Microscopy

A confocal laser scanning microscope system in a research laboratory. The microscope is enclosed in a clear protective chamber with lasers and imaging components visible inside. Next to the instrument is a workstation with two computer monitors displaying colorful microscopic images of cells and tissue samples. A keyboard, mouse, and control panel sit on the desk. The system is used for high-resolution fluorescence imaging and analysis of biological samples.
 

FAQ

   

Internal

NEXT Lab

External

LSM 880 Inverted

Unassisted

$35.00

NA

NA
 

Assisted

$65.00

$96.00

$192.00

Zeiss Axioscan 7

Unassisted

$20.00

NA

NA
 

Assisted

$40.00

$60.00

$192.00

Keyence BZ-X800

Unassisted

$0.00

NA

NA
 

Assisted

$30.00

$45.00

$90.00

Workstation

Unassisted

$10.00

$15.00

$30.00

Off-hours (7 p.m. to 7 a.m. -- Authorized Experienced Internal Users) pricing

LSM 880 Inverted

Unassisted

$25.00

Trained users have 24/7 access to Microscopy Core. Technical assistance is available on weekdays from 8 a.m. to 5 p.m.

Training is mandatory for internal users who wish to use the microscopes on their own.
 
Training on confocal will cost $110 per person and include 3 hours of demonstration and 3 hours of hands-on training.Training on Zeiss Axioscan 7(Slidescanner) can take from 2-4 hours and depends on the training objectives and costs same as assisted imaging ($40/Hr). Please contact Kishor Kunwar.
 
Training on Zeiss LSM 880 with 2-Photon can take from 2-4 hours and depends on the training objectives. Please contact Kishor Kunwar with questions or for scheduling training.
 
Inverted Zeiss LSM 880 Airyscan Confocal Training
 

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: Please contact Kishor Kunwar 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 Initial (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 two 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 two 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

Open-source image processing and analysis software

  • Fiji  : A distribution of ImageJ which includes many useful plugins contributed by the community.
  • Icy  : Used for a wide range of processing and analysis applications.
  • CellProfiler : Used for high-throughput image analysis and data mining.
  • KNIME : Used for high-throughput image analysis and data mining.
  • QuPath : Used for digital pathology and whole slide image analysis.
  • ilastik : Consists of powerful machine learning features needed to identify or classify challenging structures.
  • napari : It is a fast, interactive, multi-dimensional image viewer for Python. It is designed for browsing, annotating and analyzing large multi-dimensional images.

Bio-image processing and analysis learning resources

Bio-imaging Networking

Bio-imaging Sample Preparation 

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 and 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

Zeiss Axioscan 7 (slidescanner) 

  • 100 slide capacity magazine plus Continuous Loading capability with auto sample detection and auto focus.
  • High End Brightfield imaging with Axiocam 705 color camera and Fluorescent imaging with Axiocam 712 monochrome camera for high-resolution color acquisition
  • High Performance Objectives: 5x, 10x/.45, 20x/0.8, 40x/.95 and circular polarizer set with 20x/0.5 POL objective
  • Colibri 7 LED light source: (Excitation Wavelengths: 385nm, 430nm, 475nm, 555nm, 590nm, 630nm, 735nm) with continuous brightness adjustment for imaging of up to 7 channels
  • High Efficiency Multi-band Filter: 110 and 112 for imaging of: DAPI, GFP, Cy3, mCherry, Cy5, Cy7 and similar dyes (Alexa 488, 555, 594, 647, 730)
  • High Efficiency Single Pass Filter Sets: 38, 43, 50 and 96 for specific imaging of GFP, Cy3, Cy5 and similar dyes (Alexa 488, 555, 647)
  • Premium HP Z6 Workstation: for large data handling with Windows 10 Enterprise

Zeiss LSM 880 Confocal with 2-Photon excitation laser

This system is based on a fully motorized upright Microscope with Motorized Scanning Stage includes the following:

LSM Detectors (Spectral detection of 4 channels with two GaAsP detectors) 

  • 2 multi-anode and 1 GaAsP PMT 
  • Transmitted light detector T-PMT for detection of DIC, IR-DIC with laser illumination

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
  • 690-1040nm tunable Mai-Tai/ Spectra-physics 2 photon laser

Objectives

  • 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
  • 20X/1 clarity dipping objective
  • 40X/0.8 water dipping objective WD = 3.2mm
  • 60X/0.7 Olympus LUC Plan FLN WD=1.5 – 2.2mm

Microscopy Core houses a Zeiss LSM confocal microscope, Keyence BZ-X800 fluorescence microscope and a data analysis station. Core provides access to state-of-the-art instruments for light microscopy imaging and software for image processing and analysis. A full-time staff provides training and consultation as well as assisted imaging on a fee-for-service basis.  

Zeiss LSM 880 inverted confocal microscope with Airyscan super resolution capability is a primary microscope which is capable of simultaneous capture of four channel images using 4 laser lines which can produce 6 different laser wavelengths. It is equipped with 5 detectors, a GaAsP detector, an airyscan GaAsP detector, 2 multi anode PMT detectors and a T-PMT detector. The Airyscan feature can achieve 1.7X resolution (140nm XY, 400nm Z) and 4-8X sensitivity compared to standard confocal. Airyscan Fast is a unique feature that provides high resolution, sensitivity and speed up to 100fps that enables gentle live cell imaging with super resolution. This equipment is also equipped with definite focus, a piezo stage and a stage top live cell incubation system which maintains temperature, humidity and CO2. 

Keyence BZ-X800 fluorescence microscope is a fully motorized control system with advanced imaging and analysis functions. This inverted fluorescence microscope has a built-in darkroom and environment control system for live cell imaging. It is equipped with a high-sensitivity, cooled CCD camera that can be switched between monochrome and color imaging and supports fluorescence, brightfield and phase contrast imaging.  

Confocal Microscope is connected with 10GB Ethernet cable to an image analysis workstation which is a high-performance computer with full feature Zen Black, Zen Blue and ImageJ/FIJI, Napari and Cell profiler image analysis software installed. 

Room housing the microscope has a CO2 incubator and a refrigerator to provide users with flexibility in maintaining their live cell or tissue sample during the facility use.

The purpose of the bio-safety assessment is to identify potential hazards and hence control measures that might be needed. This is to help ensure the health and safety of the researcher and of any other person that might be affected by the work. New researchers are required to submit a bio-safety assessment form during initial consultation.

  • How can I start accessing the Microscopy Core? 
    • Email Kishor Kunwar to:
      • Schedule a training with Core staff, get trained and use microscopes by yourself.
      • Schedule assisted imaging session with Core staff, who will coordinate with you to image your sample.
  • How can I get access to microscopy instruments after hours?
    • Only trained users are authorized to operate instrument independently after hours.
  • How can I get access to data analysis software?
    • Schedule Imaging Workstation which is a HP Z6 high performance computer with software installed and located in RES111. Or you can use open source software on your own device.

Recommended Alexa Dyes for IHC/ICC
Available Laser Excitation wavelength/Channel Compatible Alexa Dyes (In order of compatibility with laser) 
405 Alexa 405 | DAPI / Hoechst
458 Alexa 430 
488 Alexa 488
514 Alexa 514 | Alexa 532
561 Alexa 546 | Alexa 555 | Alexa 568 | Alexa 594
633 Alexa 635 | Alexa 647 

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 such as 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 35 mm round dishes with #1.5 coverslip bottoms: Part number P35G-1.5-14-C.

Cover Glass

All our microscopes are tuned to image optimally with No. 1.5 (~170um) coverglass, always use the No. 1.5 coverglass.

Mounting Medium

Mounting Media with DAPI is not recommended as it adds background to the fluorescence signal of your image.

Contributions to scientific output is an important metric for Core Labs, as it enables us to obtain financial and other support to ensure continuous high standards of operations to foster innovative research.

Users are encouraged to acknowledge Microscopy Core in presentations, posters, papers, and all other publications.

For example: “We acknowledge [name of staff] in the UNT Health Microscopy Core for training and assistance on [following]”. 

When to acknowledge or provide co-authorship:

  • Include an acknowledgement any time the staff member or the Core provides services that support your research.
  • If a staff member has made a significant intellectual contribution beyond routine sample analysis, please consider co-authorship.