Microscopy Update
Our NIH-NCRR Shared Instrumentation Grant has been funded! (NIH NCRR grant S10 RR023432 01) Installation and training have been completed: the new DVRT is up and running with live-cell environental chamber, lasers and TIRF modules available.
Microscopy Services
We specialize in live-cell, real-time fluorescence microscopy using the Applied Precision DeltaVision™, image restoration system and a Bio-Rad 1024 laser scanning confocal microscope. We advise on protocols with hints and tricks acquired through much experience.
- Deltavision Image Restoration: High-resolution wide-field fluorescence microscopy in up to four colors at a time and a transmitted light as reference
- Time-lapse: Controled environment of temperature, humidity, carbon dioxide to optimize long term imaging of living cells
- Colocalization: Image analysis to determine fluorescence overlap in time and place
- DIC: differential interference contrast, can be combined with fluorescence
- FRAP: Laser-based Flourescent Recovery after Photobleaching
- FLIP: Laser-based Fluorescent Loss after Photobleaching
- FRET: Fluorescence Resonance Energy Transfer, aka (sensitized) Forster Resonance Energy Transfer
- TIRF: Total Internal Resonance Flourescence (background & theory)
- CLSM: Confocal laser scanning (fluorescence) microscopy
Service Rates
- Deltavision Image Restoration Microscopy: $110 per hour
- Confocal Microscopy: $75 per hour
- Professional Consultation: $60 per hour
- Live cell imaging on DVRT by Approved User: $60 per hour
- Bitplane Image Processing: $35 per hour
- Fixed cell imaging on Deltavision ("old") by Approved User: $25 per hour
- Off hours, live-cell, time-lapse experiments: $20 per hour
We have a wide range of services to accomodate the needs and research budget of your lab: from "do-it-yourself", where we train you to do most of the prep and analysis, to full-service where you give us your sample and we give you ready to publish data. You can save money by doing extended live-cell imaging during off hours. Talk to us about it.
Image Restoration (Deconvolution) Microscopy
The Applied Precision DeltaVision™ Image Restoration System, aka "Deconvolution Microscope" is designed for high-resolution fluorescence microscopy of living cells.
The Core DV system is a high resolution wide field microscope for acquiring images of live or fixed cells and tissues. The basic system is based on the Olympus IX71 microscope with differential interference contrast (DIC) transmitted light and 3 lasers (402nm, 488nm, 532nm) for photoactivation (PA), photobleaching (FRAP) and total internal reflection (TIRF). The light source is a short arc Xenon lamp and the excitation filter wheel has 10 places while the emission has 6. Both wheels are exchangeable for fluorescent protein-specific or user-specific filters. More info about standard and live cell filter sets here. The camera is a Nikon Coolpix HQ for fast, sensitive acquisition. The patented stage is controlled by extremely accurate XYZ nanomotors for exact z-stack and point-visiting functions.
The stage area is enclosed by a Weatherstation™ environment cabinet providing controlled heat, moisture and carbon dioxide to facilitate live cell imaging over very short ( a few minutes) to very long (days) times. Deconvolution, using an algorithm based on Sadat and Agard's work, provides image restoration processes to the appropriate data sets. Additional software (Imaris™ from Bitplane) is available for in depth analysis of the images for many variables such as intensity, direction and speed vectors, structure volume and colocalization.
DeltaVision CoreDV with WeatherStation and QLT laser accessories
Within one year the core facility will add fluorescent lifetime imaging (FLIM) to the abilities of this system. The image processing can be done on either the core computer or an additional stand alone. Up to three users can be accessing the image data bank at one time initially and more licenses can be purchased to facilitate user access to their data.
Confocal Microscopy
We perform confocal microscopy on the Oregon Hearing Research Center's BioRad 1024 ES™. This is a laser scanning confocal imaging system attached to an inverted Nikon Eclipse TE300 microscope. The acquisition system (LaserSharp) uses a krypton/argon laser with excitation lines at 488, 568 and 647 nm. Simultaneous or sequential detection can be used with one, two or three 8-bit channels (PMT detectors).
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The confocal scanning microscope allows optical sectioning of samples up to 150 microns thick. Optical sections can be processed to render two and three dimensional images with low out of focus bacground fluorescence and resolution that is near the theoretical limit. |
Help with Experimental Design and Image Analysis
Consultation is free for introduction to general image concepts, such as specimen preparation, appropriate acquisition parameters and image manipulation for publication. Consultation on a per hour basis is offered for data deconvolution or analysis and such things as image conversion to your desired format (e.g. TIFF, QuickTime, AVI and MOV movies). We provide training of authorized clients to offer the systems. Contact the microscopist for the requirements. There is a charge for the training, but these trained users can operate the system at a reduced, do-it-yourself, rate.
The MMI Core offers analysis tools through our software programs, SoftWoRx Suite and Bitplane Imaris, of iterative image restoration (deconvolution), 4D volume rendering, isosurface, tracking, measurements, colocalization (Pearson's Coefficients or the Costes Method) and ion or FRET calculations through channel ratioing.
We recommend at least an initial conversation with us to verify your needs and the capabilities of the systems available to us. Empirical determination is usually best but we have made note of things that did NOT work or can't for some reason.
Certified Users
Share-holder Labs with heavy demand for high-resolution live-cell microscopy can designate a person to be trained and certified to operate the deconvolution system on their own. This saves money and improves throughput. Coordination with the microscopist is necessary. Contact Tom Keller for information.
Your Data
Please bear in mind that immunofluorescense is extremely rewarding but does require attention to all of the parameters relavent to your specimen. We can help you to be successful. Note also, high-resolution and time-lapse microscopy generate lots of good data, but you need to move it to your own storage location or device. We do not provide data storage services at this time.
Tools
- Bioptech microenvironment accessories for live-cell work
- Bitplane Imaris software is the best available for analysis on 3 and 4 dimensional volumes for colocalization ratios and direction vectors.
- SoftWoRx Suite from Applied Precision for iterative, constrained deconvolution (image restoration)
| Objectives | (interface) Type† | Microscopy Method |
|---|---|---|
| 10X | (air) Planapo | confocal |
| 20X | (air) Planapo | confocal and deconvolution |
| 40X | (oil) Planapo | confocal and deconvolution |
| 40X | (oil) S-Fluoro | deconvolution |
| 60X | (oil) Planapo | confocal and deconvolution |
| 60X | (water) Planapo | deconvolution |
| 60X | (air) Planapo | deconvolution |
| 100X | (oil) Planapo | confocal and deconvolution |
| 100X | (oil) S-Fluoro | deconvolution |
| †Planapo lenses are quartz and do not pass UV light >340 nm. | ||
| S-Fluor lenses are fluorite and do pass 340 nm light. We use them for applications such as Ca+2 ratio imaging | ||
Scheduling
CalendarWe've set up a WebEvent™ Microscopy Calendar to handle scheduling. We try to accommodate your lab's needs (grant deadlines, meetings, etc.), but communication is essential. See the contact information on the right for email addresses and phone numbers.
The most convenient way to schedule microscopy work is the on-line Microscopy Calendar, or email Aurelie