Genomics
Microscopy
Inverted Zeiss LSM 880 Airyscan Confocal Training
Training will generally be held on the 2nd and 4th Wednesday of each month at 1pm.
Q: What is this about?
A: Opportunity to participate in a training session on the Inverted Zeiss LSM 880 AiryScan, Confocal Microscope (Super resolution image capture capability).
Q: When will the next sessions be held?
A: Generally held on Second and Fourth Wednesday every month @ 1:00 pm. Please contact Kishor.Kunwar@unthsc.edu to schedule the training.
Q: How do I book my training?
A: Request Training through iLab.
♦ Login with your EUID and Password
♦ Go to Request Services tab
♦ Go to Request Training section and Initiate Request
♦ Fill out the form, complete the payment information and submit
Q: How much will it cost?
A: $110 which will cover participation in ~2.5 hours of Demonstration Training and 3 hours of MCF staff monitored Hands-on use imaging your own samples (schedule with MCF staff within 2 months of initial Demonstration training).
Q: What will the training format be?
A: Training is divided into two parts, Demonstration, and hands-on use.
♦ Demonstration: In person: one session of ~2.5 hours.
♦ Hands-on use: In-person: one session lasting 3 hours or two sessions of 1.5 hours each.
Q: What if I am not comfortable to use the microscope on my own after initial training?
A: Please request further hands-on training. Within first 2 months after demonstration training, you will be charged $35/hour.
Training Format
Training will take place in at least two sessions, the initial Demonstration session followed by Hands-on Use.
♦ Demonstration– Core staff will operate the microscope and trainees will observe and interact with staff.
♦ Hands-on Use: After completing Demonstration, Trainees will schedule with MCF staff to operate the microscope with core staff guidance using their own samples.
Questions?
Contact:
Kishor Kunwar, MCF Research Associate, at Kishor.Kunwar@unthsc.edu
Sharad Shrestha, Core Labs, Director at Sharad.Shrestha@untshc.edu
Zeiss LSM 880 Confocal with AiryScan and AiryScan Fast Mode
This system is based on a fully motorized Axio Observer 7 Inverted Microscope with Motorized Scanning Stage with Piezo Z and includes the following:
LSM Detectors (Spectral detection of 4 channels with two GaAsP detectors)
- 2 multi-anode and 1 GaAsP PMT
- Includes additional Airyscan GaAsP detector for super-resolution and Fast Mode
- Transmitted light detector T-PMT for detection of DIC, IR-DIC with laser illumination
Incubator Enclosure – Maintains user set CO2 level, temperature & humidity around the sample stage & objectives. For live cell experiments.
Installed Lasers
- 405 nm Diode Laser 11.5 mW
- Argon Laser: 458/488/514 nm 15.4 mW
- 561 nm DPSS Laser 13 mW
- 633 nm HeNe Laser 2.3 mW
Objectives
- 5x/0.16 EC Plan-Neofluar 5x/0.16 WD=18.5
- 10x/.30 EC Plan-Neofluar 10x/0.30 WD=5.2
- 10X/0.5 W Plan-Apochromat 10x/0.5 WD=3.7mm
- 20x/0.8 Plan-Apochromat 20x/ 0.8 WD = 0.55
- 40x/1.1 W Corr FWD=0.62mm at CG=0.17mm
- 60X/0.7 Olympus LUC Plan FLN WD=1.5 – 2.2mm
- 63x/1.4 Oil Plan-Apochromat 63x/1.40 Oil
| Available Laser/Channel | Compatible Alexa Dyes (In Order of compatibility with laser) |
| 405 |
|
| 458 |
|
| 488 |
|
| 514 |
|
| 561 |
|
| 633 |
|
Microscopy Core Facility Recommended Alexa Dyes for IHC
3 Color Imaging laser and Dye combination
Lasers: 405, 488, 561 Fluorophores: Alexa 405/ DAPI/ Hoechst | Alexa 488 | Alexa 546/Alexa 568
405, 488, 633 Fluorophores: Alexa 405/ DAPI/ Hoechst | Alexa 488 | Alexa 635/Alexa 647
488, 561, 633 Alexa 488 | Alexa 546/Alexa 568 | Alexa 635/Alexa 647
4 Color Imaging laser and Dye combination
Lasers: 405, 488, 561, 633
Fluorophores: Alexa 405/ DAPI/ Hoechst | Alexa 488 | Alexa 546/Alexa 568 | Alexa 633/Alexa 635
Coverglass:
Always use coverglass No. 1.5 (~0.17mm)
Live Cell Imaging tools:
Chamber slides as well as Cover-glass bottom plates can be used for confocal imaging.
Fixed cell ICC can be done by growing cells on coverglass and mounting the coverglass on the slide or growing cells on the slide (Chamberslides) and mounting with coverglass or any other plate or slides where live cell imaging can be done.
Live cell imaging will however require,
cover glass chamber slide Lab-Tek™ II, Part numbers 155409 (8 wells), 155382 (4 wells), 155379 (2 wells), and 155360 (1 well) or
cover glass bottom plate MatTek, Part number P06G-1.5-14-F (6 wells), P12G-1.5-14-F (12 wells), P24G-1.5-13-F (24 wells),
Cover glass bottom dish MatTek Corporation 35 mm round dishes with #1.5 coverslip bottoms: Part number P35G-1.5-14-C
IHC Sample preparation guidelines from ThermoFisher Sci
https://www.thermofisher.com/us/en/home/life-science/cell-analysis/cellular-imaging/ihc.html
Cover Glass
All our microscopes are tuned to image optimally with No. 1.5 (~170um) coverglass, always use the No. 1.5 coverglass
- VWR Micro Cover Glasses, No. 1.5:
- Warner Instruments , No. 1.5
- Part numbers 64-0721(22 x 22 mm), 64-0716 (22 x 30 mm), 64-0717 (22 x 40 mm)
- For High resolution Airyscan imaging use: Warner Instruments High Tolerance #1.5 (0.17 ± 0.01 mm) Round Coverslips: Part numbers 64-0732 (12 mm), 64-0733 (15 mm), 64-0734 (18 mm), and 64-0735 (25 mm).
Mounting Media:
Open-source Image Processing and Analysis Software
Image J https://imagej.nih.gov/ij/download.html
FIJI https://imagej.net/Fiji/Downloads
ICY http://icy.bioimageanalysis.org/download/
Cell Profiler https://cellprofiler.org/
QuPath https://qupath.github.io/
BioImageXD https://www.bioimagexd.net/index.php/download
KNIME https://www.knime.com/downloads/download-knime
Napari https://napari.org/
Bio-Image processing & Analysis Learning Resources
Collection of Bio-Image analysis videos by UNTHSC Microscopy core.
https://www.youtube.com/channel/UCDMBgxTxZLpDO2FMaZ36d9w/playlists
Image Processing and Analysis for Life Scientists by EPFL
Digital Images, Colors, Operating on Images, Filtering, Image Segmentation, ROI, Colors, and dimensionality reduction and ImageJ Macro Programming.
https://www.edx.org/course/image-processing-and-analysis-for-life-scientists
Bioimage Analysis Course: The Life Cycle of an Image Data Set
Introduction to bioimage analysis, microscopy digital image handling, life cycle of an image data set, from acquisition to analysis
https://www.ibiology.org/online-biology-courses/bioimage-analysis-course
Quantifying-microscopy-images-top-10-tips-for-image-acquisition/
Quantitative Imaging for Colocalization Analysis
https://www.biodip.de/w/images/f/fa/QuantitativeColocAnalysis-10-2011.pdf
Analyzing fluorescence microscopy images with ImageJsub
https://petebankhead.gitbooks.io/imagej-intro/content/
“Digital Image Processing: An Algorithmic Introduction Using Java” by Wilhelm Burger and Mark J. Burge
https://link.springer.com/book/10.1007%2F978-1-4471-6684-9
The Center for Open Bioimage Analysis (COBA)
https://openbioimageanalysis.org/
Avoiding Twisted Pixels: Ethical Guidelines for the Appropriate Use and Manipulation of Scientific Digital Images
The international network of cutting-edge bioimaging facilities and communities: (Exchange of Experience, Training, Shadowing, Working Groups)
Sample Preparation guidelines from Thermo Fisher Sci
https://www.thermofisher.com/us/en/home/life-science/cell-analysis/cellular-imaging/ihc.html
Flow-Cytometry
- Cytek Aurora – User guide
- Amnis Imagestream Mk II – User guide
- Channel Guide
- Resources @ Luminex website
- BD LSR II – User guide
- Channel Guide
- Resources @ BD website
- Sony Cell Sorter – User guide
