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The role of cyclooxygenase (COX)-2 as a driving force in early tumourigenesis and the current interest in the combination of COX-2 inhibitors with standard therapy in clinical trials creates an urgent need to establish clinically relevant diagnostic tests for COX-2 expression. Molecular imaging using small-molecule probes radiolabelled for both positron emission tomography (PET) and single photon emission computed tomography (SPECT) offers the potential to meet this need, providing a minimally invasive readout for the whole disease burden. This review summarises current approaches to the radiolabelling of small-molecule COX-2 inhibitors and their analogues for PET and SPECT imaging, and gives an overview of their biological evaluation and likely success of clinical application. [ABSTRACT FROM AUTHOR]

Background and Purpose: Positron emission tomography (PET) has the potential to improve our understanding of the preclinical pharmacokinetics and metabolism of therapeutic agents, and is easily translated to clinical studies in humans. However, studies involving proteins radiolabelled with clinically relevant PET isotopes are currently limited. Here we illustrate the potential of PET imaging in a preclinical study of the biodistribution and metabolism of ¹⁸F-labelled IL-1 receptor antagonist ([¹⁸F]IL-1RA) using a novel [¹⁸F]-radiolabelling technique.Experimental Approach: IL-1RA was radiolabelled by reductive amination on lysine moieties with [¹⁸F]fluoroacetaldehyde. Sprague-Dawley rats were injected intravenously with [¹⁸F]IL-1RA and imaged with a PET camera for 2 h. For the study of IL-1RA metabolites by ex vivoγ-counting of samples, rats were killed 20 min, 1 h or 2 h after injection of [¹⁸F]IL-1RA.Key Results: [¹⁸F]IL-1RA distribution into the major organs of interest was as follows: kidneys >> liver > lungs >> brain. In lungs and liver, [¹⁸F]IL-1RA uptake peaked within 1 min post-injection then decreased rapidly to reach a plateau from 10 min post-injection. In the brain, the uptake exhibited slower pharmacokinetics with a smaller post-injection peak and a plateau from 6 min onward. IL-1RA was rapidly metabolized and these metabolites represented ∼40% of total activity in plasma and ∼80% in urine, 20 min after injection. CONCLUSIONS AND IMPLICATIONS Preclinical PET imaging is a feasible method of assessing the biodistribution of new biological compounds of therapeutic interest rapidly. The biodistribution of [¹⁸F]IL-1RA reported here is in agreement with an earlier study suggesting low uptake in the normal brain, with rapid metabolism and excretion via the kidneys. [ABSTRACT FROM AUTHOR]

Background: The extracellular localisation of the Heat shock protein 90 (Hsp90) is associated with the diseased state and wound healing and presents a promising opportunity for cancer targeting using Positron Emission Tomography (PET) imaging and molecularly targeted radiotherapy. The aim of this work is to develop a radiotracer with low nanomolar binding affinity to target the extracellular and particularly membrane pool of Hsp90, evaluate it in vitro, and conduct preliminary PET studies in vivo in mouse tumour models. Variable Heavy domain of Heavy chain antibodies, often referred to as Nanobodies, are suitable targeting vectors for the extracellular targets due to their favourable pharmacokinetic properties and low nanomolar target affinities. The main objective of the study is to target tumours expressing extracellular and membrane Hsp90 phenotype with minimal tracer accumulation in the non-target organs, which limited the translation of previously studied small molecule cytosolic Hsp90 tracers suffering from high non-Hsp90 specific background in the abdominal area., Results: Six nanobodies were obtained after llama immunization with recombinant Hsp90α and ELISA biopanning, produced in E. coli and screened for stability and affinity. We selected one nanobody, 4DAM26, with good thermal stability, no aggregation at elevated temperatures, and low nanomolar affinity towards Hsp90α and Hsp90β isoforms for translation as a PET radiotracer. The nanobody was bioconjugated to p-NCS-NODAGA and radiolabeled with gallium-68 with 75 ± 11% radiochemical yield and > 99% radiochemical purity and remained stable up to 3 h in phosphate buffered saline and mouse serum. Pilot in vivo evaluation using µPET/CT and ex vivo biodistribution demonstrated a favourable pharmacokinetic profile, but the tumour uptake was non-distinguishable from the background tissue., Conclusion: Compared to the small molecule Hsp90 tracers, the studied Nb-based tracer has improved pharmacokinetics properties including renal clearance and almost no accumulation in the non-target organs. Tumour uptake, on the other hand, was minimal and could not be differentiated from the background in µPET/CT. Our experiments indicate that in the studied models, membrane and extracellular expression of Hsp90 is majorly an artifact of cellular death, as only dead/dying cells had accessible pools of Hsp90 by flow cytometry, a consequence of a leaky membrane. More fundamental research is required to reassess the role of extracellular Hsp90 in cancer, and our future efforts will be focused on improving our inventory of cytosolic Hsp90 tracers with proven Hsp90-specific tumour accumulation., Competing Interests: Declarations. Ethics approval and consent to participate: Not applicable. Consent for publication: Not applicable. Competing interests: The authors declare that they have no competing interests., (© 2025. The Author(s).)

Background: Therapeutic antibodies for the treatment of neurological disease show great potential, but their applications are rather limited due to limited brain exposure. The most well-studied approach to enhance brain influx of protein therapeutics, is receptor-mediated transcytosis (RMT) by targeting nutrient receptors to shuttle protein therapeutics over the blood-brain barrier (BBB) along with their endogenous cargos. While higher brain exposure is achieved with RMT, the timeframe is short due to rather fast brain clearance. Therefore, we aim to increase the brain half-life of antibodies by binding to myelin oligodendrocyte glycoprotein (MOG), a CNS specific protein., Methods: Alpaca immunization with mouse/human MOG, and subsequent phage selections and screenings for MOG binding single variable domain antibodies (VHHs) were performed to find mouse/human cross-reactive VHHs. Their ability to increase the brain half-life of antibodies was evaluated in healthy wild-type mice by coupling two different MOG VHHs (low/high affinity) in a mono- and bivalent format to a β-secretase 1 (BACE1) inhibiting antibody or a control (anti-SARS-CoV-2) antibody, fused to an anti-transferrin receptor (TfR) VHH for active transport over the BBB. Brain pharmacokinetics and pharmacodynamics, CNS and peripheral biodistribution, and brain toxicity were evaluated after intravenous administration to balb/c mice., Results: Additional binding to MOG increases the C max and brain half-life of antibodies that are actively shuttled over the BBB. Anti-SARS-CoV-2 antibodies coupled with an anti-TfR VHH and two low affinity anti-MOG VHHs could be detected in brain 49 days after a single intravenous injection, which is a major improvement compared to an anti-SARS-CoV-2 antibody fused to an anti-TfR VHH which cannot be detected in brain anymore one week post treatment. Additional MOG binding of antibodies does not affect peripheral biodistribution but alters brain distribution to white matter localization and less neuronal internalization., Conclusions: We have discovered mouse/human/cynomolgus cross-reactive anti-MOG VHHs which have the ability to drastically increase brain exposure of antibodies. Combining MOG and TfR binding leads to distinct PK, biodistribution, and brain exposure, differentiating it from the highly investigated TfR-shuttling. It is the first time such long brain antibody exposure has been demonstrated after one single dose. This new approach of adding a binding moiety for brain specific targets to RMT shuttling antibodies is a huge advancement for the field and paves the way for further research into brain half-life extension., Competing Interests: Declarations. Ethics approval and consent to participate: All animal experiments were approved by the KU Leuven Animal Ethics Committee (project P091/2022 and P038/2024) following governmental and EU guidelines. Data availability: The data presented in this study are openly available in KU Leuven Research Data Repository (RDR) at 10.48804/JT11HB [46]. MTA statement: The VHH protein sequences can be provided by the laboratory of therapeutic and diagnostic antibodies pending scientific review and a completed material transfer agreement. Requests for the VHH protein sequences should be submitted to: maarten.dewilde@kuleuven.be. Competing interests: M.C., T.J., B.D.S, and M.D. declare to be holder of patent EP23198043.4. Apart thereof, the authors declare that they have no further competing interests, (© 2025. The Author(s).)

Background: [ 68 Ga]PentixaFor detects C-X-C chemokine receptor type 4 (CXCR4) overexpression in various malignancies, such as multiple myeloma and non-Hodgkin lymphomas, as well as in endocrine and inflammatory disorders. This study aimed to develop an Al 18 F-labeled radiotracer derived from LY2510924 for CXCR4-targeted imaging, leveraging the physical and logistical advantages of fluorine-18., Methods: We designed a CXCR4-specific radioprobe, [ 18 F]AlF-NOTA-SC, based on LY2510924 by incorporating a triglutamate linker and NOTA chelator to enable Al 18 F-labeling. The in vitro CXCR4 affinity was assessed using cell-based binding assays. Subsequently, in vivo pharmacokinetics and tumor uptake of [ 18 F]AlF-NOTA-SC were assessed in naïve mice and mice with xenografts derived from U87.CD4/U87.CD4.CXCR4 and MM.1 S cells. Finally, biodistribution was determined in a non-human primate using PET-MR., Results: Compared to Ga-PentixaFor, AlF-NOTA-SC demonstrated similar in vitro affinity for human CXCR4. [ 18 F]AlF-NOTA-SC was produced with a decay-corrected radiochemical yield of 21.0 ± 7.1% and an apparent molar activity of 16.4 ± 3.6 GBq/µmol. In [ 18 F]AlF-NOTA-SC binding assays on U87.CD4.CXCR4 cells, the total bound fraction was 7.1 ± 0.5% (58% blocking by AMD3100). In naïve mice, the radiotracer did not accumulate in any organs; however, it showed a significant CXCR4-specific uptake in xenografted tumors (SUVmean U87.CD4  = 0.04 ± 0.00 (n = 3); SUVmean U87.CD4.CXCR4  = 3.04 ± 0.65 (n = 3); SUVmean MM.1 S  = 1.95 ± 0.11 (n = 3)). In a non-human primate, [ 18 F]AlF-NOTA-SC accumulated in CXCR4 expressing organs, such as the spleen and bone marrow., Conclusion: [ 18 F]AlF-NOTA-SC exhibited CXCR4-specific uptake in vitro and in vivo, with fast and persistent tumor accumulation, making it a strong candidate for clinical translation as an 18 F-alternative to [ 68 Ga]PentixaFor., Competing Interests: Declarations. Research involving human participants and/or animals: All of the herein discussed animal studies were conducted according to the international, national, and institutional regulations governing the care and use of animals. Each experiment was approved by the KU Leuven ethical review board (Reference P010/2023 (mice studies) and P112/2019 (non-human primate study)). This research did not involve any human material or human participants. Informed consent: Not applicable. Consent for publication: All the authors approved the publication of the article. Conflict of interest: The authors have no relevant financial or non-financial interests to disclose., (© 2024. The Author(s).)

Developing devices and instrumentation in a bioimaging core facility is an important part of the innovation mandate inherent in the core facility model but is a complex area due to the required skills and investments, and the impossibility of a universally applicable model. Here, we seek to define technological innovation in microscopy and situate it within the wider core facility innovation portfolio, highlighting how strategic development can accelerate access to innovative imaging modalities and increase service range, and thus maintain the cutting edge needed for sustainability. We consider technology development from the perspective of core facility staff and their stakeholders as well as their research environment and aim to present a practical guide to the 'Why, When, and How' of developing and integrating innovative technology in the core facility portfolio. Core facilities need to innovate to stay up to date. However, how to carry out the innovation is not very obvious. One area of innovation in imaging core facilities is the building of optical setups. However, the creation of optical setups requires specific skill sets, time, and investments. Consequently, the topic of whether a core facility should develop optical devices is discussed as controversial. Here, we provide resources that should help get into this topic, and we discuss different options when and how it makes sense to build optical devices in core facilities. We discuss various aspects, including consequences for staff and the relation of the core to the institute, and also broaden the scope toward other areas of innovation., (© 2024 The Authors. Journal of Microscopy published by John Wiley & Sons Ltd on behalf of Royal Microscopical Society.)

Modern life science research is a collaborative effort. Few research groups can single-handedly support the necessary equipment, expertise and personnel needed for the ever-expanding portfolio of technologies that are required across multiple disciplines in today's life science endeavours. Thus, research institutes are increasingly setting up scientific core facilities to provide access and specialised support for cutting-edge technologies. Maintaining the momentum needed to carry out leading research while ensuring high-quality daily operations is an ongoing challenge, regardless of the resources allocated to establish such facilities. Here, we outline and discuss the range of activities required to keep things running once a scientific imaging core facility has been established. These include managing a wide range of equipment and users, handling repairs and service contracts, planning for equipment upgrades, renewals, or decommissioning, and continuously upskilling while balancing innovation and consolidation., (© 2024 The Authors. Journal of Microscopy published by John Wiley & Sons Ltd on behalf of Royal Microscopical Society.)

Nuclear DNA is the canonical target for biological damage induced by Auger electrons (AE) in the context of targeted radionuclide therapy (TRT) of cancer, but other subcellular components might also be relevant for this purpose, such as the energized mitochondria of tumor cells. Having this in mind, we have synthesized novel DOTA-based chelators carrying a prostate-specific membrane antigen (PSMA) inhibitor and a triphenyl phosphonium (TPP) group that were used to obtain dual-targeted 111 In-radioconjugates ( [ 111 In]In-TPP-DOTAGA-PSMA and [ 111 In]In-TPP-DOTAGA-G 3 -PSMA ), aiming to promote a selective uptake of an AE-emitter radiometal ( 111 In) by PSMA+ prostate cancer (PCa) cells and an enhanced accumulation in the mitochondria. These dual-targeted 111 In-radiocomplexes are highly stable under physiological conditions and in cell culture media. The complexes showed relatively similar binding affinities toward the PSMA compared to the reference tracer [ 111 In]In-PSMA-617 , in line with their high cellular uptake and internalization in PSMA+ PCa cells. The complexes compromised cell survival in a dose-dependent manner and in the case of [ 111 In]In-TPP-DOTAGA-G 3 -PSMA to a higher extent than observed for the single-targeted congener [ 111 In]In-PSMA-617 . μSPECT imaging studies in PSMA+ PCa xenografts showed that the TPP pharmacophore did not interfere with the excellent in vivo tumor uptake of the "golden standard" [ 111 In]In-PSMA-617 , although it led to a higher kidney retention. Such kidney retention does not necessarily compromise their usefulness as radiotherapeutics due to the short tissue range of the Auger/conversion electrons emitted by 111 In. Overall, our results provide valuable insights into the potential use of mitochondrial targeting by PSMA-based radiocomplexes for efficient use of AE-emitting radionuclides in TRT, giving impetus to extend the studies to other AE-emitting trivalent radiometals (e.g., 161 Tb or 165 Er) and to further optimize the designed dual-targeting constructs.

Background: [ 18 F]AlF-NOTA-octreotide is an 18 F-labeled somatostatin analogue which is a good clinical alternative for 68 Ga-labeled somatostatin analogues. However, radiolabeled somatostatin receptor (SSTR) antagonists might outperform agonists regarding imaging sensitivity of neuroendocrine tumors (NETs). No direct comparison between the antagonist [ 18 F]AlF-NOTA-JR11 and the agonist [ 18 F]AlF-NOTA-octreotide as SSTR PET probes is available. Herein, we present the radiosynthesis of [ 18 F]AlF-NOTA-JR11 and compare its NETs imaging properties directly with the established agonist radioligand [ 18 F]AlF-NOTA-octreotide preclinically., Methods: [ 18 F]AlF-NOTA-JR11 was synthesized in an automated synthesis module. The in vitro binding characteristics (IC 50 ) of [ nat F]AlF-NOTA-JR11 and [ nat F]AlF-NOTA-octreotide were evaluated and the in vitro stability of [ 18 F]AlF-NOTA-JR11 was determined in human serum. In vitro cell binding and internalization was performed with [ 18 F]AlF-NOTA-JR11 and [ 18 F]AlF-NOTA-octreotide using SSTR2 expressing cells and the pharmacokinetics were evaluated using μPET/CT in mice bearing BON1.SSTR2 tumor xenografts., Results: Excellent binding affinity for SSTR2 was found for [ nat F]AlF-NOTA-octreotide (IC 50 of 25.7 ± 7.9 nM). However, the IC 50 value for [ nat F]AlF-NOTA-JR11 (290.6 ± 71 nM) was 11-fold higher compared to [ nat F]AlF-NOTA-octreotide, indicating lower affinity for SSTR2. [ 18 F]AlF-NOTA-JR11 was obtained in a good RCY (50 ± 6 %) but with moderate RCP of 94 ± 1 %. [ 18 F]AlF-NOTA-JR11 demonstrated excellent stability in human serum (>95 % after 240 min). 2.7-fold higher cell binding was observed for [ 18 F]AlF-NOTA-JR11 as compared to [ 18 F]AlF-NOTA-octreotide after 60 min. μPET/CT images demonstrated comparable pharmacokinetics and tumor uptake between [ 18 F]AlF-NOTA-JR11 (SUV max : 3.7 ± 0.8) and [ 18 F]AlF-NOTA-octreotide (SUV max : 3.6 ± 0.4)., Conclusions: [ 18 F]AlF-NOTA-JR11 was obtained in good RCY, albeit with a moderate RCP. The cell binding study showed significant higher binding of [ 18 F]AlF-NOTA-JR11 compared to [ 18 F]AlF-NOTA-octreotide, despite the higher IC 50 value of AlF-NOTA-JR11. However, pharmacokinetics and in vivo tumor uptake was comparable for both radiotracers. Novel Al 18 F-labeled derivatives of JR11 with higher SSTR2 affinity should be developed for increased tumor uptake and NET imaging sensitivity., Competing Interests: Declaration of competing interest The authors have declared that no competing interest exists., (Copyright © 2023 KU leuven. Published by Elsevier Inc. All rights reserved.)

Multimodal imaging provides rich biological information, which can be exploited to study drug activity, disease associated phenotypes, and pharmacological responses. Here we show discovery and validation of a new probe targeting the endocannabinoid α/β-hydrolase domain 6 (ABHD6) enzyme by utilizing positron emission tomography (PET) and matrix-assisted laser desorption/ionization (MALDI) imaging. [ 18 F]JZP-MA-11 as the first PET ligand for in vivo imaging of the ABHD6 is reported and specific uptake in ABHD6-rich peripheral tissues and major brain regions was demonstrated using PET. A proof-of-concept study in nonhuman primate confirmed brain uptake. In vivo pharmacological response upon ABHD6 inhibition was observed by MALDI imaging. These synergistic imaging efforts used to identify biological information cannot be obtained by a single imaging modality and hold promise for improving the understanding of ABHD6-mediated endocannabinoid metabolism in peripheral and central nervous system disorders.

Background: Somatostatin receptor-2 (SSTR2) is expressed on cell surface of neuroendocrine neoplasias; its presence is exploited for the delivery of peptide receptor radionuclide therapy (PRRT). Patients with no or low expression of SSTR2 are not candidates for PRRT. SSTR2 promotor undergoes epigenetic modification, known to regulate gene expression. We investigated whether the demethylation agent, guadecitabine, could enhance the expression of SSTR2 in NET models, using radioligand uptake/PET imaging as a biomarker of epigenetic modification., Methods: The effects of guadecitabine on the transcriptional, translational, and functional regulation of SSTR2 both in vitro and in vivo using low (QGP-1) and high (BON-1) methylated neuroendocrine neoplasia models was characterised. Promotor region methylation profiling of clinical samples (n = 61) was undertaken. Safety of combination guadecitabine and PRRT was assessed in vivo., Results: Pyrosequencing of cell lines illustrated differential methylation indices - BON: 1 94%, QGP: 1 21%. Following guadecitabine treatment, a dose-dependent increase in SSTR2 in BON-1 at a transcriptional, translational, and functional levels using the SSTR2-directed radioligand, 18 F-FET-βAG-TOCA ([ 18 F]-FETO) (150% increase [ 18 F]-FETO uptake, p < 0.05) was observed. In vivo, guadecitabine treatment resulted in a 70% increase in [ 18 F]-FETO uptake in BON-1 tumour models compared models with low baseline percentage methylation (p < 0.05). No additive toxicity was observed with the combination treatment of PRRT and guadecitabine in vivo. Methylation index in clinical samples was 10.5% compared to 5.2% in controls (p = 0.03) and correlated with SSTR2 expression (Wilcoxon rank sign -3.75,p < 0.01)., Conclusion: Guadecitabine increases SSTR2 expression both in vitro and in vivo. The combination of demethylation agents with PRRT warrants further investigation., Competing Interests: Conflict of interest statement The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: RS: Astex pharmaceuticals, MSD, Bayer, Incyte pharmaceuticals, AAA, Boston Scientific, SIRTEX No other authors have any interests to declare., (Crown Copyright © 2022. Published by Elsevier Ltd. All rights reserved.)

Background : Radiolabeled somatostatin analogues ( e.g. [ 68 Ga]Ga-DOTATATE and [ 177 Lu]Lu-DOTATATE) have been used to diagnose, monitor, and treat neuroendocrine tumour (NET) patients with great success. [ 18 F]AlF-NOTA-octreotide, a promising 18 F-labeled somatostatin analogue and potential alternative for 68 Ga-DOTA-peptides, is under clinical evaluation. However, ideally, the same precursor (combination of chelator-linker-vector) can be used for production of both diagnostic and therapeutic radiopharmaceuticals with very similar ( e.g. Al 18 F-method in combination with therapeutic radiometals 213 Bi/ 177 Lu) or identical ( e.g. complementary Tb-radionuclides) pharmacokinetic properties, allowing for accurate personalised dosimetry estimation and radionuclide therapy of NET patients. In this study we evaluated 3p- C -NETA, as potential theranostic Al 18 F-chelator and present first results of radiosynthesis and preclinical evaluation of [ 18 F]AlF-3p- C -NETA-TATE. Methods : 3p- C -NETA was synthesized and radiolabeled with diagnostic ( 68 Ga, Al 18 F) or therapeutic ( 177 Lu, 161 Tb, 213 Bi, 225 Ac and 67 Cu) radionuclides at different temperatures (25-95 °C). The in vitro stability of the corresponding radiocomplexes was determined in phosphate-buffered saline (PBS) and human serum. 3p- C -NETA-TATE was synthesized using standard solid/liquid-phase peptide synthesis. [ 18 F]AlF-3p- C -NETA-TATE was synthesized in an automated AllinOne® synthesis module and the in vitro stability of [ 18 F]AlF-3p- C -NETA-TATE was evaluated in formulation buffer, PBS and human serum. [ 18 F]AlF-3p- C -NETA-TATE pharmacokinetics were evaluated using µPET/MRI in healthy rats, with [ 18 F]AlF-NOTA-Octreotide as benchmark. Results : 3p- C -NETA quantitatively sequestered 177 Lu, 213 Bi and 67 Cu at 25 °C while heating was required to bind Al 18 F, 68 Ga, 161 Tb and 225 Ac efficiently. The [ 18 F]AlF-, [ 177 Lu]Lu- and [ 161 Tb]Tb-3p- C -NETA-complex showed excellent in vitro stability in both PBS and human serum over the study period. In contrast, [ 67 Cu]Cu- and [ 225 Ac]Ac-, [ 68 Ga]Ga-3p- C -NETA were stable in PBS, but not in human serum. [ 18 F]AlF-3p- C -NETA-TATE was obtained in good radiochemical yield and radiochemical purity. [ 18 F]AlF-3p- C -NETA-TATE displayed good in vitro stability for 4 h in all tested conditions. Finally, [ 18 F]AlF-3p- C -NETA-TATE showed excellent pharmacokinetic properties comparable with the results obtained for [ 18 F]AlF-NOTA-Octreotide. Conclusions : 3p- C -NETA is a versatile chelator that can be used for both diagnostic applications (Al 18 F) and targeted radionuclide therapy ( 213 Bi, 177 Lu, 161 Tb). It has the potential to be the new theranostic chelator of choice for clinical applications in nuclear medicine., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Optical mesoscale imaging is a rapidly developing field that allows the visualisation of larger samples than is possible with standard light microscopy, and fills a gap between cell and organism resolution. It spans from advanced fluorescence imaging of micrometric cell clusters to centimetre-size complete organisms. However, with larger volume specimens, new problems arise. Imaging deeper into tissues at high resolution poses challenges ranging from optical distortions to shadowing from opaque structures. This manuscript discusses the latest developments in mesoscale imaging and highlights limitations, namely labelling, clearing, absorption, scattering, and also sample handling. We then focus on approaches that seek to turn mesoscale imaging into a more quantitative technique, analogous to quantitative tomography in medical imaging, highlighting a future role for digital and physical phantoms as well as artificial intelligence., (© 2022 The Authors. Journal of Microscopy published by John Wiley & Sons Ltd on behalf of Royal Microscopical Society.)