications.

The introduction of genetically expressed photosensitive proteins to optically control and monitor neuronal activity has opened new avenues for minimally invasive investigation of the brain, giving rise to the field of optogenetics.

Fully harnessing the potential of these tools has required the development of dedicated optical strategies. In particular, the use of two-photon infrared excitation combined with light-shaping techniques has enabled the precise manipulation of neuronal circuits in living tissue.

In this talk, I will introduce methods for tailoring infrared laser beams through wavefront modulation and temporal shaping of femtosecond pulses, allowing targeted excitation of single or multiple neurons within large volumes, deep inside scattering tissue. I will then highlight recent advances aimed at improving the speed and efficiency of neuronal activity manipulation, achieving kilohertz-rate interrogation of large neuronal populations. Finally, I will showcase applications of these approaches, focusing on in vivo mapping of neuronal connections, as a step toward fully optical interrogation of brain structure and function.

This work presents an integrated, high-throughput microscope on a

chip designed for rapid and automated circulating tumor cells imaging based on

cytomorphological features. The system employs a modified time-stretch imag-

ing technique, utilizing a single nanosecond laser pulse split into a sequence of

temporally and spatially separated pulses to illuminate the whole cell at different

moments. Fabricated using femtosecond laser micromachining, the device inte-

grates optical circuits, delay lines, and a microfluidic chip, enabling high-speed

image acquisition with a single-pixel detector. The system is validated using

calibration beads and tumor cells, demonstrating high resolution and stability.

Fully compatible with machine learning algorithms, this platform represents a

scalable, cost-effective solution for advancing real-time liquid biopsy and can-

cer diagnostics.

OptoRheo is a new microscopy platform that allows for live imaging of cells in 3D cultures over long-time courses, combined with micromechanical sensing of the material local to the cells. This is achieved by combining light sheet microscopy, multiplane imaging, optical trapping, and passive particle tracking micro-rheology in a single optical platform. A novel light sheet configuration allows cells to remain undisturbed during imaging, with no dipping objectives or sample scanning involved, allowing delicate samples to grow on the microscope stage over several days. This talk will demonstrate the capabilities of OptoRheo by studying two different cell culture systems, cell cultures grown in hydrogel and spheroid samples.

Wide-field imaging Mueller polarimetry has already demonstrated its potential for the accurate, non-contact, and cost-effective optical diagnosis of tissue in such diverse fields as gastroenterology, gynaecology, obstetrics, neurosurgery and digital histology. The recent developments and perspectives on translating this technique to clinics will be discussed, as well as the additional possibilities for health risks identification and management.

Fast and sensitive detector arrays make Image Scanning Microscopy (ISM) the natural successor of

confocal microscopy. Indeed, ISM enables super-resolution at an excellent signal-to-noise ratio. Optimizing

photon collection requires large detectors and so more out-of-focus light is collected. Nonetheless, the ISM

dataset inherently contains information on the axial position of the fluorescence emitters. We exploit such information

to directly invert the corresponding physical model with s2ISM, a maximum-likelihood algorithm

that reassigns the signal in the three dimensions, improving the signal-to-background ratio (SBR) and resolution.

Those kinds of algorithms show semi-convergent behaviour concerning the loss function of the problem

along the iteration routine. We regularize our s2ISM algorithm through inherent end deep-learning denoisers,

letting users analyze data with SNR levels that before were deemed to be unuseful in specimen structure or

dynamic revealings.

Mid-infrared sensor technology plays an increasingly important role in modern biodiagnostics, in particular in (pre)clinical screening and monitoring scenarios. Non-invasive exhaled breath analysis based on mid-infrared (MIR; 3-20 µm) photonics ranges among the most flexible sensing solutions for addressing molecular constituents and biomarkers within the exhaled breath matrix. In particular, with the emergence of quantum and interband cascade laser (QCL, ICL) technology along with interband cascade LEDs (IC-LEDs) along with

advanced waveguide concepts such as substrate-integrated hollow waveguides (iHWGs) integrated MIR sensing solutions for portable usage are on the horizon. The discussion of latest MIR photonic technologies in this presentation will be augmented by highlight biomedical applications including testing for long-COVID and bacterial infections in exhaled breath underlining the utility of next-generation mid-infrared biophotonics.

We present a four-dimensional (4D) Surface Plasmon Resonance (SPR) Imaging biosensor for real-time, array based high-through biomolecular bindings detection. The sensor measures the sensitive phase change induced spectral colour variation at plasmonic resonance with imaging device. It captures two-dimensional biomolecular bindings signal in the time-domain with spectral colour space information (4D imaging data), while no complex phase extraction is required. In the experiment, measurements for refractive index (RI) samples in 1.3330-1.3455 RIU were performed and the sensor RI resolution was found to be 7.2 x 10-6 RIU. The sensor was further demonstrated for antibodies molecular bindings detection in a 5 x 5 array format and 25 biomolecular binding interactions were monitored in real-time. In on-going works, we apply the imaging biosensor for Monkeypox virus neutralizing antibodies detection, which can contribute to Monkeypox virus vaccination development.

Genetically encoded voltage indicators (GEVIs) allow high-throughput and high-resolution measurements of electrical activity in mammalian cells through membrane voltage modulated fluorescence. Recently, bright, fast and photostable GEVIs have been developed, mostly though directed evolution. However, low voltage sensitivity still severely limits the signal to noise ratio of voltage measurements with GEVIs. This impedes massive parallel recording of membrane voltages and places high demands on imaging hardware. Through rational protein engineering, we have developed a novel rhodopsin-based voltage sensor with a sensitivity of over 200%, far surpassing current state of the art voltage sensors. By optimizing the kinetics of this new sensor, we hope to enable voltage recordings with unprecedented fidelity.

An optical fibre sensor with a long period grating (LPG) is described for the binding protein FKBP12 detection. An ad-hoc synthesized recognition element is immobilised on the fibre surface in correspondence of the LPG by using a home-made microfluidic flow-cell. Measurement is performed by flowing solutions in the flow-cell with increasing FKBP12 concentration and measuring the shift of the LPG resonant peak.

A nanoplasmonic biosensor is presented based on periodic arrays of gold nanoprisms, engineered for high-sensitivity and label-free detection of biomolecular interactions. Integrated within a microfluidic platform, the biosensor is designed to operate under well-controlled culture conditions, enabling real-time analysis of cytokine secretion from live cells. Our system offers superior sensitivity compared to conventional techniques while simplifying the detection workflow by eliminating the need for fluorescent markers. The optical configuration is fully compatible with standard inverted microscopes, making it ideal for widespread adoption in biological laboratories. Furthermore, the compact and modular design facilitates integration into organ-on-chip systems for monitoring the dynamic behaviour of healthy and diseased cells in co-culture environments, paving the way for advanced biomedical research and personalized diagnostics

We developed an array of quantum detectors based on superconducting nanowires to allow two-photon-excited fluorescence in-vivo imaging of mouse brain vasculature working completely in the short wave infrared (SWIR) region of the spectrum, achieving an imaging depth of up to 900 um.

Optical trapping with structured optical fibers is reported for five species of the Pseudomonas genus. Contactless trapping at low intensities was realized with 3D printed Fresnel lens fibers and an original fiber emitting a tightly focused annular beam. Specific swimming features and the behavior of trapped bacteria of the investigated species are compared applying different numerical methods.

Light-based technologies, including lasers and LEDs, are widely utilized in various medical applications. Among these, low-fluence and prolonged irradiation in the visible (VIS) and near-infrared (NIR) spectral regions have demonstrated significant clinical benefits, such as reducing inflammation and pain, as well as stimulating regenerative processes. To investigate the underlying mechanisms of these therapeutic effects, we examined the responses behaviour of fibroblasts and keratinocytes following non-contact irradiation with a 420 nm LED at varying fluences (4–40 J/cm²). Post-treatment analysis was conducted using confocal and electron microscopy, Micro-Raman spectroscopy, cell metabolism and proliferation assays, as well as patch-clamp recordings. Our findings revealed fluence- and cell type-dependent modulation of metabolism, cytochrome C redox state, and membrane ionic currents. In conclusion, these findings pave the way for the design of photonics-based medical devices capable of achieving targeted effects on specific cell groups, thereby addressing particular medical challenges [1-4].

Addressing the limitations of animal models in biomedical research, we introduce a hierarchical manufacturing method for creating anatomical phantoms using water-in-elastomer micro-emulsions.

The building blocks are water-in-elastomer micro-emulsions made of a continuous phase of hydrophobic polydimethylsiloxane (PDMS) and micro-droplets of hydrophilic solutions of various dyes and other contrast agents. This material inherits some properties from the elastomeric matrix, such as the speed of sound and acoustic attenuation coefficient, some from the hydrophilic inclusions, such as the optical absorbance, and some from their overall ultrastructure, such as the intensity of optical scattering. The final material

provides independently tunable, tissue-mimicking characteristics in the relevant ranges of optical excitation and acoustic detection for PAI.

This approach addresses the cost, ethical, and technical limitations of animal models, providing a reliable alternative for multimodal imaging and artificial intelligence development, particularly in photoacoustic imaging.

Reducing invasiveness is key in developing modern endoscopes for imaging samples in hard-to-reach areas. This can be achieved using multimode fibers. Compressive sensing algorithms can be applied to multimode fiber imaging to reduce the imaging speed. These algorithms utilize a sub-Nyquist set of patterns while allowing to super-resolve the sample. The patterns must be uncorrelated, which is typically achieved by mechanically scanning the input beam across a multimode fiber. We demonstrated a setup combining a super-continuum laser and a monochromator to obtain wavelength-dependent uncorrelated speckles, removing the need for mechanical scanning. With compressive sensing algorithms, binary samples were reconstructed up to 95% faster compared with traditional methods. We expand our setup to incorporate broadband illumination, which is expected to further increase imaging speed and spatial resolution, paving a way for a compact, real-time imaging device.

Interferometric diffuse optics (iDO) enables non-invasive measurement of deep tissue blood flow without requiring photon-counting detectors. Due to hardware constraints, achieving both optical properties and depth-dependent dynamics within a single modality remains a challenge for iDO. We present a simple method based on frequency-modulated light scattering that overcomes this limitation.

Joining the rich photo-physics of organic materials with the exquisite sensitivity of optical resonances to geometry and refractive index enables a plethora of devices with unusual and exciting properties. Examples from my team include flat microcavity sensors for interference-based detection of the mechanical forces exerted by cells, microlasers for real time sensing of cellular activity and long-term cell tracking, as well as the development of implants with extreme form factors that support optical stimulation of thousands of neurons deep in the brain with unprecedented spatial control. Very recently, by driving the interaction between excited states in organic materials and resonances in thin optical cavities into the strong coupling regime, we unlocked new tuning parameters which may enable a new generation of thin film optical filters with angle-independent characteristics as is required for more compact fluorescence-based sensing devices.

This study details the integration of optical fiber-based and electrochemical biosensors within a specially designed microfluidic system. It will present the results of a bioassay using an aptamer specific to tumor necrosis factor alpha (TNF-) on the hybrid opto-electrochemical system.

We demonstrate single-molecule detection of DNA using a low-cost,

miniaturized microscopy platform. Despite significant reductions in size and

cost, our system achieves performance comparable to a research-grade TIRF

microscope, with a limit of detection of 10 pM. Finally, we propose a path

toward wearable single-molecule sensors through integration with photonic cir-

cuits

Optical emitters in two-dimensional (2D) materials are emerging as ultrabright and robust optical probes for fluorescence based sensing of single molecules in physiological conditions. Controlling their spatial and spectral properties, however, remains challenging. Here, we demonstrate that thermally induced wrinkles in exfoliated hexagonal boron nitride (hBN) flakes act as nanoscale channels capable of localizing both optical emitters and biomolecules. Wrinkle formation is governed by the thermal expansion mismatch between hBN and the substrate, generating strain gradients that activate visible-range emitters. We perform structural and optical characterization of the wrinkles using atomic force microscopy (AFM), scanning electron microscopy (SEM), photoluminescence (PL) spectroscopy and single-molecule fluorescence imaging. Our results show that hBN wrinkles form optically active nanochannels that can be filled with liquid, and provide nanofluidic confinements suited for single-molecule transport and detection. These represent a promising platform for on-chip optofluidic sensing with single-molecule resolution.

The advancement of Genetically Encoded Voltage Indicators (GEVIs) has enabled real-time monitoring of voltage dynamics at subcellular resolution. Among various GEVI platforms, microbial rhodopsin-based GEVIs are particularly promising due to their superior sensitivity and spatiotemporal resolution. These indicators have the potential to enhance our understanding of complex cellular events such as synaptic transmission and neuronal plasticity. We present a set of nanofabricated tools to aid achievement of this goal. We employ Laser-Assisted 3D-Printed Nanostructured Arrays to guide neuronal development, optimizing network architecture. In parallel, we explore plasmonic enhancement strategies to boost the kinetics and fluorescence brightness of GEVIs. These top dop-down approaches dovetail with bottom-up genetic engineering of GEVI structure to optimize the experimental design for the investigation of subcellular voltage dynamics at various scales.