Fluorescence Microscopy/Image Analysis Center
The Center is housed in two rooms in MRB, one for macroscopic imaging and one for microscopic imaging.
Schedule Time: Confocal
Access data on NAS1: Web Mac OS X On Windows, map network drive to \\fmic-nas1.bmb.colostate.edu\home
E-mail Userlist: Confocal
Macroscopic Imaging Facility Supervisors: Laurie Minamide, Senior Research Associate; Tel: 1-5531 (CCD camera); Lynn Taylor, Senior Research Associate; Tel. 1-0583 (Odyssey IR Scanner); Dinaida Lopez, Research Associate; Tel. 1-5017 (Storm Imager)
The macroscopic image analysis equipment is housed in rooms 210 and 214 MRB. In room 214 are two networked PC computers, one of which controls the acquisition of images from a 16 bit (64,000 fold dynamic range and >80% quantum efficiency) Photometrics CH250 cooled CCD camera attached to a cabinet for image acquisition from chemiluminescent blots (dark mode), from a UV transilluminator, or from transmitted or overhead white light. This cabinet also has a filter wheel in front of the camera lens with four barrier filters for enhancing contrast or for fluorescence imaging. Total Lab software is available in a network version for analysis of densities from digital images. The second computer controls an Odyssey infrared dual laser scanning for fluorescence detection of proteins on blots and gels. This system permits use of samples (western blots or gels) stained with two different fluorescent probes for precise two color overlays. Secondary antibodies for western blots need to be those for 600 to 800 nm excitation. In Room 210 is a Storm phosphorimager/fluorescence detector for quantitative radiographic analysis of gels/blots and for chemifluorescence detection of proteins on western blots.
Microscope Imaging Facility Supervisor: Barbara Bernstein, Senior Research Scientist; Tel: 1-0430
The fluorescence microscopes in this Microscope Imaging Center are part of the CSU Microscope Imaging Network
The Microscope Imaging Center is housed in room 224 MRB. In the front room, separated by surround black curtains that allow independent illumination control of each area, are two Nikon Diaphot inverted phase/epifluorescence microscopes, one of which is equipped with a stage incubation system and less sensitive camera used mainly for phase contrast studies of cell behavior (motility assays). The second microscope is on a vibration isolation table and has a color CCD camera (Roper CoolSnap cf) for imaging stained samples (or tissue sections) which can also be operated in a more sensitive b/w mode for fluorescence image capture. Both microscopes are interfaced to networked computers running Metamorph V7.1 for image acquisition and analysis. A third Nikon Diaphot phase/DIC/epifluorescence microscope is available in the very back room of the center (accessed through the confocal room). This microscope has a stage incubator a Coolsnap ES 20 mHz transfer rate digital camera, a computer controlled x,y,z stage, 6-place excitation filter wheel, and brightfield and fluorescence shutters with manual control overrides. All the Nikon Diaphot microscopes have a wide range of filter cubes (near UV to near IR), stage adapters, and oil immersion and air objectives for phase, fluorescence, and DIC (4x-100x). Besides a custom-built plexiglass enclosure for stage heating, there is a Peltier heater/cooler and a Medical Systems temperature controller for live cell microscopy. Available for microinjection work are a Camden micropipette puller, a Narishige micromanipulator, and a Medical Systems microspritzer for directional application of guidance cues. A side illumination unit for dark field viewing microscope slides is available, especially designed for in situ hybridization with radiolabelled probes and silver grain counting.
Spinning Disk Confocal Microscope for Live Cell Imaging
The FMIAC has a spinning disk confocal microscope for live cell imaging.
Microscope:
Base: The base is a fully automated Olympus IX81 with DIC and fluorescence optics. A computer controlled turret contains a cube with polarizing filter for DIC, a Quad cube for DAPI, FITC, TxRed, and Cy5, a cube for argon laser photobleaching (488 nm), a cube for Hg illumination and photoactivation (405 nm), a cube for FURA ratio imaging (350/380 excitation) and an open position for spinning disk confocal imaging. The automated turret allows for the acquiring of DIC images along with combinations of fluorescence images. The six objectives mounted on the microscope are: 4x Fluorite. 10X UPlan Fluorite, 20X UPlan Fluorite, 40X Oil UApo340/DIC (for FURA), 60X PlanApo 1.42na DIC, 100X PlanApo 1.4na DIC. All oil objectives are designed for use with #1.5 coverslips (nominal 0.17 mm).
Stage: The ASI computer controlled XY Piezo Z stage has excellent return (within a fraction of a micron across a large XY distance). Up to 65000 different stage positions can be recorded, but for most uses 20 or fewer are selected for multiple imaging series. The Piezo Z control allows precise and reproducible Z stacks (max of 100 μm) to be obtained over long periods of imaging.
Incubator: A custom made incubation chamber surrounds the entire microscope head, including the objectives, to keep them all within 0.1° C. The incubator should be turned on at least 4 h before (and preferably overnight) imaging animal cells at 37° C. A humidified chamber for holding 35 mm dishes in a 95% air/5% CO2 atmosphere is also available. For DIC optics, the cell holding chamber being used should have an exclusively glass optical path since DIC imaging is degraded when the polarized light goes through plastic.
Wide Field Microscopy (left side of microscope):
Illumination: The microscope has an external xenon light source and a liquid light guide for delivering coherent illumination for equal excitation of samples at any depth. Kohler Illumination, which is bright in one plane but falls off out of the plane of focus, is used from the microscope’s internal source. The xenon source has a dual filter wheel set up for fluorescence excitation selection and neutral density filter selection to control illumination intensity.
MID-SAC: The microscope has a spherical aberration correction system on the output to the camera which, if adjusted correctly, should give the sharpest resolution of deconvolved images that can be achieved. This device introduces a 2X magnification into the image. It can be applied to fixed samples at every plane of image acquisition, but it takes about one second for the MID-SAC to reset for each focal plane and thus this is not useful for rapid image acquisition studies. However, the MID-SAC can be set just once at the midpoint of the image stack and then the entire stack can be captured over time without requiring any additional adjustments. The images, especially at the extremes of the stack may be slightly less well resolved after deconvolution than the center planes, but you can collect Z-stacks at a much faster rate (rate is determined by exposure time because piezo drive move stage to new position within microsconds and exposures are generally 10 or more milliseconds. The 4D deconvolution software allows an image stack to be displayed within 5-10 seconds.
Camera: The camera is the CoolSnap HQ with a 1392 x 1040 imaging array in a 6.45×6.45 μm pixel, 12 bit digitization with flexible binning. Full chip image transfer rates are 10 frames per second, but rates are 19/ sec using 512×512 array and 30/ sec using 256×256 at 1×1 binning. Maximum rates are 56 frames per second using 4×4 binning at a 256×256 pixel array. The QE is above 50% from about 400 nm to almost 700 nm and above 60% from 480 to 620 nm.
Spinning Disk Microscopy (right side of microscope)
Yokogawa Spinning Disk head: The CSU-22 spinning disk head operates at user selected speeds up to 5500 rpm and allows for rapid image acquisition. A custom multipass filter set allows for selection of either single or double laser lines.
Dualview: Between the spinning disk head and the camera is a dualview beam splitter that allows simultaneous acquisition of two images excited by different laser lines on two halves of the camera chip. These can be displayed independently or in an overlay mode. For full chip imaging the dualview can be easily removed from the imaging path with a slider that pulls up the mirrors.
Laser Launch: Current lines available from the diode laser launch, which rapidly switches between lasers with an acousto-optical tunable filter (AOTF), are 445 (CFP), 473 (FITC/GFP), 561 (RFP) and 658 (Cy5).
Camera: A Cascade II EMCCD 16 bit camera with a back thinned chip that has over 90% QE from 480 to 700 nm is installed. This camera has 1024x 1024 imaging array on 13 x 13 μm pixels. This camera has dual amplifiers for optimal high speed/high sensitivity (non-linear) or for optimal wide dynamic range (quantitative linear imaging).
Mosaic Laser Ablation/Photoactivation System: The mosaic digital diaphragm system uses thousands of adjustable mirrors to reflect a light beam simultaneously onto single or multiple user selected regions of interest on the image. These regions are identified by drawing around the spots of interest on the image screen.
Argon laser: a 488 nm 325 mW laser provides sufficient power to photobleach spots of GFP in about 100 ms while simultaneously imaging using the spinning disk system with the 473 laser line. Thus very rapid recovery rates (in the tens or hundreds of ms) can be measured.
Mercury light source: A mercury light source can replace the laser input into the Mosaic system and is required for photoactivation at 405 nm utilizing the same mirror system and on screen selection of the region(s) of interest. Utilizing filters inserted into the Hg light source, this source can be used for photobleaching other fluors that are not photobleached by the 488 nm laser. By focusing the Hg lamp in the center of the field, sufficient power can be delivered by this lamp to give good photobleaching of smaller regions of interest in 1-2 s. Photobleaching larger regions of interest will take longer so fluorescein/GFP tagged molecules should be used when short half lives of FRAP are anticipated.
Software
The image acquisition and analysis software is SlideBook 4.1, which drives all automated components of the microscope, laser line selection, mosaic system and cameras. The SlideBook has a fast Fourier transform (FFT) deconvolution package for making 4D movies of the image stacks over time. It also has particle tracking and many other types of image processing capabilities. An off-line processing computer running SlideBook and Metamorph is available in the FM/IAC. Individual software licenses are $1000. Data can also be transferred as tif files and analyzed off-line using MetaMorph software.
Data Storage
Users must bring their own portable 250-500 Gbyte drives for downloading image files as soon as the experiments are complete. These drives are now available for less than $100 each at electronics stores.
Fees For Use of Macroscopic Imaging Equipment
1. Costs of CCD camera imaging is $3.50/ hour with 1 h minimum charge.
2. Odyssey costs are billed at $235/month divided by hours of use, averaged over 6 months. Typical hourly costs are between $25-30.
3. Storm costs are billed at $450/month divided by hours of use, averaged over two month periods. Typical hourly costs range from $30-70.
Fees For Use of Microscope Facility
Costs of Nikon Microscopes are billed at $3.50 per hour (min use is 1 h).
For use of the spinning disk confocal microscope, two methods of billing are available. Infrequent users can pay hourly costs from the following scale: 1 h or less: $40/h; Second hour: $20; Third hour: $10; Fourth and additional hours: $5 to a maximum charge of $120 per 24 h period. Frequent users can pay an annual upfront charge of $2,500 for “unlimited use.” Charges will be prorated at $210 per month with annual billing due in advance on or before June 30 for the following year.