Confocal Microscopes

Zeiss LSM 510-Meta Laser Scanning Confocal Microscopes

There are two Zeiss LSM 510-Meta Laser Scanning Confocal Microscopes in the MIN Core Facility. The first, in room W5 A/Z has a META multi-spectral analyzer and a dual laser scanning system. Wavelengths available are 458, 488, 514, 543 and 633nm. It does not have a stage incubator and is used primarily for fixed samples unless the operator provides an incubation chamber. Zeiss imaging software is used to capture and process images. The microscope  in room B318 Microbiology also has a META multi-spectral analyzer and a dual laser scanning system. Wavelengths available are 405, 458, 488, 514, 543 and 633nm. This instrument has a stage incubator for live cell imaging and Zeiss imaging software to capture and process images.

The Zeiss LSM 510 Meta Laser Scanning Confocal Microscope in Biomedical Sciences was purchased in 2004 and has been maintained by Zeiss. The microscope provides capabilities for confocal analysis of cultured cells, tissue sections and other biological specimens. However, since an incubation chamber or heated stage is not available, this system is not well suited for imaging at other than room temperature. Live cell imaging at physiological temperatures is best performed with the Olympus spinning disk confocal system or the Zeiss LSM 510 Meta in room B318 Microbiology.

The 510 Meta is well equipped for basic quantitative fluorescence microscopy. Laser lines for excitation are 405 , 458, 488, 514, 543, and 633 nm. The meta spectral detector provides emission wavelength spectra as well as serving as a variable bandpass filter. Two additional filter-based PMT detectors allow for the simultaneous separation of multiple fluorescent signals. The DIC equipped objectives include a 10X, 0.5NA, 40X, 1.3NA and 63X, 1.4 NA. The system has been used for both FRAP and FRET studies. The facility is overseen by Associate Professor Shane Hentges, a member of the Microscope Imaging Network steering committee, which sets the policies on use, training, fee structure and fee-for-service access.

Access/Sign-up Process

Access is limited to individuals that have passed a basic class in operation and care of the system. Time reservation will be via the link provided below. Only those individuals approved by the Director will be given access to the reservation calendar.

Cost

The user fees for this instrument are as follows:

  • Hourly use for trained users: $40/h

  • Fee-for-service: $25/h (in addition to hourly use fee)

  • Note that this service is pending hiring of the required personnel but is currently available on the Zeiss LSM 510 Meta confocal microscope in room B318 Microbiology.

  • Computer log-in will keep track of use time and billing will be done bi-monthly by electronic billing of pre-registered account numbers.

  • Costs for users external to CSU will be at $80/h plus $50/h fee-for-service.


Olympus IX81 Inverted Confocal Laser Scanning Microscope

The Olympus IX81 Inverted Confocal Laser Scanning Microscope is located in the Research Innovation Center, Foothills Campus has widefield fluorescence visualization, brightfield and differential interference contrast. Four channel spectral and filter-based confocal scanning includes a transmitted light channel for DIC. This feature provides sensitivity and flexibility for imaging with and without fluorescent fluorophores and will allow users to image up to four compatible fluorophores simultaneously.  Objectives on this system are 10X, 20X, 40X, and 100X. This instrument also has a second set of galvanometer mirrors allowing simultaneous photo-activation/stimulation/bleaching during imaging experiments. The second scan unit is a laser point scanner with the capacity to target defined regions using a Tornado (helical) scan. This feature allows activation and depletion of fluorescent probes for analysis of temporal and spatial activity in live samples. A spectral detector based PMT system allows for specific bandwidth selection in co-localization experiments plus spectral scanning and separation of overlapping fluorophores. The FV1000 also has a motorized, programmable stage that can be used for multipoint time-lapse and mosaic imaging. The four lasers with AOTF control allow six different wavelengths to be selected: 405 nm, 457 nm, 488 nm, 514 nm, 559 nm and 635 nm. Other features include multimode imaging in six dimensions including: XYZ, time, wavelength, mosaic scanning, and multipoint high content imaging.  Fluorescence Recovery after Photobleaching (FRAP) and Photoactivation (PA) is facilitated by the easy software wizard with any laser line. The system also includes co-localization software module for complete analysis including scattergrams and statistical data ready for easy export.

The FV1000 is an Olympus IX81 Inverted Microscope with widefield fluorescence visualization, brightfield and differential interference contrast. The system allows for four channel spectral and filter-based confocal scanning including transmitted light channel for DIC. This feature provides sensitivity and flexibility for imaging with and without fluorescent fluorophores and will allow users to image up to four compatible fluorophores simultaneously. We have 10X, 20X, 40X, and 100X objectives for use on this microscope.

A key feature of this instrument that was not available from other manufacturers is the incorporation of a second set of galvanometer mirrors for simultaneous photo-activation/stimulation/bleaching during imaging experiments. The second scan unit is a laser point scanner with the capacity to target defined regions using a Tornado (helical) scan. This feature allows activation and depletion of fluorescent probes for analysis of temporal and spatial activity in live samples.

This spectral detector based PMT system allows for specific bandwidth selection in colocalization experiments plus spectral scanning and separation of overlapping fluorophores. The FV1000 also has a motorized, programmable stage that can be used for multipoint timelapse and mosaic imaging. The six line laser combiner with AOTF control includes the following lines that will provide us the flexibility required to look at multiple fluorophores:

405                                                                 DAPI, Hoescht, Alexa 405

mArgon (457nm, 488nm, 514nm)              CFP, GFP, FITC, Alexa 488, YFP

559                                                                 TRITC, CY3, mCherry, DsRed

635                                                                 Cy5, Alexa 647

Other features that this model includes multimode imaging in six dimensions including: XYZ, time, wavelength, mosaic scanning, and multipoint high content imaging.The Fluorescence Recovery after Photobleaching (FRAP) and Photoactivation (PA) that is facilitated by the easy software wizard with any laser line. The system also includes colocalization software module for complete analysis including scattergrams and statistical data ready for easy export.

For access to this microscope, contact the facility supervisors, Prof. Brad Borlee: brad.borlee@colostate.edu and Grace Borlee: grace.borlee@colostate.edu.


Olympus IX81 Inverted Spinning Disk Confocal Microscope

The Olympus IX81 Inverted Spinning Disk Confocal (SDC) Microscope has environmental control chamber with CO2 control for live cell work, an X,Y, piezo Z stage for rapid image stack acquisition across many predetermined fields (4 D imaging), a CSU 22 head with quad dichroic and additional emission filter wheel to eliminate spectral crossover, four high power diode lasers (405 nm, 488 nm, 561 nm and 647 nm) with rapid (microsecond) switcher and a phasor holographic photobleaching/photoactivation/photoconversion system for intracellular molecular dynamic measurements. Phasor can also be used for ion uncaging and other applications. System has differential interference contrast (DIC) optics with 10, 20, 40, 60 and 100X objectives, built in correction for spherical aberration for all objectives, and a wide field Xenon light source.  A cascade II EMCCD camera (confocal imaging) and a Photometrics HQ camera (wide field imaging) are both integrated for image capture using Slidebook software.  Laser selection and power are software controlled.  System is connected to RAID terabyte storage. 

Data Storage

Users must bring their own drives for downloading image files as soon as the experiments are complete. Storage capacity for a longer time may be purchased on the Terabyte drive system.

Cost

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; Maximum charge of $120 per 24 h period.

Scheduling

To schedule time on the SDC, please contact the facility supervisor, Dr. Barbara Bernstein, Senior Research Scientist, Tel: 1-0430.

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