Apotome 2

Manufactured by Hamamatsu Photonics

The Apotome 2 is an advanced optical imaging system designed to provide high-quality, high-resolution images. It utilizes a structured illumination technique to optically section samples, improving image quality and contrast. The Apotome 2 is capable of producing optical sections with increased resolution and reduced background noise, allowing for improved visualization of fine details within a sample.

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8 protocols using apotome 2

Autophagy Quantification in C. elegans

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Animals were synchronised by egg lay for 1 hour at 20°C. Once animals reached the L4 stage, they were transferred onto NGM plates with or without food for 24 hours. After this period, worms were moved back to plates containing food for 24 hours, at which point, fed and fasted animals were heat-shocked in a water bath for 4 hours at 35°C, or kept at 20°C for an equivalent period of time. Immediately following heat shock, worms were mounted onto slides for image acquisition. Worms were mounted on 2% agarose pads in 5 mM levamisole and imaged immediately. For image acquisition, a Zeiss Imager.Z2 microscope with a Hamamatsu C13440 ORCA-Flash4.0 V3 digital camera, Apotome.2 for Z-stack images and ZenBlue software were used. Z-stacks were acquired at 0.6 μm slice intervals using 100x objective and processed as maximum intensity projections. Based on previous studies,⁶⁸ (link) a Z-position was selected where the nucleus could be seen in the intestine and posterior pharyngeal bulb in the pharynx. The area of the first two intestinal cells was used for the quantification of visible puncta in the intestine, whereas the area of the posterior pharyngeal bulb was used for the quantification of visible puncta in the pharynx. Green or yellow puncta were scored as autophagosomes while red puncta were scored as autolysosomes.

Tataridas-Pallas N., Aman Y., Williams R., Chapman H., Cheng K.J., Gomez-Paredes C., Bates G.P, & Labbadia J. (2024). Mitochondrial clearance and increased HSF-1 activity are coupled to promote longevity in fasted Caenorhabditis elegans. iScience, 27(6), 109834.

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Confocal Microscopy Cell Counting

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Confocal images were taken on Zeiss Axio-Observer Z1 with Apotome 2 and Hamamatsu Orca Flash 4.0LT camera and Zen Pro 2015 software. For cell counting we imported Zeiss z-stack images into Metamorph software (ver 7.8.13.0) and manually counted cells by scrolling through the z-stack and labeling individual cells.

Molotkov A., Mazot P., Brewer J.R., Cinalli R.M, & Soriano P. (2017). Distinct Requirements for Fgfr1 and Fgfr2 in Primitive Endoderm Development and Exit from Pluripotency. Developmental cell, 41(5), 511-526.e4.

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Quantifying Axonal TrkB Receptor Exocytosis

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Chimeric FLAG-TrkB:A receptors in distal axon compartments of compartmentalized cultures were live-labeled with a mouse anti-FLAG antibody (1:500). Excess antibody was washed off, and axons were treated with BDNF (100 ng/ml, 1 hr, 37°C) or left un-stimulated. Neurons were fixed, and incubated with anti-mouse Alexa-546 secondary antibody without permeabilization. Images were acquired using a Zeiss AxioImager Z1 imaging microscope with Hamamatsu Orca Flash 4.0 CMOS camera and Apotome 2. The same acquisition settings were applied to all images taken from a single experiment. Axon-derived Trk receptors exocytosed to soma surfaces were quantified as the number of FLAG-immunopositive punctae per soma. 20 neurons were analyzed per condition per experiment.

Yamashita N., Joshi R., Zhang S., Zhang Z.Y, & Kuruvilla R. (2017). Phospho-regulation of soma-to-axon transcytosis of neurotrophin receptors. Developmental cell, 42(6), 626-639.e5.

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Evaluating Mtb rv0500A Mutant Morphology

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Erdman(P₆₀₆’::His-rv0500A-tetON, smyc’::mCherry) or Erdman(P₆₀₆’::His-rv0500A-M39A/T40A-tetON, smyc’::mCherry) Mtb were grown in standing, filter-capped T-25 flasks in 10 ml 7H9, pH 7 medium or 7H9, pH 5.7 medium supplemented with 250 mM NaCl. Cultures were started at an OD₆₀₀ = 0.05 and treated after 6 days of growth with either 200 ng/ml of ATC or ethanol as a carrier control. At indicated timepoints, Mtb was fixed in 4% PFA overnight and subsequently resuspended in PBS with 0.1% Tween 80. For examination of WT, Δrv0500A, and rv0500A* bacterial lengths, each of the strains carrying a constitutively expressed smyc’::mCherry reporter were grown for 6 days in standing, filter-capped T-25 flasks in 10 ml 7H9, pH 7 medium, or 7H9, pH 5.7 medium supplemented with 250 mM NaCl, from a starting OD₆₀₀ = 0.05, before samples were fixed in 4% PFA overnight and subsequently resuspended in PBS with 0.1% Tween 80. Samples were mounted using ProLong Diamond antifade (Invitrogen), and bacteria were imaged using a Zeiss Axio Observer Z1 microscope equipped with Colibri LED and HXP metal halide illumination, a Hamamatsu ORCA-R2 CCD camera, and an Apotome 2 for structured illumination microscopy. Image reconstruction was carried out with ZEN software (Zeiss) and Volocity software (Quorum Technologies), and bacterial lengths measured in Volocity.

Kevorkian Y.L., MacGilvary N.J., Giacalone D., Johnson C, & Tan S. (2022). Rv0500A is a transcription factor that links Mycobacterium tuberculosis environmental response with division and impacts host colonization. Molecular microbiology, 117(5), 1048-1062.

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Subcellular Localization of TAZ by IF

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MDA-MB-231 cells plated on Lab-Tek II chamber slides (Thermo Fisher Scientific) were fixed with 4% paraformaldehyde (Sigma), permeabilized with 0.1% Triton X-100 (Fisher) for 5 min, blocked with PBS supplemented with 10% BSA for 20 min, and stained with anti-TAZ antibody (Novus Biologicals) overnight at 4°C, followed by incubation with FITC-conjugated goat anti-rabbit IgG (Novus Biologicals). F-actin and nuclear DNA were stained using Alexa Fluor 568-conjugated phalloidin (Life Technologies) and 300 nM DAPI (Invitrogen), respectively. Fluorescent imaging was performed using a 63× (1.4 NA), oil immersion objective on an AxioObserver microscope (Carl Zeiss), which was equipped with ApoTome.2 and an ORCA-ERG digital camera (Hamamatsu). Images were processed with ZEN software 2012 (Carl Zeiss). Fluorescence intensities of TAZ in nucleus and cytoplasm were determined using ImageJ software (NIH). Fifty cells were randomly imaged and scored for each experiment, and data from three independent experiments were plotted as mean + SEM.

Xiang L., Gilkes D.M., Hu H., Takano N., Luo W., Lu H., Bullen J.W., Samanta D., Liang H, & Semenza G.L. (2014). Hypoxia-inducible factor 1 mediates TAZ expression and nuclear localization to induce the breast cancer stem cell phenotype. Oncotarget, 5(24), 12509-12527.

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Colocalization Analysis of SQST-1 and LGG-1 in C. elegans

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For images of higher magnification (including for colocalization of SQST-1::GFP and tdTOMATO::LGG-1/Atg8), animals were mounted on a 2% agarose pad in M9 medium containing 0.1% NaN₃ and imaged on a Zeiss Imager Z1 including apotome.2 with a Hamamatsu orca flash 4LT camera and Zen 2.3 software at ×1000 magnification. For colocalization, images were analyzed using the colocalization plugin Coloc2 in Fiji-ImageJ (National Institutes of Health). The correlation between the red and green fluorescence is given as intensity correlation quotient (ICQ) values^{68 (link)}. For random or mixed patterns of fluorescence intensity of the red and green channel this number will tend toward 0, for segregated localization of the red and green fluorescence signal the ICQ score will tend toward −0.5, and for colocalization of the fluorescent signals, the ICQ score will tend toward +0.5.

Kumsta C., Chang J.T., Lee R., Tan E.P., Yang Y., Loureiro R., Choy E.H., Lim S.H., Saez I., Springhorn A., Hoppe T., Vilchez D, & Hansen M. (2019). The autophagy receptor p62/SQST-1 promotes proteostasis and longevity in C. elegans by inducing autophagy. Nature Communications, 10, 5648.

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Monitoring TrkB Receptor Trafficking

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Surface chimeric receptors in cell body compartments were live labeled with a mouse anti-FLAG antibody, and then treated with PTP1B inhibitor (200 nM) added to cell body compartments. Neurons were stimulated with BDNF on axons for 2 hr, then fixed and immunostained for anti-LAMP1 antibody (1:500) to assess the co-localization with lysosomes. To investigate the co-localization with Rab11, neurons were infected with FLAG-TrkB:A and mCherry-Rab11a adenovirus followed by antibody feeding. Images were acquired using a Zeiss AxioImager Z1 imaging microscope with Hamamatsu Orca Flash 4.0 CMOS camera and Apotome 2. The number of punctae double-positive for FLAG and LAMP1 or Rab11 were counted and expressed as a percentage of the total FLAG signal in cell bodies.
The co-localization signal, visualized in white color, in Figures 3G, 3J, 3M, 3P, 5B, 5C, S1I, S1L, S1O, S1R, S3E, and S3F were presented using co-localization highlighter (ImageJ).

Yamashita N., Joshi R., Zhang S., Zhang Z.Y, & Kuruvilla R. (2017). Phospho-regulation of soma-to-axon transcytosis of neurotrophin receptors. Developmental cell, 42(6), 626-639.e5.

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Cell Viability Assay in Hydrogels

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At days 0 (6 hr post encapsulation) and 15, cell-laden hydrogels were rinsed with PBS for 15 min and labeled with a Live/Dead® cell viability kit (ThermoFisher C#L3224) according to the manufacturer’s instructions. The hydrogels were rinsed with PBS and placed between coverslips as previously described.^{[22 (link)]} Fluorescent z-stacks (z-height: 150 μm) were acquired using structured illumination on a Zeiss AxioObserver Z1 inverted fluorescent microscope equipped with a Zeiss Apotome2 imaging system and a Hamamatsu ORCA-Flash 4.0LT camera as previously described.^{[22 (link)]} To obtain representative images, z-stacks were captured at the approximate center of each hydrogel, between its top and bottom surfaces. We did not encounter limitations in oxygen transport as the hydrogel height (500 μm) was too low to form oxygen/transport gradients.^{[22 (link)]} Using FIJI software (NIH), the number of live and dead cells were counted to quantify cell viability. A minimum of 5 z-stacks from 5 individual hydrogels were imaged and quantified at each time point for each hydrogel formulation for the four cell lines.

Farino C.J., Pradhan S, & Slater J.H. (2021). The Influence of Ligand Density and Degradability on Hydrogel Induced Breast Cancer Dormancy and Reactivation. Advanced healthcare materials, 10(11), e2002227.

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