Module 3: Microscopy and Animal Phenotyping

The Microscopy and Animal Phenotyping (MAP) Module provides equipment, infrastructural support, expertise and technical assistance to vision scientists to enable detailed, quantitative imaging of cells and tissues, as well as equipment and support for structural and functional assessment of the visual system in vivo. This advanced imaging facility includes several pieces of high-end hardware for image and data collection, dedicated analysis workstations, and the expertise needed to support and enhance ongoing research and explore new research questions posed by vision scientists.

MAP Module 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 and visual function data for both current and future applications

The specific services and technologies provided within the MAP Module are:

• Cell and tissue processing, labeling and immunocytochemistry
• Light and fluorescent microscopy
• Laser scanning confocal microscopy
• Multiphoton microscopy and time-lapse imaging
• Slit lamp imaging and in vivo confocal microscopy
• Optical coherence tomography (OCT), angiography, autofluorescence imaging (BAF), infrared imaging (IR), and fundus imaging
• Electroretinography (ERG/PERG/VEP)
• Behavioral testing (optokinetic response, looming object, and pupillometry)
• Quantitative image and data analysis

The MAP Module supports a range of imaging technologies for participating investigators and facilitates easy access to equipment, training, and technical support that will expand experimental capabilities, open up new research directions, and result in more cost-effective, time-efficient, and rigorous imaging studies. An additional goal is to facilitate collaborative studies using these advanced imaging techniques among vision scientists at UTSW and with other outside institutions.

Facilities and Resources

The MAP Module laboratories are in the Department of Ophthalmology on the 7th floor of the Florence (E) Building. The Facility is housed in nine dedicated rooms with a total of 1,561 square feet of space, plus a portion of the Director’s laboratory that provides an additional 150 square feet. Desk space for the technical support staff is in an adjacent room (E7.228). The labs contain resources for sample preparation, animal anesthesia and recovery, as well as several pieces of high-end imaging hardware. All animal imaging and phenotyping space is approved by the Institutional Animal Care and Use Committee (IACUC) at UT Southwestern, which is AALAC accredited.

• Tissue processing and immunocytochemistry lab (E7.232, 150 ft²)

  Equipment includes a Leica Cryostat CM3050S for sectioning of frozen tissue samples, a Leica M28 Dissecting microscope for processing of tissue samples, a Leica VT1000 S vibrating blade microtome (Vibratome) for generating thicker sections of organoids and other tissues, 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 three lasers for visible light excitation, a UV laser, and three photodetectors for simultaneous detection of three 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. The microscope also has a multiphoton imaging system 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.

• Anterior segment imaging 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. It also houses a Haag Streit BM 900 slit lamp biomicroscope equipped with an LED light source for clinical characterization of the cornea, optical surface, and lids. The slit lamp has two magnifications (10x and 16x) and a yellow barrier filter for fluorescein applications. The room also has bench space for animal handling and monitoring.

• Optical coherence tomography, scanning laser ophthalmoscopy, and behavioral response lab (E7.128 and E7.128A, 587 ft²)

  This lab houses the Heidelberg Engineering Spectralis HRA + OCT scanning laser ophthalmoscope. This is a multimodal imaging platform that allows for optical coherence tomography (OCT) of both the posterior segment as well as the anterior segment (an upgrade purchased by the Department of Ophthalmology). The Spectralis also has infrared reflectance and blue light autofluorescence fundus imaging, and angiography. The Department of Ophthalmology has multiple lenses for the Spectralis including normal and widefield imaging, and a platform designed for its use with animals that is connected to an isoflurane anesthesia machine. The Spectralis is connected to a computer with software for full image acquisition and analysis. This lab also houses the CerebralMechanics Inc. OptoMotry system for rodents, with multiple platform sizes for use with rodents, including both rats and mice. The OptoMotry equipment is connected to a computer dedicated to the machine, carrying the software required for running the programs and data analysis. For additional behavioral testing of visual processing, we are also purchasing a Sarl iMetronic Automated Looming Test for Mice and a NeurOptics A-2000 Pupillometer System. The lab also contains bench space for set-up, housing animal cages in preparation for experiments, as well as a heating source for animal recovery after anesthesia.

• Electroretinography (ERG) lab (E7.134, 103 ft²)

  The ERG lab is an IACUC-approved dark room space with red lighting for investigators to use while working in the room. It contains a dark adaptation cabinet for housing up to nine animal cages overnight, bench space for animal anesthesia and pupil dilation, and a heating pad/space for animal recovery. The ERG room houses the Pheonix MICRON Ganzfeld ERG, an electrophysiology tool optimized for the retinal response of rodent photoreceptors and allowing for specific excitation of S-Cones, M-Cones, and rods. The MICRON is connected to its own computer equipped with the ERG software. This room also houses the Diagnosys LLC Celeris Rodent ERG system connected to its own computer with software. The Celeris ERG system is equipped with two standard stimulators for scotopic and photopic ERG, as well as a pattern ERG stimulator and electrodes for recording visually evoked potentials (VEPs). A small bench-top isoflurane machine and oxygen tank holder is present in this lab for the use of either injectable or gas anesthetics.

• Fundus imaging and optical coherence tomography lab (E7.212B, 105 ft²)

  An additional IACUC-approved dark room space with red lighting and a large dark adaptation cabinet that can hold up to 18 rodent cages is available to investigators. This lab houses the Pheonix MICRON IV system, an ophthalmic imaging system for in vivo eye and eye-brain research using small animals. This equipment allows for retina resolutions below three microns, and performs color fundus and fluorescent imaging with brightfield, angiography and fluorescent modalities. The MICRON IV is also equipped with OCT imaging. This equipment is connected to its own computer and software for data acquisition and analysis. The lab also contains bench space for animal preparation, anesthesia, and recovery.

• Image analysis lab (E7.230A, 200 ft²)

  In addition to those integrated with the microscopes and animal phenotyping equipment, three high-end PC Workstations and two dedicated color printers will be 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. They also contain the Celeris ERG and Spectralis HRA + OCT software. 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. This 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 and Methodologies

The MAP Module provides vision scientists access to high-end imaging and animal phenotyping equipment that would otherwise be difficult, inconvenient, or costly to use or is not otherwise available. It will also provide training, guidance, and hands-on assistance with these technologies and approaches.

• Immunocytochemistry

  This service will provide 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.

• Light and fluorescence microscopy

  Digital images from fluorescently labeled tissue sections or cultured cell monolayers will be collected using the Leica DMI 3000B microscope.

• Laser scanning confocal microscopy

  This service will be 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 can be collected to determine the localization and interrelationships between structures in 3-dimensional space. The Leica SP8 can also be used for fluorescence resonance energy transfer (FRET) and fluorescence recovery after photobleaching (FRAP) to more precisely co-localize two or more proteins. The super-resolution module uses real-time deconvolution 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 conventional or visible confocal microscopic techniques, so that thicker tissues can be analyzed. The system can also be 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 by Dr. Petroll’s lab.

• Scanning laser ophthalmoscopy and optical coherence tomography

  This service provides support for SLO modalities that include infrared imaging, blue autofluorescence imaging, angiography, and OCT imaging of the anterior and posterior eye. Widefield and ultra-widefield lenses are available for imaging, and quantitative measurements of anterior or posterior eye structures can be assessed using the Spectralis software. Example applications include autofluorescence imaging to assess the localization of green fluorescent-tagged gene therapy viral vectors after injection into the eye, or quantification of the total retinal thickness to assess for degeneration over time in the same animal. This service allows for the assessment of retinal vasculature. It provides insight into the progression of degeneration, as well as the ability to capture regions of rescue in models for therapeutic treatments.

• Optokinetic response testing

  This service provides for the testing of the optokinetic response and visual acuity in rodents. Users can adjust both contrast and frequencies of gradients during testing to assess the visual pathway between the eye and the brain.

• Pupillometry

  This service will provide a binocular dual-camera system to measure both eyes at once for small rodents. It can work for multiple pupil sizes and allows for multi-chromatic four-color light stimulus (white, green, blue, and red). Automatic tracking and pupil detection studies pupil latency, diameter during light stimulation, constriction and dilation velocity, as well as recovery time. This is a newer piece of equipment for the department based on interest from incoming faculty and investigators in the O’Donnell Brain Institute at UT Southwestern who want to meet top standards for behavioral testing of the visual processing pathway between the retina and the brain.

• Automated looming test

  This automated service will provide real-time locomotor activity of the rodent, as well as freezing behavior. This is also a new piece of equipment for the Department based on interest from incoming faculty and investigators in the O’Donnell Brain Institute that want to meet top standards for behavioral testing of the visual processing pathway between the retina and the brain.

• Electroretinography

  This service provides the ability to test the function of the visual processing pathway, both overall and specific to individual cells in this pathway. The equipment has the capability to assess individual photoreceptors (S-cones, M-cones, and rods), as well as overall rod and cone photoreceptor function. Scotopic and photopic ERG examine retinal signaling after light sensing by the photoreceptors, and pattern stimulators are available to examine the function of the retinal ganglion cells, those that signal to the optic nerve. Both flash and pattern visually evoked potentials can be tested to assess the function of the optic nerve.

• Fundus imaging and optical coherence tomography

  This service provides a high-resolution option for fundus imaging coupled with OCT. This equipment has modalities for various fluorescent channels, broadening the capabilities of the scanning laser ophthalmoscope to include excitations at different wavelengths and brightfield fundus imaging. This allows for the assessment of drusen-like deposits for animal models of macular degeneration, and to track multiple drugs or viral vectors that are fluorescently tagged after injection into the eye. Additionally, this service can be used to cause light-induced retinal degeneration for the study of retinal responses to injury, using a protocol developed by Rafael Ufret-Vincenty, M.D., in the Department of Ophthalmology.

• Quantitative image and data analysis

  This service provides access to 3 high end 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. These PC workstations are also equipped with ERG and scanning laser ophthalmoscopy/OCT software. The Facility will provide training and assistance on using the various software programs and selecting the appropriate analysis procedures.