Apotome module

Manufactured by Zeiss

Sourced in Germany, France, India, United States

The ApoTome module is a hardware accessory developed by Zeiss for use with their microscopy systems. It is designed to enhance optical sectioning and improve image contrast by utilizing a structured illumination technique. The ApoTome module can be integrated with various Zeiss microscope models to provide an efficient means of acquiring high-quality, optically sectioned images.

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Apotome (432 citations) Apotome.2 module (28 citations) ApoTome slide module (5 citations) Apotome slider module (3 citations) ApoTome optical sectioning module (2 citations) Apotome module microscope (2 citations) ApoTome fluorescence module (1 citations)

53 protocols using apotome module

Quantifying Malaria Ookinete Conversion

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Hybridoma cells expressing anti-Pfs25 IgG (clone 4B7) were obtained from BEI Resources. IgG fraction was purified using Protein G Sepharose columns (GE Healthcare) and labeled to Alexa Fluor 568 (Life Technologies) in the Central Laboratory Facility (Deutsches Rheuma-Forschungszentrum, Berlin). Ookinetes were stained with anti-Pfs25-Alexa Fluor 568 antibody (7.5 μg/ml at 2 hpi and 1.5 μg/ml at 23 hpi) and Hoechst 1:500 (Molecular Probes) for 30 min at 4°C in PBS. Samples were washed (300 g, 4 min) and resuspended in PBS. The numbers of Pfs25-positive parasites were enumerated in a counting chamber (Neubauer) using a Leica DMRB fluorescence microscope and classified into stages using an Axio Observer Z1 fluorescence microscope equipped with an Apotome module (Zeiss) (Figure 1).
Ookinete conversion was defined as follows:
The proportion of mature ookinete was defined as follows:
Representative pictures were acquired with the Apotome module, and images were deconvoluted and corrected for phase contrast and local bleaching using ZEN 2012 software (Zeiss).

Siciliano G., Costa G., Suárez-Cortés P., Valleriani A., Alano P, & Levashina E.A. (2020). Critical Steps of Plasmodium falciparum Ookinete Maturation. Frontiers in Microbiology, 11, 269.

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Immunofluorescence Staining for Intracellular Protein Analysis

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Immunofluorescence staining was carried out essentially as described earlier^{13 (link),58 (link),59 (link)}. Briefly, cells at 36 h post-transfection were fixed using 4% paraformaldehyde in 1X phosphate-buffered saline (PBS) for 20 min. The fixed cells were permeabilized using 0.05% Triton X-100 and blocked using 5% equine serum and 5% fish gelatin for 45 min. Cells were subsequently incubated with the primary and secondary antibody according to the manufacturer’s protocol. To visualize the nuclei, the cells were counter stained with DAPI for 5 min. Fluorescence images were captured using the Axiovision Epifluorescence microscope fitted with the ApoTome module (Carl Zeiss, Bangalore, India) and the images were assembled using Adobe Photoshop. For quantifying the LC3-positive cytoplasmic puncta (red-green signal overlap), the captured images from three independent sets, and in each set two biological repeats were processed (approximately 30 cells in each set) using the colocalization macro in ImageJ software, as described earlier^{13 (link)}. The Huntingtin aggregates were counted manually by a blind observer from three independent sets (approximately 100 cells in each set) of experiments.

Rai A., Singh P.K., Singh V., Kumar V., Mishra R., Thakur A.K., Mahadevan A., Shankar S.K., Jana N.R, & Ganesh S. (2018). Glycogen synthase protects neurons from cytotoxicity of mutant huntingtin by enhancing the autophagy flux. Cell Death & Disease, 9(2), 201.

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Cryopreservation Effects on Mitochondrial Integrity

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For determination of mitochondrial integrity by fluorescence microscopy, cells were seeded onto collagen-coated glass coverslips in six-well plates. Before cryopreservation, cells were loaded for 40 min with 500 nM MitoTracker Green (molecular probes, #M7514) and 20 min with 200 nM tetramethyl rhodamine methyl ester (TMRM) in HBSS at 37°C. For TMRM, a maintenance concentration of 20 nM was used after loading. After thawing, the cryopreservation solutions were exchanged for Krebs-Henseleit buffer with 10 mM D-glucose and 20 nM TMRM. Fluorescence was assessed by confocal fluorescence microscopy using a Zeiss Observer.Z1 with ApoTome module (Zeiss) with filter sets for λ_exc = 546 ± 6 nm/λ_em ≥ 590 nm (TMRM) and λ_exc = 470 ± 20 nm/λ_em = 525 ± 25 nm (MitoTracker Green).

Pless-Petig G., Knoop S, & Rauen U. (2018). Serum- and albumin-free cryopreservation of endothelial monolayers with a new solution. Organogenesis, 14(2), 107-121.

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Morphometric Analysis with ApoTome

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A morphometric analysis was performed using the software of an Axiovert 200 M inverted microscope equipped with an ApoTome module and Axio Cam MRM and Axio Cam HRC (Carl Zeiss, Germany) digital cameras. The measurements were performed at 400× magnification in five randomly chosen fields of view for each area examined.

Pushchina E.V, & Varaksin A.A. (2019). Neurolin expression in the optic nerve and immunoreactivity of Pax6-positive niches in the brain of rainbow trout (Oncorhynchus mykiss) after unilateral eye injury. Neural Regeneration Research, 14(1), 156-171.

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Visualizing Phospho-Tau in Tissue Sections

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For heat‐induced epitope retrieval, floating sections were kept in sodium citrate (pH 6, 10 mM, 37°C, 30 min). After permeabilization (PBS1X/Triton X‐100 2%, 15 min), unspecific labeling was blocked (PBS1X/Triton X‐100 0.1%/horse serum 5%, 1 h at RT) and slices were incubated overnight with polyclonal anti‐CBP antibody (1/50; ab2832; Abcam), washed, and further incubated with anti‐rabbit Alexa Fluor 594 (red, 1/1,000) antibody (Invitrogen, Thermo Fisher Scientific) for 1 h. IHC was performed sequentially and the second antibody was added in a second step. Slices were incubated overnight with monoclonal anti phospho‐Tau (Thr212, Ser214) antibody (1/1,000; AT100, MN1060 Thermo Fisher Scientific), washed, and further incubated with anti‐mouse Alexa Fluor 488 (green, 1/1,000) antibody (Invitrogen, Thermo Fisher Scientific). Slices were incubated with the Hoechst dye 33342 (1 mg/ml; 5 min) and mounted in Mowiol for observation. Acquisitions were performed using a fluorescence microscope coupled with an ApoTome module (Zeiss). Photomicrographs were captured using the z‐stack mode with 0.5 μm of thickness between slices, 18 in‐depth slices were taken and flattened using the maximum intensity projection (MIP).

Chatterjee S., Cassel R., Schneider‐Anthony A., Merienne K., Cosquer B., Tzeplaeff L., Halder Sinha S., Kumar M., Chaturbedy P., Eswaramoorthy M., Le Gras S., Keime C., Bousiges O., Dutar P., Petsophonsakul P., Rampon C., Cassel J., Buée L., Blum D., Kundu T.K, & Boutillier A. (2018). Reinstating plasticity and memory in a tauopathy mouse model with an acetyltransferase activator. EMBO Molecular Medicine, 10(11), e8587.

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Zeiss Confocal Microscopy Imaging

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Images were acquired using LSM 780, LSM 880 Zeiss confocal microscopes equipped with 20×, 40× and 63× objectives or a Zeiss Imager MZ with an ApoTome module. Images were acquired in the lsm format and processed with ImageJ for export as tiff files. Figures were compiled using Adobe Photoshop and Illustrator.

Kastriti M.E., Faure L., Von Ahsen D., Bouderlique T.G., Boström J., Solovieva T., Jackson C., Bronner M., Meijer D., Hadjab S., Lallemend F., Erickson A., Kaucka M., Dyachuk V., Perlmann T., Lahti L., Krivanek J., Brunet J., Fried K, & Adameyko I. (2022). Schwann cell precursors represent a neural crest‐like state with biased multipotency. The EMBO Journal, 41(17), e108780.

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Time-lapse Microscopy of Live Cells

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Time-lapse recordings with phase contrast or double fluorescence applications were performed on a Zeiss Axio Observer Z1 inverted microscope with Zeiss Plan Neofluar 10x, 0.3 NA objective coupled to a Zeiss Axiocam MRM CCD camera and equipped with a Marzhauser SCAN-IM powered stage. Zeiss Colibri LED illumination system was used for fluorescent excitation. For structured illumination microscopy and fluorescent optical sectioning we used Zeiss Apotome module. During time-lapse imaging the cell cultures were kept in a stage-top incubator (CellMovie) providing for required temperature and CO₂ atmosphere. Power stage positioning, illumination, focusing, optical sectioning and primary image collection were controlled by Zeiss Axiovision 4.8 software and a custom-made experiment manager software module on a PC. Images were further processed using Zeiss Axiovision 4.8 and NIH ImageJ software.

Méhes E., Mones E., Varga M., Zsigmond Á., Biri-Kovács B., Nyitray L., Barone V., Krens G., Heisenberg C.P, & Vicsek T. (2023). 3D cell segregation geometry and dynamics are governed by tissue surface tension regulation. Communications Biology, 6, 817.

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Intravital Imaging of Pancreatic Vasculature

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Twelve week old RIP1-Tag2 mice were anesthetized, the chest was opened, and the vasculature was perfused by injection of 150 μl FITC-conjugated dextran (containing 7.5 mg dextran, 2000 kDa, Sigma, Schnelldorf, Germany) into the left heart chamber. Circulation was allowed for 5 minutes before pancreas preparation, followed by fixation in 4% PFA for 2 hours, incubation in 20% sucrose overnight, embedding into Tissue-Tek and freezing on dry ice. Cryosections (50 μm) were stained as described below and Z-series acquisitions were performed with a Zeiss Axio Imager.Z2 microscope equipped with the ApoTome module and AxioVs40 software (Carl Zeiss Microimaging) with a 3D analysis tool.

Spenlé C., Gasser I., Saupe F., Janssen K.P., Arnold C., Klein A., van der Heyden M., Mutterer J., Neuville-Méchine A., Chenard M.P., Guenot D., Esposito I., Slotta-Huspenina J., Ambartsumian N., Simon-Assmann P, & Orend G. (2015). Spatial organization of the tenascin-C microenvironment in experimental and human cancer. Cell Adhesion & Migration, 9(1-2), 4-13.

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Microscopic Imaging and Morphometric Analysis

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For visualization and morphological analysis, we used a motorized inverted microscope of a research class with a fluorescent module and an enhanced contrast attachment when working with Axiovert 200 M luminescence with an ApoTome module (Carl Zeiss, Oberkochen, Germany). Microphotographs of the preparations and material analysis were performed using the Axio Vision program (Carl Zeiss, Oberkochen, Germany). The measurements were carried out with magnification 100×, 200×, 400× and 630× in several randomly selected fields of view for each area of study. The number of immunolabeled cells in the field of view was counted at a magnification of 200×. Morphometric analysis of the parameters of cell bodies (measurements of the greater and lesser diameters of the neurons’ soma) was performed using the Axiovert 200 M microscope software (Carl Zeiss, Oberkochen, Germany). Micrographs of the brain sections were taken with an Axiovert 200 digital camera. The material was processed using the Axioimager program (Carl Zeiss, Oberkochen, Germany).

Pushchina E.V., Zharikova E.I., Varaksin A.A., Prudnikov I.M, & Tsyvkin V.N. (2020). Proliferation, Adult Neuronal Stem Cells and Cells Migration in Pallium during Constitutive Neurogenesis and after Traumatic Injury of Telencephalon of Juvenile Masu Salmon, Oncorhynchus masou. Brain Sciences, 10(4), 222.

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Whole-Mount In Situ Hybridization of Embryos

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HCR v3 was performed using the protocol suggested by Molecular Technologies (Choi et al., 2018 ). Briefly, the embryos were cultured to desired stages and fixed in 4% paraformaldehyde overnight at 4°C. On day 2, the fixed embryos were washed in 0.1% PBS-Tween and dehydrated using a series of 25%, 50%, 75%, and 100% methanol. Following an overnight incubation at −20°C in 100% methanol, the embryos were rehydrated, treated with proteinase-K for 2.5 minutes, and incubated with 5–10pmol of probe mixture overnight at 37°C. On day 3, excess probe was washed off and the embryos were incubated with 30pmol of both hairpins H1 and H2 at room temperature overnight. On day 4, the embryos were washed in 0.1% 5x-SSC-Tween and imaged on a Zeiss Imager M2 with an ApoTome module and/or Zeiss LSM 880 confocal microscope at the Caltech Biological Imaging Facility.

Gandhi S., Ezin M, & Bronner M.E. (2020). Reprogramming axial level identity to rescue neural crest-related congenital heart defects. Developmental cell, 53(3), 300-315.e4.

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