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.
Colibri led illumination system
Manufactured by Zeiss
Sourced in United States
The Colibri LED illumination system is a compact and versatile lighting solution designed for microscopy applications. It provides bright, uniform illumination across a wide range of magnifications. The system utilizes high-performance LED technology to deliver consistent and reliable illumination, ensuring optimal image quality for a variety of microscopy techniques.
Automatically generated - may contain errors
Lab products found in correlation
4 protocols using colibri led illumination system
1
Time-lapse Microscopy of Live Cells
2
Microscopic Imaging of Fluorescent Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cultures for microscopy were grown overnight at 23 °C, to optimise folding and maturation of the fluorophores, in relevant dropout or drug selectable media plus 100 mg/L adenine. Cells were imaged either directly from overnight cultures, or by embedding the cells into 0.7% low melting point agarose, with a Zeiss Axioimager Z2 microscope (Carl Zeiss AG, Germany), using a 63 × 1.4NA oil immersion lens. Illumination was from a Zeiss Colibri LED illumination system (GFP = 470 nm, YFP = 505 nm, RFP = 590 nm, CFP = 445 nm and Azurite = 385 nm). Bright field contrast was enhanced with differential interference contrast (DIC) prisms. The resulting light was captured using a Hamamatsu Flash 4 Lte. CMOS camera containing a FL-400 sensor with 6.5 μm pixels, binned 2 × 2. Exposure times were set to ensure that pixels were not saturated and were identical between control and experimental images. All images were acquired using either Axiovision or Zen software from Zeiss, then images to be shown in the publication were prepared using Icy [6 (link)] and Illustrator (Adobe).
3
Cellular Localization of FP-tagged Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
We used epifluorescence microscopy to determine the cellular localisation of FP-tagged proteins. Cells were grown to log-phase and mixed with 0.7% low melting point agarose in growth medium on glass microscope slides. A Zeiss Axioimager Z2 microscope (Carl Zeiss AG, Germany) was used to image cells using a 63x 1.4NA apochromatic oil immersion lens. Fluorescence was excited using a Zeiss Colibri LED illumination system (GFP=470 nm, YFP=505 nm, and RFP=590 nm) and differential interference contrast (DIC) prisms were used to enhance the contrast in bright field. The emitted light was captured using a Hamamatsu Flash 4.0 Lte CMOS camera with FL-400 (6.5 µm pixels, binned 2x2). Exposure times were adjusted to ensure that signal intensities remained below saturation and remained identical between control and experimental images. Images were acquired using the Zen software (Zeiss) and analysed and prepared using the Icy BioImage Analysis unit (version 2.0.3.0) [87 (link)] and FIJI/imageJ [88 (link)].
4
Fluorescence Microscopy of Bacterial Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Bacteria were incubated in 5 mL of fresh LB medium and harvested at an optical density at 600 nm (OD600) of 0.4 for samples in exponential phase or an OD600 of 2.0 for samples in stationary phase. After washing twice with M63B1 medium, cells corresponding to 1 mL of the bacterial culture were pelleted by centrifugation and resuspended into 0.1 mL of M63B1 medium for exponential samples or 1 mL of the medium for stationary samples. Ten-microliter aliquots of the cell suspension were immobilized on glass slides previously covered with freshly made M63B1 medium-0.8% agarose pads. Cells were observed using a Zeiss Definite focus fluorescence microscope (Carl Zeiss, Oberkochen, Germany), equipped with an oil-immersion lens objective microscope (Pln-Apo 63×/1.4 oil Ph3). GFP or mCherry fluorescence was excited with a Zeiss Colibri LED illumination system, and the fluorescence signal was detected with Zeiss FS38 HE (Carl Zeiss) or Semrock HcRed (Semrock, Rochester, NY, USA) filters. GFP and mCherry fluorescence images were taken at 1,000- and 2,000-ms exposure, respectively. Image processing was performed using ImageJ and Adobe Photoshop. For each tested strain, the subcellular localization patterns of 50 randomly selected bacteria were evaluated four times (a total of 200 cells), and each frequency was expressed as a percentile.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!