Module 3: Microscopy and Digital Imaging

The Microscopy and Digital Imaging Module provides equipment, infra-structural support, expertise and technical assistance to vision scientists to enable detailed, quantitative imaging of cells and tissues. This state-of-the-art imaging facility includes several pieces of high-end image acquisition and analysis hardware, and the expertise needed to support and enhance ongoing research and explore new research questions posed by vision scientists. Facility operations include:
1) Maintaining and servicing the equipment
2) Managing usage of the equipment
3) Educating investigators on the capabilities of the equipment
4) Advising investigators on the design of imaging experiments for vision research projects
5) Providing training on the proper usage of the equipment
6) Helping researchers collect, process and analyze high quality microscopy data for both current and future applications

The specific services and technologies provided within the Microscopic and Digital Imaging Facility are:

Cell and tissue processing, labeling and immunocytochemistry
Light and fluorescent microscopy
Laser scanning confocal microscopy
Multiphoton microscopy and time-lapse imaging
In vivo confocal microscopy
Quantitative morphometry and image analysis

Overall, the Microscopy and Digital Imaging Module supports a range of imaging technologies for participating investigators, and facilitates easy access to equipment, training and technical support that expand experimental capabilities, open up new research directions, and result in more cost-effective, time-efficient and rigorous imaging studies. This module also facilitates collaborative studies using these cutting edge imaging techniques between vision scientists at UTSW and other institutions in the region.

Facilities

The MDI Module laboratories are in the Department of Ophthalmology on the 7th floor of the Florence (E) Building. The Facility is housed in 4 dedicated rooms (all on the same hallway) having a total of 616 square feet of space, plus a portion of the Director’s laboratory which provides an additional 150 square feet. The labs contain resources for sample preparation, as well as several pieces of high-end imaging hardware. Desk space for the Technical Support staff is in an adjacent room (E7.228).

Tissue Processing and Immunocytochemistry (E7.232, 150 ft²)

Equipment includes a Leica Kryostat CM3050S for sectioning of frozen tissue samples, a Leica M28 Dissecting microscope for processing of tissue samples, a Revco Ultralow Freezer and other conventional refrigerators and freezers for storage of samples and labeling reagents, and a fume hood for formaldehyde fixed tissues.
Light and Fluorescence Microscopy Lab (E7.212A, 80 ft²)

This room houses a Leica DMI 3000B inverted microscope equipped with standard brightfield, epifluorescent (FITC, TRITC, Texas Red, DAPI, CY-5), phase and DIC capabilities. The system has a Hamamatsu Flash 4.0 camera for high sensitivity, high resolution image capture of fluorescent samples, and a Leica color camera (DFC295) for histological imaging. A Leica LAS AF Imaging Workstation is used for image capture and storage.
Laser Scanning Confocal Microscopy Lab (E7.226B, 168 ft²)

This lab houses the Leica SP8 Confocal Microscope. This system has 3 lasers for visible light excitation, a UV laser, and 3 photodetectors for simultaneous detection of 3 separate fluorophores. Sequential scanning allows separate excitation and detection of triple or quadruple labeled samples without cross-talk between fluorochromes. The system is equipped for both standard and high speed resonant scanning modes. Resonant scanning gives a higher fluorescence quantum yield and reduced photobleaching. The microscope also has an environmental chamber suitable for long-term time-lapse imaging, and a software controllable scanning stage which can be used to create wide field montages from high magnification scans. In 2015, a multiphoton imaging system was added which consists of a Coherent Chameleon Vision II, ultrafast Ti:Sapphire laser with precompensation which is controlled directly through the Leica software. To optimize signal detection during multiphoton imaging, the microscope has two non-descanned detectors. In 2018, the Leica Super-resolution module was incorporated. This provides seamless real-time deconvolution using a parallel GPU processor, and can provide 120nm resolution.
In Vivo Confocal Microscopy Lab (E7.230, 168 ft²)

This lab houses an HRT II confocal microscope with Rostock Corneal Module, which is dedicated for in vivo confocal microscopy of rabbits and mice. This instrument has hardware modifications developed by Dr. Petroll’s group that allow quantitative confocal microscopy through-focusing. The room also has bench space for animal handling and monitoring.
Image Analysis Lab (E7.230A, 200 ft²)

In addition to those integrated with the microscopes, three high-end PC Workstations and two dedicated color printers are available to users. Each PC runs the latest version of MetaMorph, Image J and Imaris software for quantitative morphometric analysis and 3-D reconstruction of digital images obtained with the various microscope systems. Specialized programs developed “in-house” by Dr. Petroll for visualization and analysis of 3-D confocal microcopy are also installed on each computer workstation, as is the off-line version of the Leica LAS imaging software and Lightning deconvolution software. Matlab software is available for the development of custom image analysis programs. The suite of programs provides a full range of image processing, reconstruction, visualization and analysis functions. In addition, Adobe Photoshop can be used for cropping and labeling of images for publication.

Services Provided

Immunocytochemistry

This service provides assistance with the sectioning of tissue, and the processing and staining of cultured cells, frozen sections, or en bloc tissue samples using antigen specific antibodies or other fluorescent labels.

Digital images from fluorescently labeled tissue sections or cultured cell monolayers are collected using the Leica DMI 3000B microscope.
Laser Scanning Confocal Microscopy

This service is used to more precisely co-localize two or more structures that are labeled using specific sub-cellular probes or antibodies. Up to four fluorescent probes can be imaged either simultaneously or sequentially depending on the excitation and emission characteristics of the probes. Additionally, z-series of optical sections can be collected to determine the localization and interrelationships between structures in 3-dimensional space. The Leica SP8 also has the capability to perform more advanced techniques such as fluorescence resonance energy transfer (FRET) and fluorescence recovery after photobleaching (FRAP) to more precisely co-localize two or more proteins. The super-resolution module provides real-time deconvolution using a parallel GPU processor, and can provide 120nm resolution.
Multiphoton Microscopy and Time-Lapse Imaging

This service is designed to address questions requiring either deep tissue penetration or the temporal study of live cells and tissues. Multiphoton confocal microscopy provides non-invasive tissue sectioning deeper within the tissue than is available with standard conventional or visible confocal microscopic techniques so that thicker tissues can be analyzed. The system is also used for second harmonic generation (SHG) imaging, which allows imaging of collagen structure within a tissue without an exogenous label. Since multiphoton confocal microscopy uses infrared light for excitation, there is less cellular phototoxicity associated with imaging of live cells. Using the environmental chamber, time-lapse multiphoton imaging can be used to investigate the spatial organization or temporal distribution of fluorescently tagged proteins within living cells and tissues. Furthermore, the resonant scanner on the SP8 provides higher fluorescence quantum yield and reduced photobleaching, and is thus ideally suited for live cell imaging.
In Vivo Confocal Microscopy

This service provides support for in vivo confocal imaging of the cornea in animal models. Example applications include temporal studies of corneal wound healing following injury or surgery, assessment and monitoring of infectious keratitis and epithelial toxicity, and analyzing changes in the density and morphology of sub-basal nerves. Quantitative measurement of corneal sublayer thickness and stromal backscattering can also be assessed using custom software developed in Dr. Petroll’s lab.
Quantitative Morphometry and Digital lmage Analysis

This service provides access to 3 PC workstations with a suite of programs that can be used for quantitative morphometric analysis and 3-dimensional reconstruction of digital images obtained with the various microscope systems. The facility provides training and assistance on using the various software programs and selecting the appropriate analysis procedures for their images.