The Time-Lapse 1 widefield fluorescence microscope is enclosed in a polycarbonate incubator system and has an active gas mixer and temperature controller and is suitable for long term live cell imaging such as wound healing , tube formation . Wound healing can be conveniently quantitated in Image Pro Premier.
The Time-Lapse 2 widefield fluorescence microscope can be equipped with a heating stage insert for 35mm plates, humidifier and 5% CO2 gas cylinder and is suitable for short term live cell imaging.
Cell migration is essential in a number of clinical processes such as embryonic development, tissue regeneration, immune response, and pathological conditions. The wound healing (scratch) assay is an easy, low cost method to measure cell migration in vitro. A scratch is created in a monolayer capturing phase contrast images at regular intervals, typically at our Time-Lapse 1 widefield fluorescence microscope supporting multi well dishes with different treatments. The wound healing application of the Image Pro Premier software uses interactive thresholding to process and analyze the images (single assay or batch) iteratively and deliver the data as an excel file.
Protocol for acquisition setup
Protocol for quantitation
The in vitro formation of capillary-like tubes by endothelial cells on a basement membrane matrix is a powerful in vitro method to screen for angiogenic regulators. Endothelial cells can be seeded in multi well dishes with different treatments and imaged at a few minute intervals overnight at our Time-Lapse 1 widefield fluorescence microscope .
Calcium imaging can be used to optically probe intracellular calcium using calcium indicators, fused proteins composed of a calcium sensing element coupled with autofluorescent molecules such as circularly permuted eGFP, eYFP, eCFP that alter their fluorescent intensity as a result of conformational changes in the calcium sensing element.
The Time-Lapse 2 widefield fluorescence microscope can be equipped with a heating stage insert for 35mm plates, humidifier and 5% CO2 gas cylinder and is suitable for short term live cell imaging. It has a high speed Axiocam HS camera. The inverted LSM 510 confocal system and the upright LSM 700 confocal system can also use the heating stage insert.
Shinnawi R, Huber I, Maizels L, Shaheen N, Gepstein A, Arbel G, Tijsen Anke J, Gepstein L. Monitoring Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes with Genetically Encoded Calcium and Voltage Fluorescent Reporters. Stem Cell Reports. 2015;5(4):582-96.
Fluorescence recovery after photobleaching (FRAP) technique follows the mobility of fluorescent molecules within a living cell membrane by photodestroying a small region and plotting over time the repopulation into the bleached area, revealing information about protein interaction partners, organelle continuity, mitochondrial network and protein trafficking.
Ben-Shachar D, Suss-Toby E, Robicsek O. Analysis of Mitochondrial Network by Imaging: Proof of Technique in Schizophrenia. Mitochondrial Medicine: Springer New York; 2015. p. 425-39.
Förster (or fluorescence) resonance energy transfer (FRET) in optical microscopy permits determination of the proximity between two molecules within up to 10 nanometers. Typically, an excited chromophore emits a virtual photon that is absorbed by a receiving chromophore where the radius of interaction is much smaller than the wavelength of the emitted light. FRET can be used to measure distances between domains in a single protein and provide information about protein conformation.
One common pair of fluorophores for biological use is a cyan fluorescent protein (CFP) – yellow fluorescent protein (YFP) pair. Our inverted LSM 510 confocal system is equipped with a multiline Argon laser (458, 477, 488, 514nm) which enables differential excitation of CFP and YFP and suitable filters at the detectors. Our Time-Lapse 2 widefield fluorescence microscope has (optionally) a CFP/YFP FRET filterset (Zeiss FSet 48) and a dual CFP/YFP filterset (Zeiss FSet 59HE) at the colibri LED lines 455 and 505.
Fluorescent proteins photoconvert when following exposure to UV they switch to different excitation and emission wavelengths. Photoconversion facilitates monitoring of protein dynamics in transgenic cells.