Dora Mahecic

Event-driven acquisition for content-enriched microscopy

Juliette Griffie, Dora Mahecic, Suliana Manley, Willi Leopold Stepp, Martin Weigert, Chen Zhang

Event-driven acquisition uses neural-network-based recognition of specific biological events to trigger switching between slow and fast super-resolution imaging, enriching the capture of interesting events with high spatiotemporal resolution. A common goal of fluorescence microscopy is to collect data on specific biological events. Yet, the event-specific content that can be collected from a sample is limited, especially for rare or stochastic processes. This is due in part to photobleaching and phototoxicity, which constrain imaging speed and duration. We developed an event-driven acquisition framework, in which neural-network-based recognition of specific biological events triggers real-time control in an instant structured illumination microscope. Our setup adapts acquisitions on-the-fly by switching between a slow imaging rate while detecting the onset of events, and a fast imaging rate during their progression. Thus, we capture mitochondrial and bacterial divisions at imaging rates that match their dynamic timescales, while extending overall imaging durations. Because event-driven acquisition allows the microscope to respond specifically to complex biological events, it acquires data enriched in relevant content.

NATURE PORTFOLIO2022

Characterization of flat-fielding systems for quantitative microscopy

Khalid Ashraf Mohie Ibrahim, Dora Mahecic, Suliana Manley

Optical flat-fielding systems, such as field-mapping or integration-based beam shapers, are used to transform nonuniform illumination into uniform illumination. Thus, flat-fielding paves the way for imaging that is independent of position within a field of view and enables more quantitative analysis. Here, we characterize and compare three systems for homogenizing both widefield and multitbcal illumination. Our analysis includes two refractive field-mapping beam shapers: PiShaper and TopShape, as well as one integration-based: Kohler integrator. The comparison is based on figures of merit including ISO-standard values, such as the plateau uniformity and edge steepness, transmission efficiency, stability of the beams along propagation and multifocal intensity, pitch, and point width. By characterizing and comparing existing beam shapers, we facilitate the choice of the appropriate flat-fielding solution and increase their accessibility for different applications. (C) 2020 Optical Society of America under the terms of the OSA Open Access Publishing Agreement

OPTICAL SOC AMER2020

High-throughput and adaptive super-resolution fluorescence microscopy of organelles

Dora Mahecic

Fluorescence super-resolution microscopy has allowed unprecedented insight into the workings of biological systems below the diffraction limit of light. Over the past decade, it has overcome several challenges to deliver 3D, multi-color and faster imaging techniques, which have found many applications in deciphering the workings and structure of organelles â specialized cellular compartments whose shape and dynamics serve their specific functions. However, most super-resolution techniques are laborious and slow, and there are existing challenges in achieving sufficient throughput for collection of large datasets and capturing dynamic processes in living cells. The aim of this thesis is to address issues of limited throughput, temporal resolution and live-cell compatibility, with application to centriole architecture and mitochondrial dynamics. The design of a novel flat-fielding module for multi-focal excitation which â once integrated into an instant structured illumination microscope â allows the capture of larger fields of view with uniform illumination and hence results in increased throughput. This flat-fielded microscope allows multi-color, 3-dimensional imaging at double the diffraction-limited resolution of up to 100 mammalian cells within 1-2 minutes. Furthermore, combined with advanced sample preparation using expansion microscopy, the setup achieves an effective resolution of ~35 nm, comparable to some of the more powerful super-resolution techniques, but at 100-1000 times increased throughput. To showcase this improvement, we study the distribution of post-translational modifications within the centriole, revealing previously unobserved molecular organization. Second, with fast, live-cell imaging, we investigate the role of membrane bending energy and tension in mitochondrial division. While many molecular factors involved in this process have been identified, little is known about its physical requirements. By analyzing mitochondrial constrictions enriched in Drp1 that divide successfully or reverse, we establish that mitochondrial constrictions with higher bending energy are more likely to divide. Furthermore, by analyzing changes in mitochondrial shape and employing a novel membrane tension sensor, we observe that conditions under which mitochondrial membrane tension is increased, result in more successful divisions. These observations are supported by a model in which membrane bending energy increases the elastic energy at the constriction site, bringing it closer to the energy barrier to fission, while membrane tension acts as a fluctuation to overcome the remaining residual energy. Finally, to overcome existing trade-offs between temporal resolution and imaging duration, we develop an acquisition framework for smart and adaptive temporal sampling. By adapting the temporal resolution is in response to events of interest, they are captured at high imaging speed, while preserving the sample during a lack thereof. Using the accumulation of Drp1 to detect mitochondrial constriction sites allowed us to adapt the imaging speed to capture mitochondrial constrictions at high temporal resolution while preserving the sample when such events are lacking. Overall, this framework provides both high temporal resolution and long imaging times when needed, decreasing resulting phototoxicity, photobleaching and amount of redundant data.

EPFL2020

Homogeneous multifocal excitation for high-throughput super-resolution imaging

Niccolo Banterle, Kyle Michael Douglass, Denis Fortun, Davide Gambarotto, Pierre Gönczy, Paul Guichard, Virginie Hamel, Khalid Ashraf Mohie Ibrahim, Dora Mahecic, Suliana Manley

Super-resolution microscopies have become an established tool in biological research. However, imaging throughput remains a main bottleneck in acquiring large datasets required for quantitative biology. Here we describe multifocal flat illumination for field-independent imaging (mfFIFI). By integrating mfFIFI into an instant structured illumination microscope (iSIM), we extend the field of view (FOV) to >100 x 100 mu m(2) while maintaining high-speed, multicolor, volumetric imaging at double the diffraction-limited resolution. We further extend the effective FOV by stitching adjacent images for fast live-cell super-resolution imaging of dozens of cells. Finally, we combine our flat-fielded iSIM with ultrastructure expansion microscopy to collect three-dimensional (3D) images of hundreds of centrioles in human cells, or thousands of purified Chlamydomonas reinhardtiic entrioles, per hour at an effective resolution of similar to 35 nm. Classification and particle averaging of these large datasets enables 3D mapping of posttranslational modifications of centriolar microtubules, revealing differences in their coverage and positioning.

NATURE PUBLISHING GROUP2020

Koehler integrator device and application thereof in a multi-focal confocal microscope

Kyle Michael Douglass, Dora Mahecic, Suliana Manley

A Koehler integrator device (10) comprises a collimating lens (11) being arranged for collimating a light field created by an incoherent or partially coherent light source, a pair of planar first and second micro-lens arrays (12, 13) being arranged for relaying portions of the collimated light field along separate imaging channels, wherein all micro-lenses of the first and second micro-lens arrays (12, 13) have an equal micro-lens focal length and pitch and the micro-lens arrays (12, 13) are arranged with a mutual distance equal to the micro-lens focal length, and a collecting Fourier lens (4) having a Fourier lens diameter and a Fourier lens focal length defining a Fourier lens front focal plane and a Fourier lens back focal plane, wherein the Fourier lens (14) is arranged for superimposing light from all imaging channels in the Fourier lens front focal plane and wherein the second micro-lens array (13) is arranged in the Fourier lens back focal plane, wherein a third micro-lens array (15) is arranged in the Fourier lens front focal plane for creating a wavelength independent array of illumination spots. Furthermore, a confocal microscope apparatus, which comprises the Koehler integrator device, and a method of using the confocal microscope apparatus are described..

2020

Mechanosensitive Fluorescent Probes to Image Membrane Tension in Mitochondria, Endoplasmic Reticulum, and Lysosomes

Dora Mahecic, Suliana Manley, Stefan Matile

Measuring forces inside cells is particularly challenging. With the development of quantitative microscopy, fluorophores which allow the measurement of forces became highly desirable. We have previously introduced a mechanosensitive flipper probe, which responds to the change of plasma membrane tension by changing its fluorescence lifetime and thus allows tension imaging by FLIM. Herein, we describe the design, synthesis, and evaluation of flipper probes that selectively label intracellular organelles, i.e., lysosomes, mitochondria, and the endoplasmic reticulum. The probes respond uniformly to osmotic shocks applied extracellularly, thus confirming sensitivity toward changes in membrane tension. At rest, different lifetimes found for different organelles relate to known differences in membrane organization rather than membrane tension and allow colabeling in the same cells. At the organelle scale, lifetime heterogeneity provides unprecedented insights on ER tubules and sheets, and nuclear membranes. Examples on endosomal trafficking or increase of tension at mitochondrial constriction sites outline the potential of intra-cellularly targeted fluorescent tension probes to address essential questions that were previously beyond reach.

AMER CHEMICAL SOC2019

Strategies for increasing the throughput of super-resolution microscopies

Juliette Griffie, Dora Mahecic, Suliana Manley

Fluorescence microscopy methods have been developed to circumvent the diffraction limit by exploiting nonlinearities in the interactions between light and fluorophores. Initially, these methods were up to orders of magnitude slower than standard microscopies. In recent years, a wide array of technological advances have increased the throughput of super-resolution microscopies, through parallelization, smart scanning or data processing, and sample expansion. Here, we review re cent innovations for increased throughput, some applications that have benefitted from them, and how they could be improved in the future.

ELSEVIER SCI LTD2019

High-throughput and adaptive super-resolution fluorescence microscopy of organelles

Dora Mahecic

EPFL2020