OMC News

July 2011:

New BSL2 microscopy facility at MRB.

Live Cell Confocal and TIRF Microscope.

OMC-TIRF

banner

MRB-BSL2, Prairie Technologies/Nikon MultiModal Live cell imaging system, Swept Field confocal (SFC), TIRF and wide field:

SFC-TIRF
SFC-launch
6-line laser launch with dual AOTF-driven output. (405, 440, 488, 514, 561 and 640 nm)
SFC-incubator Motorized stage, laser-based autofocus and stage-top incubator with CO2 and humidity control

This microscope is a new addition to the core. It is intended to be an ideal solution for high resolution time lapse experiments in live cells and is capable of long term studies (many hrs to days) including the use of multi-chamber slides. This setup is a true multidimensional imaging system that combines confocal, Total Internal reflection fluorescence (TIRF) and wide-field fluorescence microscopy with fully automated image capture capabilities. The setup is configured with a Nikon Ti inverted fully motorized epifluorescence microscope equipped with Nikon’s ‘perfect focus ™‘ laser based auto-focus system and a high resolution x-y motorized stage. The perfect focus system is essential for focus stability during long term incubation experiments and, in combination with the motorized stage and specialized software control, allows for automatic imaging though multiple locations including multiwell format dishes. Wide field fluorescence illumination is provided by an Exfo Excyte Metal Halide illuminator. For multicolor fluorescence the microscope is equipped with a motorized filter turret and automated switching between epifluorescence and transmission modes. Transmission modalities available are bright-field, phase contrast and DIC. For confocal imaging a Prairie Technologies Swept Field Confocal (SFC) scanhead is installed in the left port of the microscope. The SFC system uses a row of 32 pinholes (with different sizes available) and a piezo electric translator to scan the sample at high resolution optical true confocal sectioning at speeds several times faster than conventional confocals. In addition, the SFC is also equipped with a series of confocal slits for even faster optical sectioning when full confocal resolution is not absolutely required. The microscope is also equipped with a Nikon motorized TIRF illuminator which allows for precise switching between laser lines and automated switching between TIRF and epifluorescence modes. TIRF imaging is performed using a specialized TIRF plan apochromat 60x 1.49 NA lens allowing the imaging of events occurring at the region of the plasma membrane directly in contact with the glass coverslip. TIRF microscopy is a well established technique used for high resolution studies of fusion and signaling events at the plasma membrane. The high resolution of TIRF microscopy in the z axis (100-200 nm) is far better than the axial resolution of confocal or multiphoton microscopy. The SFC and TIRF illuminators share a single laser launch equipped with 5 lasers capable of excitation lines at 405, 440, 457, 488, 514, 561 and 633 nm. These wavelengths allow the use of this microscope with virtually any dye available except some of the new extreme far red dyes. Automatic selection of wavelength and excitation power is provided by a Neos AOFT. Image acquisition is performed by two Photometric cameras, a QuantEM EMCCD back-thin illuminated cameras and a CoolSnap ES camera. The QuantEM camera has a 512x512 detector array of 16 µm pixels and is designed for really high sensitive and fast image capture, while the CoolSnap camera has an array of 1300x1040 6.4 µm pixels and is intended for high resolution imaging of brighter samples. The QuantEM camera is used for SFC experiments in live cells while the CoolSnap camera is dedicated to wide field studies in live or fixed samples such as immunofluorescence stains. Incubation is provided by a Tokai Hit stage top closed-style incubator with CO2 and humidity control, which can accept a variety of samples including small petri dishes, chambered coverslips and histological slides. Software control is provided by Prairie View (Prairie Technologies) and Nikon Elements (Nikon).

Instrumentation List