Support
Need Help?
If you need help with any core instrumentation, or even if you have questions about using your own lab microscopes please reach out to the OiVM Core! We are happy to help in any way we can!
You can contact us via our website here:
3D Reconstructions / Image Analysis
The OiVM Core provides 2 custom built, high-end computer workstations for 3D volume reconstruction and analysis. Each machine is a Windows 10 box configured with Fiji, Zeiss’ ZEN software, Bruker CTVox, CTVol & CTAn, and MATLAB. Workstation 1 has a license for Imaris while Workstation 2 has a license for Vision4D from Arivis. Each workstation is attached to OiVM’s 10GbE internal network for high speed data transfer from our instruments as well as BCM’s domain for quick data transfer throughout the College.
Workstations are free for use regardless of your core access. They are located just outside the core in Rm 113A of the Cullen building at BCM Main.
Workstation 1: Imaris
Imaris is a 3D rendering and analysis software package for processing confocal datasets. The strength of this software is the three dimensional quantification environment. 3D segmentation, surfaces, cell counting can all be accomplished.
Workstation 2: Arivis Vision4D
arivis Vision4D is a modular software for working with large volume multi-channel 2D, 3D and 4D images, independent of RAM. This software is fantastic for rendering and analyzing lightsheet and super-resolution datasets.
Sample Preparation
Please find some useful information on mounting/preparing samples for use on OiVM Core microscopes.
The objectives on all of our microscopes are designed to use coverslips that are precisely 170 microns thick. In the United States – this typically equates to a #1.5 coverslip.
Coverslips can be purchased through a variety of vendors and most #1.5 lots fall within 160-190 microns in thickness. If you are using low NA objectives (NA < 0.8) – the variation in thickness will not have a profound effect on overall image quality.
Using immersion optics with an NA > 1.0 will be adversely affected by these variations. If you require resolution to be better than 300nm, coverslip choice will be critical to achieving the objective’s theoretical resolution.
For high resolution confocal microscopy with immersion objectives with an NA > 0.8 (including super-resolution with Airyscan), we recommend high performance coverslips that have a ±5μm tolerance.
For tissue sections, preparation for high resolution imaging is critical. For imaging with an objective NA > 0.8, sections must be mounted to the coverslip (NOT THE SLIDE) prior to the addition of mounting media. Mounting sections to a slide followed by mounting solution and the addition of the coverslip will introduce a variable gap between the coverslip and the tissue. This gap can vary wildly depending on tissue morphology and the volume of the mounting solution used.
High NA objectives are designed to work best at the interface between the coverslip and the media/tissue. The further you move away from this interface (toward the slide) the more you increase both spherical and chromatic aberration, which will reduce your signal and resolution.
In addition, high NA objectives have a very short working distance, many < 200μm and if you artificially introduce a gap between your tissue and coverslip, you may be unable to focus the objective on your specimen.
Many investigators choose to mount sections to slides because the slides come pre-coated (or ‘charged’) to enhance adhesion. This same treatment can be applied to glass coverslips, and we have a protocol for this in the ‘Protocols’ section of our website.
For fixed tissue, we recommend using a high refractive index mounting solution combined with a strong anti-fade reagent. Matching the refractive index of the mounting medium with the fluid the objective is designed to use will maximize the resolution of the microscope.
ProLong Glass and ProLong Gold are the two we recommend for most mounting. Prolong Glass has a cured refractive index of 1.52 which is as close to oil as you can get.
These are curing mounting mediums – and they will tend to ‘flatten’ tissue when cured – so if your experiments require precise measurements of 3D morphology, use the non-curing variants – SlowFade Glass and SlowFade Gold.
Be sure to use cell culture media that has NO PHENOL RED.
Phenol Red media will increase background fluorescence for many fluorophores in the 450-620nm range.
Reference: https://link.springer.com/protocol/10.1385/1-59259-826-9:395
Information for Publications
The Optical Imaging & Vital Microscopy Core Facility is subsidized by the Advanced Technology Cores group at the Baylor College of Medicine. Scientific publications that contain images or data derived from core instrumentation or intellectual input from core staff should be acknowledged by the authors. Acknowledgements are a crucial metric for evaluating core performance and value – so please acknowledge your cores!
To acknowledge the OiVM Core facility, please find the following example statement to be included in the acknowledgement section of your publication for your convenience:
This project was supported by the Optical Imaging & Vital Microscopy Core at the Baylor College of Medicine.
If you had support from a specific staff member, please include them in the acknowledgement:
This project was supported by the Optical Imaging & Vital Microscopy Core at the Baylor College of Medicine, with the expert assistance of (Name of Staff Member).
