1 mL of T. cruzi cells expressing Cas9 and the T7 polymerase at 1×107 cells/mL density were centrifuged at 800×g for 5 min at RT. Cells were resuspended in 1 mL fresh LDNT media with 100 μM reduced L-glutathione (Sigma-Aldrich, St. Louis, MO). 125 μL of cells containing 1.25×106 cells were plated in a Lab-Tek II chambered coverglass #1.5 Borosilicate chamber (Nunc International, Rochester, NY). A cut-to-fit agarose grid was inverted onto the cells and the chamber sealed as previously described. Imaging chamber was placed into a live-cell imaging insert (Oko labs) set to 27 °C and mounted on a Zeiss Axio Observer Z1 microscope using a 20×/0.8 NA Plan Aprochromat objective lens. Cells were imaged every 10 minutes for 48 h with 30 ms exposures. In order to compensate for focus drift over long-term imaging, a Definite Focus 2 system (Carl Zeiss Microscopy) was employed. Images were captured using an ORCA-Flash 4.0 V2 sCMOS camara with Slidebook 6 microscopy software.
Definite focus 2 system
Manufactured by Zeiss
Sourced in Azerbaijan
The Definite Focus.2 system is a digital autofocus module designed for microscopy applications. It provides a stable and consistent focus during long-term imaging and time-lapse experiments. The system continuously monitors and adjusts the focus position in real-time to compensate for drift or other focus-related issues.
Automatically generated - may contain errors
Lab products found in correlation
Axio observer z1 microscope, by Zeiss (2 mentions)
Prime 95b back illuminated scientific cmos camera, by Teledyne (2 mentions)
L glutathione reduced, by Merck Group (1 mentions)
Lsm800 point scanning confocal microscope, by Zeiss (1 mentions)
Zen 2, by Zeiss (1 mentions)
Plan apo objective, by Zeiss (1 mentions)
Matlab, by MathWorks (1 mentions)
4 protocols using definite focus 2 system
1
Live-cell imaging of T. cruzi cells
2
Time-Lapse TIRF Microscopy Imaging
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Images were acquired at RT with the same Zeiss Axio Observer.Z1 microscope described above, equipped with a spinning illumination ring VectorTIRF system which includes a 405/488/561/640 quad band dichroic filter (Intelligent Imaging Innovations, Inc.—Denver, CO) and an Alpha Plan-Apochromat 100X/1.46 NA oil TIRF objective. Samples were excited using 488 nm 150mW and 647 nm 120mW lasers at 20% power. Images were acquired every 10 sec for 20 min using a Prime 95B Back Illuminated Scientific CMOS camera (Teledyne Photometrics—Tuscon, AZ) and a Definite Focus.2 system (Carl Zeiss Microscopy) for automatic focus correction.
SlideBook 6 digital microscopy software (Intelligent Imaging Innovations, Inc.) was used to manipulate the microscope and acquire images. Images were analyzed with ImageJ (National Institutes of Health—Bethesda, MD) and exported to Adobe Photoshop and Illustrator (CC 2020) for publication.
SlideBook 6 digital microscopy software (Intelligent Imaging Innovations, Inc.) was used to manipulate the microscope and acquire images. Images were analyzed with ImageJ (National Institutes of Health—Bethesda, MD) and exported to Adobe Photoshop and Illustrator (CC 2020) for publication.
3
Live Imaging of Cellular Structures
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All in vitro imaging experiments were performed using LSM800 point-scanning confocal microscope (Zeiss) equipped with 20× air immersion objective (for sphere imaging) and 63 × oil immersion Plan Apo objective (for all other experiments), as well as 405, 488, 563, and 630 nm lasers. Temperature, CO2, and humidity were controlled throughout live imaging using an automated temperature control system and a gas mixer. For time-lapse imaging, Zeiss Definite Focus.2 system was used to ensure image stabilization. Images were acquired using Zen 2 software (Zeiss) at 16-bit depth, and the acquisition settings were kept constant for the same type of experiments to aid statistical analysis.
4
Multimodal Microscopy for Microtubule Dynamics
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