Scanning the Body: Biomedical Imaging
Correlative fluorescence and soft X-ray microscopy in an integrated laboratory-based setup (FSU Jena and HIJ, Germany)
Correlative imaging is a very useful method for combining complementary imaging techniques with different con- trast mechanisms. A good example is the correlation of the functional contrast of fluorescence microscopy (FLM) with the structural contrast of soft X-ray (SXR) microscopy in the water window (WW). The WW is a spectral region defined by the absorption edges of carbon (282 eV) and oxygen (533 eV), which offers label-free structural contrast in biological samples and a relatively high penetration depth in water.
This correlation has already been demonstrated at synchrotron sources, but a laboratory-based solution, as pre- sented here, was needed to make the method more widely accessible. The setup used (Figure 1) combined a wide- field SXR microscope with a bright-field epi-fluorescence microscope. The required SXR radiation was generated using a laser-produced gas plasma source, based on a gas puff target (GPT) developed by the Institute of Optoelectronics at the Military University of Technology, Warsaw, with nitrogen as the working gas. The condenser optic was an ellipsoidal mirror, with a Fresnel zone plate (FZP) used for imaging. Due to the strong absorption of the SXR radia- tion in air, the microscope was operated in a vacuum.
Figure 1: Sketch of the setup. A) The top figure shows the operating SXR microscope with the zone plate imaging the sample structure on the detector, while the fluorescence microscope remains inactive in this mode. B) For imaging with the fluorescence microscope, the zone plate, objective and the first mirror behind
it are moved sideways, while the sample remains in place. The laser and plasma source are not operating in this mode.
The major advantage of this setup was the integrated FLM, which allowed switching between the two imaging methods without having to move the sample, ensuring identical measurement conditions and avoiding sample alteration or destruction. To facilitate this, the FLM objective was integrated into the vacuum chamber of the SXR microscope, allowing easy switching with the FZP. All other components of the FLM, except for the sample, were located outside the vacuum chamber. This approach enabled easy changing of filter sets and illumination LEDs, enabling multi-colour fluorescence imaging.
With this setup, a resolution of 50 nm half pitch was achieved, measured with a Siemens star test target, as shown in Figure 2. 3T3 cells were successfully imaged with labelled cytoskeleton and nuclei, as well as fluorescent nanobeads, autofluorescent cyanobacteria, and labelled COS-7 cells.
Figure 2: Different samples measured with the correlative microscope. A) SXR image of a Siemens star test target, showing 50 nm half pitch resolution. B) SXR image of NIH-3T3 cells. Four nuclei and the cytoskeleton are visible. C) Composite image of SXR image B and fluorescence images, with labelled actin skeleton and nuclei. SXR contrast was inverted for better visibility of the fluorescence image. D) Correlative image of autofluorescent cyanobacteria. E) SXR image of COS-7
cells. Two nuclei, the cytoskeleton and surrounding mitochondria are presented. F) Composite image of SXR image E and fluorescence images, with labelled mitochondria (upper left) and cytoskeleton (lower right). SXR contrast was inverted for better visibility of the fluorescence image.
In addition to the imaging of biological samples, this setup also allowed the degradation of the fluorescence signal by SXR radiation to be studied, which is of particular interest for the further development of correlative experi- ments.
Sophia Kaleta and Julius Reinhard (IOQ, University Jena)
J. Reinhard et al., Microsc. Microanal.: ozad123 (2023)
Non-linear and photoacoustic microscopy reveal fundamental biological mechanisms in model organisms (FORTH, Greece)
The development of non-invasive microscopic techniques as new tools in biomedical research is extremely important. At IESL-FORTH, non-linear optical microscopy (NLOM) has been used for sub-cellular imaging of biological samples, and to provide new insights into fundamental biological phenomena, such as cell differentiation, embryogenesis, and fat metabolism during ageing [1] or in the context of disease [2]. Alongside label-free high resolution, high-contrast imaging, NLOM offers increased biological sample penetration depth, and permits precise quantitative analysis and testing of specific mechanisms and biological processes. IESL-FORTH has also developed novel, low-cost photoacoustic (PA) microscopy platforms [3], integrating intensity-modulated continuous wave laser sources, to provide accurate optical absorption mapping of cells and tissues with diffraction-limited spatial resolutions and excellent molecular specificity levels. As the laser-induced ultrasonic waves present significantly higher transmissivity than pure optical signals, PA imaging approaches may offer enhanced penetration depths for in vivo diagnostic applications without the use of labelling agents. The costs of such imaging systems are also around one third of those of conventional PA microscopes utilising Q-switched nanosecond lasers.
Left side: NLOM imaging of Caenorhabditis elegans nematode. Right side: Label-free PA microscopy for monitoring the development of Parhyale hawaiensis emerging model organism.
In recent interdisciplinary research, NLOM (MPEF, SHG and THG modalities) was implemented to visualise the deposition and distribution of lipid droplets (LDs) in cells of the simple nematode Caenorhabditis elegans. Lipid content was discovered to accumulate progressively with age in the nuclear envelope of cells in several tissues, and importantly, genetic interventions known to delay ageing reduced the number and size of nuclear LDs. NLOM measure- ments uncovered a molecular mechanism that preserves nuclear lipid homeostasis and organismal physiology dur- ing ageing [4], and highlighted its critical role in prevent- ing age-associated nuclear LD build-up. In a further collaboration, a PA microscopy system was used for label-free live imaging of developing Parhyale hawaiensis embryos, exploiting the intrinsic optical absorption properties of the yolk’s pigments (e.g. carotenoids) [5, 6]. During the first embryogenesis stages, the yolk distribution in the cells and the membranes of the blastomeres were clearly visible. In later embryogenesis stages (soccer- ball stage – late segmentation), it was possible to observe the yolk sac location, monitor midgut development, and acquire structural information of the surrounding visceral and somatic mesoderm. At a late organogenesis stage, the spatial distribution of the signals delineated the longitudi- nal and circular muscles surrounding the gut and associated midgut glands. The findings of the study [7] pave the way for the broader adoption of inexpensive PA approaches for detailed investigations of developmental mechanisms in traditional model organisms, such as Drosophila melanogaster and zebrafish, and other emerging models without established labelling and imaging resources.
George Filippidis, George J. Tserevelakis, Meropi Mari and Giannis Zacharakis (FORTH)
[1] K. Palikaras et al., J. of Lipid Research 58: 72-80 (2017)
[2] V. Tsafas et al., J of Biophotonics 13: e202000180 (2020)
[3] G.J. Tserevelakis et al., Optics Letters 46: 4718-4721 (2021)
[4] K. Palikaras et al., Aging Cell 22: e13788 (2023)
[5] G.J. Tserevelakis et al., J. Biophotonics 15: e202200202 (2022)
[6] G.J. Tserevelakis et al., Photonics 10: 264 (2023)
[7] A collaboration among the IESL-FORTH, the Medical School of the National & Kapodistrian University of Athens (K. Palikaras) and the Institute of Molecular Biology and Biotechnology (IMBB)-FORTH (T. Pavlopoulos and N. Tavernarakis)
Carotenoid compounds identified in brain tissue from Alzheimer Disease patients (LLAMS, the Netherlands)
At LaserLaB Amsterdam, Raman imaging methodologies have been applied to post-mortem brain tissue samples from patients suffering from Alzheimer’s Disease (AD). To characterise the chemical composition, these samples were only snap-frozen, with no additional fixation or staining. The standard workflow [1] began with autofluorescence microscopy; the green emission of certain areas was found to be associated with so-called amyloid plaques, a hallmark of AD. Raman mapping was then carried out on those regions of interest. In addition, stimulated Raman scattering (SRS) microscopy of the same area was carried out to detect misfolded peptides (not shown). Subsequently plaque-specific staining was performed to verify the plaque location.
Fluorescence and Raman imaging of plaque area in post-mortem brain tissue of an Alzheimer’s Disease patient. The green autofluorescence (blue excitation; top left image) indicates the plaque area. Raman mapping of the same area (exc = 532 nm) followed by unsupervised spectral unmixing leads to three components
(endmembers): plaque (blue), lipofuscin deposits (yellow) and regular brain tissue (orange), of which the distribution is shown in the top right image. The average Raman spectra of the three tissue types are shown in the bottom image; the plaque spectrum (blue) also shows strong carotenoid signatures. (C. Keskin MSc
thesis; unpublished results)
The top left image shows the plaque area, indicated by the green emission. The unsupervised unmixing [2] of the Raman map resulted in three endmembers, corresponding with three spectrally different compound mixtures, namely plaque, regular brain tissue, and lipofuscin. Their distribution is shown in the top right image. Interestingly, all three regions show typical Raman bands of tissue components, but the blue spectrum (plaque) also shows strong Raman bands at 1150 cm-1 and 1514 cm-1 , indicative of carotenoid compounds [3]. Their Raman signatures are enhanced due to pre-resonance excitation. The presence of carotenoids (originating from food items, such as carrots, tomatoes) had not previously been observed in AD plaques and their role is as yet unclear. Being strong anti-oxidants, they may have been invoked to fight local inflammation.
Can Keskin, Robert W. Schmidt and Freek Ariese (LaserLaB Amsterdam, the Netherlands)
[1] B. Lochocki et al., Nature Commun. Biol. 4: 474 (2021)
[2] R.W. Schmidt et al., J. Optics 24: 064011 (2022)
[3] L. Ettema et al., J. Optics 24: 054005 (2022)
Translational biomedical imaging of whole organs using lightsheet microscopy (Central Laser Facility, United Kingdom)
Lightsheet microscopy is growing in popularity and proving to be a fast, efficient, and informative method of obtaining three-dimensional data of whole mouse organs, and for biopsies of human organs. Key biomedical issues, such as the differences between healthy and diseased tis- sue, are traditionally addressed through histology, which is the process of cutting thin slices of tissue and imaging each slice as a two-dimensional image. Sometimes only a few slices are chosen and used to represent the entire sample (using a process known as stereology to derive three-dimensional data from two-dimensional samples). However, after the tissue has been made transparent in a process called ‘clearing,’ lightsheet microscopy is capable of optically sectioning tissue without destroying it, using a sheet of light orthogonal to the plane of the camera. The light illuminates only a very thin plane of the transparent sample and can be scanned to collect multiple planes, building a three-dimensional representation of the sample for comprehensive analysis.
The Central Laser Facility has been collaborating with scientists from University College London, UK, to apply this technique, using a 3i Cleared Tissue Lightsheet microscope to acquire images. The ultimate aim of the work is to create a digital database (or atlas) of organs, or biopsies of organs, from different genetic backgrounds and disease states, and to preserve this data online in a central location. In early work, images have been acquired of whole mouse hearts, lungs, kidney, eyeballs, and sections of gut at a resolution of <1 μm. Knowledge gained from these early studies will be used to develop on-the-fly analysis of images as they are acquired by the microscope; for example, extracting cell positions that will reduce the burden of data storage and handling. Images and data collected are contributing to several projects, including: determining the causes of kidney rejection in human kidney transplant; understand- ing how the immune system develops in the eye and brain during disease; and understanding the structure of the lymphatic vasculature in organs.
Robert Lees (Central Laser Facility)
Renderings of three-dimensional data from a mouse eyeball (left two panels) and a mouse cystic kidney (right two panels), with an inset (blue outline) of a single optical section for each dataset. The insets show single-cell resolution features across the layer of the eye (left), and the abundance of large cysts in the kidney (right)
Illuminating the brain: Unveiling the YWHAZ gene with lightsheet fluorescence microscopy (ICFO, Spain)
An international team of scientists employed cutting-edge techniques, specifically lightsheet microscopy in whole-brain imaging, to investigate the role of the YWHAZ gene in brain development and function. This novel approach delivered unprecedented insights into neural activity and connectivity in zebrafish, a powerful model organism for studying brain disorders. Whole-brain imaging using lightsheet microscopy enables non-invasive investigation of neuronal activity and connectivity in living organisms. Using an electrically tunable lens, modulated in synchrony with the axial displacement of the illuminating light sheet, enabled high-resolution, three-dimensional images of the entire zebrafish brain to be captured. By combining the power of whole-brain imaging with genetic engineering techniques, researchers aimed to unravel the intricate neural mechanisms underlying human brain disorders associated with YWHAZ dysfunction. The ability to observe neuronal activity and connectivity in live zebrafish embryos and adults enabled the impact of YW- HAZ on neurotransmission, and behaviour across different developmental stages, to be assessed.
Image of zebra fish (ZF) whole brain showing neuronal activity. In the experiment we used LSFM to record the calcium activity of ~30 wild type ZF whose activity was compared with ~30 mutant ZF with a deficiency in the YWHAZ gene. A deficiency in the YWHAZ gene is involved in neuro-developmental disorders.
This innovative approach sought to bridge the gap between molecular studies of YWHAZ and the complex behavioural phenotypes observed in neurodevelopmental and psychiatric disorders. By visualising and analysing neural dynamics in a living organism, the studies aimed to un- cover the functional consequences of YWHAZ alterations, providing valuable insights into the pathogenesis and potential therapeutic targets for these disorders. Overall, the use of advanced techniques, such as whole-brain imaging through lightsheet microscopy, will provide a crucial step in investigations into the role of the YWHAZ gene in brain development and function. This approach has allowed the intricate neural processes involved in neu- rodevelopmental disorders to be explored, and enabled researchers to gain a deeper understanding of the underlying mechanisms at a comprehensive, whole-brain level.
Gustavo Castro and Pablo Loza-Alvarez (ICFO)
E. Antón-Galindo et al., Mol. Psychiatry 27: 3739–3748 (2022)
Drug biodistribution and pharmaco-kinetics by photoacoustic tomography (CLL, Portugal)
The most basic principle in pharmacology is that a drug must reach its target to elicit a therapeutic effect. Following the biodistribution of drugs in an organism is very important to understanding the pharmacological action, but is very challenging to achieve using non-invasive methods. Researchers from the University of Coimbra made use of photoacoustic tomography, a non-invasive method, to follow the biodistribution of the drug redaporfin in clinical trials for advanced head and neck cancer. The organisms investigated were BALB/c mice with subcutaneous colon carcinoma (CT26) or with orthotopic breast (4T1) tumours.
The locations of the tumours in the mice were identified by ultrasound, and are shown in the photographs by continuous lines encircling the tumour regions where the tumours diameters attained their maximum values. Re- daporfin was injected intravenously before the acquisition of the photoacoustic tomography signals. The colours refer to the intensities of the signals, where red is oxyhaemo-globin, blue is deoxyhaemoglobin and green is redaporfin. Redaporfin is particularly suited to photoacoustic tomography, because its absorption maximum occurs at 750 nm and its phototoacoustic spectrum is distinct from those of both forms of haemoglobin.
Photoacoustic tomography signals showing redaporfin levels in CT26 tumours (left) and 4T1 tumours (right)
Imaging revealed that CT26 tumours accumulate a higher amount of redaporfin and have more oxyhaemo-globin, which explains why these tumours respond better to treatment than 4T1 tumours. The poor oxygenation and limited redaporfin infiltration in orthotopic 4T1 tumours may be attributed to high solid stress and elevated interstitial fluid pressure.
Luis Arnaut, Fabio Schaberle, Maria Inês Mendes and Catarina Lobo (Coimbra Laserlab (CLL); University of Coimbra)
C.S. Lobo et al., Sci. Rep. 13: 11667 (2023)
Advancements in HILO microscopy for optimal single-molecule microscopy performances (LENS, Italy)
Highly inclined and laminated optical sheet (HILO) microscopy, originally introduced by Tokunaga et al. in 2008, has emerged as a revolutionary strategy for imaging samples with increased contrast.
HILO microscopy employs a single-objective inclined lightsheet illumination method that exploits refraction at the glass/water interface to produce a thin, focused sheet of light, targeting specific regions of the sample volume. This ingenious approach significantly minimises unwanted background fluorescence from out-of-focus planes, a prevalent issue in conventional microscopy.
Despite its widespread adoption and popularity, HILO’s underlying beam characteristics and mechanisms have remained poorly understood, leaving researchers without established procedures for optimal control and customisation. A recent paper, published in Optics Express [1], presented a theoretical model that precisely describes the propagation of the inclined beam. Through meticulous far-field and near-field experiments, the model has been successfully validated, providing researchers with the essential tools to predict the beam’s geometrical features at the sample level.
The paper also introduced an efficient alignment and beam shaping procedure, which will allow users to tailor the beam to their specific experimental needs with ease. By progressively reducing the inclined beam thickness, the team demonstrated a remarkable impact on image quality, both in conventional fluorescence microscopy and, for the first time, in localisation-based super-resolution microscopy. Through optimisation of the optical adaptation of the HILO configuration, it was possible to shrink the inclined beam thickness to less than three micrometres while maintaining a suitable field-of-view for cell imaging. This innovative approach resulted in a remarkable doubling of the number of single molecule localisations, effectively extending the resolution capabilities and significantly reducing the acquisition times in super-resolution PALM/STORM imaging.
The research team believes that their work addresses a widespread need for a better understanding of this powerful microscopy technique. It provides all the tools needed to achieve full control and shape the beam according to the user’s experimental need, with a simple solution that can be implemented with minimal effort on any inverted fluorescence microscope.
The implications and the possible applications of this research are broad, covering various scientific fields, including biology, medicine, and materials science.
Lucia Gardini (LENS)
[1] L. Gardini et al., Opt. Express 31, 26208-26225 (2023)
Upper panels: simulations of inclined Gaussian beam propagating at an angle of 77 degrees with decreasing thicknesses (from left to right). Lower panels: super-resolved images of actin cytoskeleton reconstructed from 10 thousand frames with decreasing inclined beam thickness (from left to right).