Tg(LysC:GCaMP3) larvae were imaged at 2 days post fertilization (dpf) for bacterial infection experiments and all other transgenic larvae were imaged at 3 dpf. For microscopy, larvae were anesthetized in 0.016% Tricaine (MS-222) and immobilized in 1% low melting point agarose and imaged with a SiMView microscope (light-sheet microscopy) or a Zeiss axio observer Z1, or Nikon TE-2000U. Additional experimental details are provided in the Supplemental Experimental Procedures .
Axio observer z1
Manufactured by Nikon
Sourced in France
The Axio Observer Z1 is a high-performance inverted microscope designed for advanced live-cell imaging and analysis. It features a stable, vibration-free platform and a wide range of optical configurations to accommodate various experimental requirements.
Automatically generated - may contain errors
Lab products found in correlation
Em ccd luca r camera, by Oxford Instruments (2 mentions)
Te2000 u, by Nikon (1 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 594 secondary antibody, by Thermo Fisher Scientific (1 mentions)
Paraformaldehyde (pfa), by Thermo Fisher Scientific (1 mentions)
Normal goat serum (ngs), by Vector Laboratories (1 mentions)
Yo pro 1, by Thermo Fisher Scientific (1 mentions)
Eclipse te2000 e, by Nikon (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 conjugate, by Thermo Fisher Scientific (1 mentions)
Fluorsave, by Merck Group (1 mentions)
G actin, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 647 phalloidin f actin, by Thermo Fisher Scientific (1 mentions)
Eclipse ti2, by Zeiss (1 mentions)
Orca flash 4, by Hamamatsu Photonics (1 mentions)
Lsm 710, by Zeiss (1 mentions)
Zen pro, by Zeiss (1 mentions)
7 protocols using axio observer z1
1
In Vivo Bacterial Infection Imaging
2
Mitochondrial Morphology and Cell Viability
3
Quantifying F-actin and G-actin in hCOs
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F-actin and G-actin measurement was performed as described.65 D28 hCOs were dissociated into single cells using the same method used for scRNA-seq. Harvested cells were resuspended in organoid maturation medium and passed through a 40 μm filter strainer to obtain single cells. Cells then seeded at a density of 0.05 million/well on 24-well size coverslips coated with hESC qualified Matrigel. After two days in culture, cells were fixed with 4% PFA for 15 min. Permeabilization and blocking of the samples are performed as mentioned in the section on immunofluorescence microscopy. After blocking, cells were stained with antibody Alexa Fluor™ 647 Phalloidin (F-actin, ThermoFisher Scientific, Cat#A22287), Deoxyribonuclease I, Alexa Fluor™ 488 Conjugate (G-actin, ThermoFisher Scientific, Cat# D12371) and DAPI (ThermoFisher Scientific, Cat#D1306) for 2 h at room temperature. Cells were then washed thrice in 1×PBS and mounted with FluorSaveTM (Merck, Cat#345789) for imaging (Nikon, Eclipse Ti2; Zeiss, Axio Observer Z1 and Nikon, Eclipse TE2000-E). The same exposure settings were used to acquire images from WT and FEZ1-null groups. Images were analyzed with ImageJ/Fiji with quantification details in the following section. At least three independent experiments were performed.
4
Time-lapse Imaging of Cell Adaptation and Aging
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All time-lapse experiments have been performed at least two times.
Freshly thawed cells were grown overnight at various final cell densities. In the morning, log phase cells were allowed to grow a few divisions and were transferred into the microfluidic device. Cells were imaged using an inverted Zeiss Axio Observer Z1 (adaptation assay) or a Nikon Tie (aging experiments). Focus was maintained using dedicated hardware throughout the assays. Fluorescence illumination was achieved using LED light (precisExcite, CoolLed or Lumencor) and light was collected using a 100× N.A. 1.4 objective and an EM-CCD Luca-R camera (Andor; adaptation experiments) or an Hamamatsu Orca Flash 4.0 (Aging experiments).
We used automated stages in order to follow up to 20 (adaptation experiments) or 60 (aging experiments) positions in parallel over the course of the experiment. Images were acquired every 3 min (adaptation experiments) or 10 min (aging experiments).
Temperature control was achieved using custom sample holder with thermoelectric modules and an objective heater with heating resistors. Temperature control was achieved using a PID controller (5C7-195, Oven Industries).
Freshly thawed cells were grown overnight at various final cell densities. In the morning, log phase cells were allowed to grow a few divisions and were transferred into the microfluidic device. Cells were imaged using an inverted Zeiss Axio Observer Z1 (adaptation assay) or a Nikon Tie (aging experiments). Focus was maintained using dedicated hardware throughout the assays. Fluorescence illumination was achieved using LED light (precisExcite, CoolLed or Lumencor) and light was collected using a 100× N.A. 1.4 objective and an EM-CCD Luca-R camera (Andor; adaptation experiments) or an Hamamatsu Orca Flash 4.0 (Aging experiments).
We used automated stages in order to follow up to 20 (adaptation experiments) or 60 (aging experiments) positions in parallel over the course of the experiment. Images were acquired every 3 min (adaptation experiments) or 10 min (aging experiments).
Temperature control was achieved using custom sample holder with thermoelectric modules and an objective heater with heating resistors. Temperature control was achieved using a PID controller (5C7-195, Oven Industries).
5
Multimodal Confocal Imaging Protocol
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For all immunofluorescence experiments, images were acquired using ×63 oil objectives on a multimodal confocal Zeiss LSM 710 or Zeiss AxioObserver Z1 equipped with ApoTome and AxioCam MRm or NIKON eclipse A1R/Ti2. Images were processed using Fiji (Schindelin et al., 2012 (link)) and Zeiss ZEN (Carl Zeiss, version 3.4). Figures for cultured cells were arranged using QuickFigures (Mazo, 2021 (link)).
6
Inverted Microscope Wide-Field Epifluorescence Imaging
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Cells were imaged using an inverted microscope (Zeiss Axio Observer Z1, or Nikon Tie). Wide-field epifluorescence illumination was achieved using an LED light source (precisExcite, CoolLed), and light was collected using a 100× N.A. 1.4 objective and an EM-CCD Luca-R camera (Andor).
7
Immunofluorescence Imaging with ApoTome
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Image were acquired by using the ApoTome optical sectioning system (Zeiss, Paris, France) with an inverted microscope (Zeiss Axio Observer Z1) for immunofluorescent images and a Nikon microscope (type 120c) for chromogenic images. Zenpro (Zeiss) and Adobe Photoshop were used for image processing. For comparing of fluorescence images, contrast and brightness were adjusted identically. All other quantifications involved using ImageJ.
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