Director: Joanne Steinauer, BS
Anesthesiology Image Core Facility utilizes a Leica CM 1800 cryostat for sectioning tissue that will further be evaluated with general or immunohistochemical stains. This cryostat will reliably cut 5-60um frozen tissue sections for slide or free floating preparation.
Whole mount work has significant advantages in defining changes in epitope expression in large voluems of tissue such as thick brain/spinal slices, DRG and peripheral organs such asbladdder and GI. In addition to the Leica TCS SP5 Confocal System, the Anesthesiology Core Imaging Facility maintains an Olympus BX51 microscope that has been equipped with the fluorescence illuminator, BX-URA2, which incorporates a multi-cube filter turret. The aspherical lens within the lamp house allows fluorescence illumination of up to double the brightness of existing models. The fluorescence illuminator utilizes a mercury burner with the following filters:
- Omega Optical XF06 filter for blue bandpass with a 365nm wide-band exciter
- Omega Optical XF100-2 filter for green bandpass with a 475nm exciter
- Olympus U-MNG2 mirror unit with a 530-550nm excitation filter for red excitation
Leica TCS SP5 Confocal System
This True Confocal Scanner (TCS) SP5 module is equipped with dichroic beam splitters on two tunable spectral detection channels, plus a third channel of transmitted light (bright field or DIC) for simultaneous or sequential fluorescence image acquisition. The SP5 has user friendly, intuitive software for ROI (Region of Interest), sequential scanning, z-stack, and 3D visualization features such as orthogonal sectioning, 3D projection and 3D rotation. There are several tools for image improvement and quantification analysis. Provisions for live cell imaging are available.
The system has a DMI 6000 inverted microscope with a fully motorized stage. This high precision stage allows the user to perform tile scans (aka stitching) and define sample positions in a mark & find mode for recall during live or time-lapse experiments. Broadband fluorescence filters for green and red dyes as well as Differential Interference Contrast (DIC) for transmitted light are available for visual inspection and focusing of samples.
Using the Leica Confocal
To gain access to the Leica TCS SP5 Confocal, each user must complete a 1-4 hr training requirement.
This training includes a basic description of the microscope and the controls, demonstration of microscope operation using fluorescence of a Leica standard sample, and basic operation of the confocal software. Users with previous confocal experience need only demonstrate proficiency to gain access.
Click here to download more information on the Leica confocal, including training & scheduling use.