Winner: Nobody Likes Me (Think I’ll Go 'Clear' Worms)
Dr Liam Rooney, University of Strathclyde
‘Big, fat, juicy ones’, as the song goes, in this case. The wax moth larva, Galleria mellonella, is a common model for studying host-pathogen interactions and infection. Researchers observe the gradual melanisation as the larva become sick and die, quantifying the colour change as a function of pathogenicity. However, to peer inside and localise the pattern of infection has remained elusive due to the larvae’s dense and highly scattering fat deposits and internal structure. We have developed an optimised optical clearing method which strips out the fat and extracellular milieu that has hindered microscopists from visualising infection dynamics. This image shows an abdominal section of the larva, measuring over 1mm thick, optically cleared, stained with propidium iodide, and imaged for 25 hours using the Mesolens configured in confocal laser scanning mode. The colour-coding of this z-projection illustrates the internal structure of the cleared larva; the three loops of the silk gland are clearly visible, forming a sad face following the ordeal of fixation, bleaching, RNA depletion, dehydration and clearing. Following these successful trials, we aim to visualise infection in cleared larvae, providing a new investigative method that sheds light on the intricacies of host-pathogen interactions across spatial scales.
Competition Entries
1. Villi Vasculature
Jade Phillips, CRUK Scotland Institute/University of Glasgow
Whole mount immunostaining of mouse small intestine at a x63 zoom on a villus. Immunostaining for Pecam-1 (cyan) to detect the vasculature and Piezo-1 (red) which is detected in neurons and smooth muscle cells. Image was taken on a Zeiss 880 confocal.
2. Viral superinfection exclusion in the airway
Cal Bentley-Abbot, University of Glasgow - MRC Centre for Virus Research
Adjacent lesions formed by two competing fluorescently tagged influenza A viruses (green and magenta) in the airway of a mouse 3 days postinfection, coinfected cells are labelled in white. Following initial infection, superinfection exclusion prevents infection of a cell by a secondary virus after around 4 hours. This process restricts coinfection to cells at the boundaries between competing infectious lesions where cells encounter both viruses within the time window prior to superinfection exclusion.
Here, 300um thick sections were taken from lungs of mice co-infected with two influenza A viruses endogenously tagged with eGFP and mCherry respectively. Samples were optically cleared using Ce3D and images captured via Zeiss LSM 710, 20x objective. An Imaris surface was rendered showing co-infected cells based on the co-expression of both fluorophores. This surface represented only 2% of infected cells, which are predominantly located at the boundary between competing lesions, aligning with previous in vitro results.
3. Neuromuscular Junction with New Super-resolution Imaging Technology
Abdullah Ramadan, University of Edinburgh
This multi-stained (pseudocolours) structure is a mouse neuromuscular junction (NMJ) which is a specialized synapse that relays signals from lower motor neurons to skeletal muscle fibres. Neurofilaments (magenta), Acetylcholine receptors (green) and Sodium channels (red). This image is a Zstack – Deconvolved (0.5 µm intervals) and has been acquired with the newly developed Nikon Spatial Array Detector (NSPARC) detector confocal microscopy, which utilizes an ultra-low noise detector array to collect a two-dimensional image at each scanned point. The lens objective that has been used is the 60×/1.4 oil immersion objective.
4. Scale Models
Dr Louise Stephen, Roslin Institute, University of Edinburgh
Zebrafish scales stained with Alizarin Red and Alcian Blue to mark mineralised ‘bone’ (red) and cartilage (blue). 5x magnification.
5. The heterogeneity of the skin
Dr Patricia Centeno, CRUK Scotland Institute
Here we can see the heterogeneity of the skin. First the suprabasal layer (magenta) formed by dead cells. Below, the heterogeneous basal layer, made by progenitors or adult stem cells (cyan) and by committed progenitors or cells that are about to differentiate and migrate to the upper layers and whose differentiation program has already started (magenta) but not completed as they retain their nuclei (dark blue). On the right bottom corner there is a portion of a hair follicle bulb. Below the basal layer, there is the adipose layer formed by fat.
Mouse skin. 21um. RFP is expressed under the Ivl promoter (committed progenitors). Krt14 marks the basal layer. DAPI marks the nuclei. Primary antibodies: KRT14 (ab7800, Abcam) and RFP (600-401-379, Rockland). Secondary antibodies: anti-rabbit IgG 488 (A11034, Invitrogen) and goat anti-mouse IgG 647 (A21236, Invitrogen diluted), and DAPI (MBD0015, Sigma-Aldrich).
Confocal images were collected on a Zeiss 710 point-scanning confocal microscope, built on an inverted Zeiss Axio Imager.Z2 stand. Images were acquired using a EC Plan-Neofluar 40x/1.30 Oil and a confocal pinhole diameter of 107 µm. Multi-channel images were captured sequentially: DAPI (nuclear marker) using 405 nm excitation and 410-481 nm emission bandwidth, IgG 488 using 488 nm excitation and 514-582 nm emission and IgG 647 using 633 nm excitation and 638-747 nm emission. Images were collected with a 1x zoom with an image size of 2320 x 2320 pixels, yielding a pixel size of 92 x 92 nm, and a 1.38 us pixel dwell time. Z-stacks were collected using a step size of 2 um. Images were acquired using the software Zen LSM 2.1 Black (Zeiss).
6. Vascular Rainbow
Jade Phillips, CRUK Scotland Institute
Confocal image of the mouse intestinal vasculature from whole mount tissue preparations. Image was aquired on a Zeiss 880 confocal microscope at x20 at the BAIR facility in the CRUK Scotland Institute and a colour coded projection produced in ZEN.
7. Liver Mets High-Plex
Dr Fiona Ballantyne, CRUK Scotland Institute
Investigation of CAF subtypes in PDAC using the Phenocycler-Fusion (Akoya Biosciences) to create a 42-plex antibody panel using custom conjugated and preconjugated antibodies that will categorise and quantify fibroblasts, hallmarks of cancer, and immune cells, combined with PDAC specific markers. Markers visualised here in Liver Mets are: DECORIN, LIF, CD68, PANCK, KI67, FAP and CD8.
8. Single vascular like tracheal cell from adult Drosophila regenerative intestine
Dr Jessica Perochon, University of Glasgow
This image focuses on a single vascular cell (in Green) from an adult Drosophila melanogaster intestine (in Blue) in homeostasis versus regenerative context. In the latest case, the fly was subjected to intestinal damage (fed with pathogenic bacteria) to address the functional role of the vascular tissue in adult intestinal regeneration. Here, we can appreciate an extensive remodelling of the vascular branches in response to intestinal injury (right panel) in comparison with the homeostasis condition (left panel). Surrounding intestinal proliferating cells are labelled in Red within the epithelium. Extensive vascular remodelling is critical for increasing oxygen, nutrients and secreted factors delivery to intestinal stem cells which were adapting their metabolic demand in order to promote their tissue repair activity via proliferation. Vascular cells are labelled with the Gal4/UAS system, a method used to express genes such as Green fluorescent protein (GFP) in a tissue-specific manner in Drosophila model. Image details: -GFP Alexa Fluor 488 (Green) labels vascular cells, while -Phospho-Histone H3 Alexa Fluor 594 (Red) labels proliferative cells and Dapi 405 (Blue) labels cells nuclei. Images were captured using Zeiss LSM 780 confocal microscopy at 20X objective lens, 1024-pixel frame size, 0.5mm Z-stack intervals. Scale bar = 25 m.
9. Acute-stage Toxoplasma parasites living in human skin cells
Dr Kseniia Bondarenko, University of Edinburgh
Here, you are observing an acute-stage, overgrown Toxoplasma parasite within human foreskin fibroblasts, presented as a maximum intensity projection of a z-stack. The parasite’s membrane complex (its inner shell) appears in light blue, stained with anti-GAP45 and AlexaFluor647, while DNA (DAPI) is depth-colour coded—upper optical sections in yellow, with deeper layers in orange, pink, and purple. The larger nuclei belong to fibroblasts, while the smaller ones indicate parasitic nuclei. This image was captured using a Zeiss LSM980 confocal microscope equipped with an alpha Plan-Apochromat 100x objective, followed by deconvolution. Image adjustments include background removal, along with tone and contrast refinements.
Toxoplasma gondii is one of the most successful intracellular parasites, capable of invading various cell types in all warm-blooded animals, humans included. The parasite’s chronic stage remains incurable, and our lab is dedicated to unravelling the biological pathways that govern its lifecycle, with the ultimate goal of identifying potential drug targets. Microscopy plays a key role in our research, allowing us to pinpoint the location of proteins of interest to determine their function. Here, you see one of the parasite mutants I developed using CRISPR/Cas9 to investigate how gene removal affects parasite replication and survival.
10. Acute-stage parasites of Toxoplasma gondii in a human skin cell
Dr Kseniia Bondarenko, University of Edinburgh
Toxoplasma gondii, a highly successful intracellular parasite, invades various cell types in all warm-blooded animals, including humans. Here, the parasites were stained to show their inner shell (inner membrane complex, GAP45+AlexaFluor647, magenta), skeleton (microtubules, tubulin+AlexaFluor488, yellow), and nuclei (DNA, DAPI, blue), and presented as a maximum intensity projection of a z-stack. Notice that the top of the yellow skeleton basket in each parasite (an apical end) has a ring-like structure called a conoid. This structure extends when the parasite is ready to infect and is used as an attachment and delivery mechanism of the hijacking machinery inside the host cell. Toxoplasma replicates daily through the process called endodyogeny, where two daughter cells form inside a mature mother parasite that is eventually consumed. You can see daughters developing in four mother parasites on the left side of the picture. The big blue blob on the left is part of the human foreskin fibroblast nuclei, the host cell of the parasite.
The sample was enlarged using 4.5x ultrastructure expansion microscopy. The expanded gel imaged with laser-scanning Zeiss LSM980 confocal microscope with an alpha Plan-Apochromat 100x objective, followed by deconvolution. Image adjustments include background removal, along with tone and contrast refinements.
11. Fibre typing of Mouse Soleus Muscle
Muhammad 'Rizwan' Farrukh, University of Edinburgh Centre for Discovery Brain Sciences
Fibre typing of mouse skeletal muscle. x20 epifluorescence image of soleus muscle dissected from a 19-days-old mouse. 10um skeletal muscle cross section is stained with anti-Laminin 1+2 and BA-D5 antibodies to label skeletal muscle laminin (red) and myosin heavy chain Type I (green), respectively. Fibre typing is performed to study fibre type switching of the skeletal muscle from fast to slow as a result of disease process.
12. Metabolic Fireworks: Visualizing Lactate Dynamics in Drosophila Intestinal Stem Cells and Enteroblasts
Yuanliangzi Tian, University of Glasgow
This image shows pseudo-coloured mTFP lifetime measurements derived from ex vivo fluorescence lifetime imaging (FLIM) of adult Drosophila midguts, where intestinal stem cells and enteroblasts express the fluorescence resonance energy transfer (FRET)-based lactate sensor Laconic. The image was produced by merging the pseudo-colour lifetime measurements of mTFP (donor fluorescence of Laconic) with the intrinsic expression intensity image of Laconic. Blue colour indicates a shorter lifetime (sensor closed, with less lactate binding), while red indicates a longer lifetime (sensor open, with more lactate binding). The image was acquired with a 60x/0.8 Plan-Apochromat objective lens using the LaVision TriM Scope equipped with a tuneable Ti: Sapphire laser (Chameleon Ultra II, Coherent) and a FLIM X16 time-correlated single photon counting (TCSPC) detector. The fluorescence lifetime pseudo-colour map was generated by FLIMfit 5.1.1 software, which performed phasor segmentation to segment only the cells of interest by lifetime and intensity and subsequent exponential pixel-by-pixel fitting to extract an average lifetime per segmented pixel.
13. Nobody Likes Me (Think I’ll Go 'Clear' Worms)
Dr Liam Rooney, University of Strathclyde
‘Big, fat, juicy ones’, as the song goes, in this case. The wax moth larva, Galleria mellonella, is a common model for studying host-pathogen interactions and infection. Researchers observe the gradual melanisation as the larva become sick and die, quantifying the colour change as a function of pathogenicity. However, to peer inside and localise the pattern of infection has remained elusive due to the larvae’s dense and highly scattering fat deposits and internal structure. We have developed an optimised optical clearing method which strips out the fat and extracellular milieu that has hindered microscopists from visualising infection dynamics. This image shows an abdominal section of the larva, measuring over 1mm thick, optically cleared, stained with propidium iodide, and imaged for 25 hours using the Mesolens configured in confocal laser scanning mode. The colour-coding of this z-projection illustrates the internal structure of the cleared larva; the three loops of the silk gland are clearly visible, forming a sad face following the ordeal of fixation, bleaching, RNA depletion, dehydration and clearing. Following these successful trials, we aim to visualise infection in cleared larvae, providing a new investigative method that sheds light on the intricacies of host-pathogen interactions across spatial scales.
14. Neon weave: A fluorescent lattice woven by Drosophila melanogaster visceral muscle fibers and their skeptical producer enteroblasts
Yuanliangzi Tian, University of Glasgow
This image depicts the visceral muscle fibre network (Magenta) and the intestinal progenitor cells enteroblasts (Cyan) in the adult Drosophila midgut after bacterial infection. Dissected fly midguts were fixed and stained with the anti-GFP antibody to label enteroblasts with endogenous GFP expression and Phalloidin to label visceral muscle fibres. The visceral muscle fibre network was severely destroyed upon bacterial ingestion. This image was captured on a ZEISS LSM710 confocal microscope with a 20x lens.
15. STEDy on: glucose transport in action
Dr Shannan Foylan, University of Strathclyde
Glucose transporters are crucial molecules for the uptake of blood glucose following a meal and the dysregulation in their trafficking pathways are implicated in metabolic diseases such as Type 2 diabetes, insulin resistance and cardiovascular disease. In fat and muscle tissue, the most studied of the 14 member family of glucose transporters, GLUT4 is responsible for maintaining healthy blood sugar levels by uptaking glucose upon an insulin signal from the pancreas. Here, I use super-resolution STED microscopy in order to probe the organisation of single molecules and clusters of molecules in the plasma membrane of fat using a 100X/1.45NA objective on an Olympus system with an Abberior STEDYCON module. The GLUT4 molecule is tagged on its N-terminus with GFP and plasma membrane bound GLUT4 molecules are tagged with AlexaFluor 594. Only the magenta AlexaFluor 594 channel is STED compatible with the system in our lab, the green GFP channel is a confocal image of total GLUT4 in the cell. We are using this system, alongside others, to characterise the changes in GLUT4 organisation at their point of binding to the plasma membrane under different conditions to probe their native versus disease state.
16. Deep in the lung, or under the sea?
Lindsey Arnott, CRUK Scotland Institute
Before cancer spreads to the lungs, collagen in the extracellular matrix can be remodelled to provide a more supportive environment for secondary tumour growth. In the lung we can image fibrillar collagen by its second harmonic generation signal using multiphoton microscopy. Precision cut lung slices were taken from wild type mice and cultured with a tumour conditioned media for two weeks prior to imaging with a ZEISS 880 multiphoton microscope using a 20X objective lens and a non-descanned detector to identify the SHG signal from fibrillar collagen. As SHG signal is intrinsically generated no fluorophores were required. This image shows fibrillar collagen (in red-orange) around a large blood vessel and near an airway. Blue shows other autofluorescent lung tissue.
17. Colourful Cell
Dr Leandro Lemgruber, Cellular Analysis Facility - University of Glasgow
Stem cell imaged using super-resolution microscopy (3D-SIM). Nucleus stained with Dapi (pink), different membrane receptors stained with secondary antibodies (blue and yellow).
18. Tackling Ageing Head On
Alistair Ozzie Gemmell, University of Glasgow, University of Strathclyde
Drosophila are key model organisms for ageing biology, where their short lifespan and genetic toolsets provide advantages over costly and time-consuming vertebrate models.
An important question remains: do tissue-specific interventions that extend lifespan also preserve the integrity of tissues at older ages? A barrier to answering this question lies in the opaque nature of the fly cuticle: because of this, most studies rely on time-consuming dissections that disrupt organ architecture or even destroy the native position and integrity of the tissue as it would exist in vivo.
To study organ architecture in otherwise opaque flies, I have optically cleared the heads and used a combination of HCS Cellmask Green and Propidium Iodide to fluorescently label the cytoplasm and nuclei respectively, and imaged heads using a Leica SP5 confocal laser scanning microscope equipped with a 4x/0.1 numerical aperture lens. My images show differences in morphology and staining in multiple fly heads simultaneously, which may reveal new insights into ageing biology.
19. Developing pupae
Dr Cat Ficken, CRUK Scotland Institute
Fly pupae fixed in formalin , paraffin embedded . Section cut at 4µm and placed onto a glass slide. Stained with hematoxylin and eosin and scanned on the Leica Aperio AT2 at 40x magnification.
20. U2OS_LifeAct_mNeonGreen
Dr Bea , CRUK Scotland Institute
Dividing U2OS cells transfected with LifeAct-mNeonGreen DNA, showing the actin cytoskeleton. The montage is made up of individual frames acquired live on an Evident FV4000 confocal microscope with a 5 minute interval over a total period of 16 hours. Beatrice Bottura, CRUK-Scotland Institute.
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