Zeiss ELYRA PS.1 Super Resolution Microscope
The UNMC Advanced Microscopy Core Facility has recently obtained a Zeiss ELYRA PS.1 Super-resolution Microscope for SIM, TIRF, PALM and dSTORM microscopy, along with a workstation for processing data.
This system will allow 3-D SIM and provide ~100 nm resolution on the lateral axis with about ~300 nm resolution axially, about 3-fold better than confocal resolution. There are 4 laser lines, several lenses and two separate cameras (for SIM and PALM/dSTORM, respectively). PALM will allow resolution to ~10-20 nm on the x axis and 50 nm axially, whereas dSTORM will provide similar resolution with endogenous proteins using antibody-coupled dyes. 2-channel PALM and STORM is also available for 3-D imaging. Read policies regarding use of the SIM.
Practical Aspects for SIM Imaging
- Prepare samples in the same manner as for confocal microscopy, using coverslips and mounting on cover glasses.
- Coverslips should always be no. 1.5 thickness, and high quality coverslips that have minimal variation in thickness can be ordered from Zeiss (cat. # 474030-9000-000).
- Zeiss recommends using Vectashield Mounting Medium from Vector Labs (Item # H-1000), which has a refractive index of 1.44.
- The system has 4 laser lines: 405 nm (blue), 488 nm (green), 561 nm (red), 643 nm (far-red), and virtually any fluorophore that works for confocal should work for SIM.
- As a rule of thumb, if the signal looks good by confocal, there is a good chance that SIM will be informative.
- If the signal is weak, noisy, diffuse, cytoplasmic, or not localized to any structure by confocal, there is no point wasting time or money on SIM.
For additional information, please talk to core facility manager Janice Taylor or Jimmy Talaska.
PALM and dSTORM Super-resolution are not ready for “roll-out” and information will be forthcoming in the next few months.
Please credit: the "Nebraska Research Initiative Grant" and the Nebraska Center for Cellular Signaling NIH CoBRE "P30-GM-103697" when citing use of this instrument.
Zeiss LSM 800 with Airyscan
The Airyscan represents new technology, that manages to obtain resolution beyond that of confocal light microscopy (about 1.7 fold higher to about ~180 nm in the x-y plane—see images below), but not as good as with SIM (~100 nm). The way this works is that the pinhole, which is typically adjusted on the confocal to allow in light (at the expense of resolution), is left open and all the light is collected by a series of detectors. Through engineering ‘tricks’ that are similar to deconvolution, the signals are subsequently reassigned from the 32 detectors and this increases the signal-to-noise ratio—and thus the resolution.
The Airyscan is very easy to use, and not too different from the Zeiss LSM 710, and I would encourage users to talk to Janice and/or Jimmy for a brief training session so that you can take advantage of this new equipment.
Please credit: Center for Cellular Signaling CoBRE "NIH P30GM106397" when citing use of this instrument.
Zeiss 710 Confocal Laser Scanning Microscope
The Zeiss 710 Confocal Laser Scanning Microscope equipped with 4 lasers: a Blue Diode 405 nm; an Argon Laser 458/488/514nm; a DPSS 561nm; and a HENe 633nm. Nomarski optics allow superimposition of signals from the fluorophores overlaid with a DIC image of cells. Zeiss software is available for reconstruction of 3-D images, presentation of image "galleries", Z-sectioning, Orthogonal sectioning, time series, etc. Zeiss software also includes data analysis software for the quantitation of the fluorescence, analysis of colocalization, etc. Images can be directly stored on CDs or DVDs. The Zeiss Axio observer inverted microscope used by the system has 10x, 20x, 40x oil, 63x oil and a Z-axis motor, as well as an xy stage motor. All functions are computer automated with Zeiss Zen software. The large incubator with heat, CO2 and humidity is available for live cell studies.
Please credit: the National Institutes of Health Shared Instrument Grant program "NIH S10-RR-027301" when citing use of this instrument.
Cell Discoverer 7
The Zeiss Cell Discoverer 7 is a fully integrated high-end automated live cell imaging system. The system has the capability to use Petri dishes, chamber slides, multiwell plates, plastic or glass, thin or thick vessel bottoms, low skirt or high skirt plates. Automatic sample recognition detects all relevant vessel features while loading your sample. Autocorr objectives adjusts the correction ring of the objective to compensate for spherical aberrations. Find Focus automatically places your sample in focus and Definite Focus keeps it there.
Equipment for processing fixed and frozen tissues/cells includes a Shandon 3 Cytospin.