MDRC investigators and their staff are provided with consultation either prior to or concurrent with training. Advice includes but is not limited to immunological staining of fixed cells/tissues; fluorescent-based dyes/proteins/sensors for live cell imaging; confocal/brightfield/epi-fluorescence microscopy; specialized optical techniques; processing and visualization/presentation of microscopic images; and quantification and analysis of morphometric features. The majority of consultation is handled by either the Core Director or the Laboratory Director. Both actively conduct research in the field of diabetes and its complications, which provides depth of experience for consultation. If the needs of the investigators are outside the expertise of the core, they will be directed to the appropriate experts within the UM community.
Consultation and Advice on Microscopy and Immunohistochemistry
Confocal microscopy is the most widely needed imaging service. The core has the following confocal microscopes: an Olympus Fluoview 500, a Leica TCS SP5 Confocal Microscope, and a Nikon A1 Confocal Microscope. Each system has individual strengths that allow investigators the ability to optimize acquisition over a variety of imaging configurations. The Laboratory Director directs investigators to the confocal system that best suits their imaging needs. Following conventional immunofluorescence to validate staining, sections will be analyzed on one of the available confocal microscopes either by Core personnel, or more generally, by users after training. All users will be provided four free hours of instruction.
Image analysis services include quantitative morphometric analysis of images and preparation of publication quality images and slides. Investigators can use the Core PC workstations on an hourly recharge or this service can be provided by the core personnel for additional fees. The Core has AutoQuant software that specializes in image restoration and includes a deconvolution module for producing high resolution images by using cutting-edge algorithms to perform image restoration by removing out-of-focus haze, blur, noise and other problems from both 3D and 2D images. AutoQuant also includes alignment software that will correct for any drifts or shifts in images that are acquired in time or z-series data sets. Quantitative analysis of static images can be performed using MetaMorph or ImageJ software on a PC for analysis of area, size, shape, distance, and relative position of defined cellular and subcellular structures. Particle counting can be performed and expressed per cell area or membrane length. Quantitative analysis including 3D reconstruction, quantification of fluorescence intensity, etc., can also be performed with either Volocity or Imaris software and carried out on the core’s high-end 64-bit PC workstations. Preparation of publication quality figures and montages can be done on the PC workstations using Photoshop, Prism, Illustrator, or PowerPoint software. Instruction in the use of these programs is available from the Laboratory Director.
Widefield Light Microscopy
Investigators have access to widefield light microscopes in their laboratories for routine brightfield and epi-fluorescent microscopy, the core maintains an inverted Nikon Diaphot 200 that is available for imaging and acquisition of standard blue, green red and far-red fluorescent signals with a sensitive CCD camera. The microscope is equipped with additional specialized excitation and emission filters to allow users to capture fluorescence based images of transfected green fluorescent protein (GFP) chimeric molecules, eCFP and eYFP signals for Förster (or Fluorescence) Resonance Energy Transfer (FRET), or ratiometric fura-2 signals for calcium imaging. Image acquisition and analysis are done with Compix SimplePCI, MetaMorph or MetaFluor software.
Live Cell Microscopy
Initial consultation with the Core Director will determine whether the study is better performed on the confocal or wide-field fluorescent microscope. High resolution work, work on thick specimens or studies requiring high speed time resolution will be performed on one of the confocal microscopes. The core has a number of specialized chambers and devices to regulate temperature and CO2 in order to maintain proper conditions for live cell microscopy. The core has experience to date using fura-2, fluo3, calcium green and Magfura dyes for Ca 2+, SBFI for Na +, BCECF for H+, SPQ for Cl-, lucifer yellow and lissarhodamine as gap junction tracers, and eCFP, eGFP, eYFP, DSRed, mCherry fluorescent proteins as markers for transfected cells. Other probes are available for potential sensing, labeling of mitochondria (MitroTracker and Bacmam MitoGFP/RFP) and following endocytosis. Most projects in living cells have a developmental component and require continued consultation with core personnel. All instruments can be set up to vary the frequency of image collection which affects both the size of data files and bleaching of probes.
Over the last 10 years, more investigators have begun using advanced imaging techniques such as FRET, fluorescence recovery after photobleaching (FRAP), fluorescent loss in photobleaching (FLIP), photoactivation and uncaging of specialized fluorophores or fluorescent proteins. Most of these techniques require the use of one of the laser scanning confocal microscopy systems. The Leica SP5 and Nikon A1 have specialized wizards or software protocols to simplify image acquisition. FRET work requires a large developmental component and so this is not offered as a routine service. The Core will provide instrumentation and training in its use. At present, the Core is able to monitor energy transfer from CFP to YFP. Other types of FRET will require purchase of appropriate filters by the investigator or jointly with the core if there is general interest. The MIAC continues to offer investigators the ability to collect and analyze FRET data from the wide-field fluorescent and confocal microscopes. The confocal is used primarily to perform acceptor (YFP) bleach protocols to measure and analyze FRET data and the wide-field system is used to perform stoichiometric FRET analysis.
Remember to cite the Michigan Diabetes Research Center (MDRC) in publications as follows:
“The project described was supported by Grant Number P30DK020572 (MDRC) from the National Institute of Diabetes and Digestive and Kidney Diseases”