Lab Services

Core A: SBI


The Montgomery Laboratory includes areas for accessioning, processing and staining specimens for histology and immunohistochemistry.  There is slide storage and paraffin block storage space in the laboratory.  The laboratory includes an AO dissecting microscope, a new (2015) VIP Miles/Sakura tissue processor, Shandon embedding center, two ThermoShandon microtomes, Sakura Tissue Tek DRS automatic stainer, Surgipath slide and block labeler, Fischer and Lipshaw slide warmers, Fisher Thermix stirrer, Emerson microwave oven, refrigerator, Olympus 2PT macrophotography center, Weiscor 7520 cytocentrifuge, and two enclosed hoods with air, vacuum, and gas nozzles. The sign-out room, immediately adjacent to the Director’s office, contains an Olympus BHTU microscope with eyepieces for up to 5 coincident observers. An Olympus BX50 photomicroscope with an Olympus PM10SP 35 mm, an Olympus Camedia 7.7 megapixel dye sublimation printer, and Dell Intel Xeon computer are located in the Director’s office. During the last cycle, the laboratory has instructed other laboratories (Nickerson lab for example) on tissue preparation and sectioning. These sections are then stained with the automatic stainer in the Montgomery lab, thus making the actual number of experimental samples in histology artifactually low. 

Confocal Scanning Microscopy

There are now two confocal scanning laser microscopes available and both of these instruments are very heavily used. One is a Nikon-C1-SI-R confocal microscope with AOTF control and six laser lines, including a 408 nm Diode and 457 nm, 477 nm, 488 nm and 514 argon lasers. The other is a Nikon A1R-HD25 confocal system on a Nikon Ti2 inverted microscope (Nikon Corporation, Tokyo, Japan).  This microscope is equipped with 6 laser lines (405, 445, 488, 514, 560 and 640 nm) and was purchased in 2019 with departmental funds. Both are available to members of AVRC for use.  There is an Arcturus Laser Capture Microdissection (LCM) system in an adjacent room. The confocal microscopist performs the imaging or trains/supervises PI lab personnel on performing the imaging. The images are ported and archived in a university file server that is backed up daily. The confocal is the workhorse of the Structural Biology and Imaging Core and it is heavily used necessitating careful scheduling and after hours use, with frequent monitoring by the Core Tech (Micah Chrenek) to alleviate potential conflicts. 

In Vivo Confocal

The in vivo confocal microscope, a Nidek Confoscan 3, is located on the 2nd floor of the Emory Eye Center.  This microscope was purchased via partial Core Center Grant support and other funds from the Edelhauser Laboratory.  The instrument includes a computer with image storage and retrieval characteristics. Corneal images (ex vivo in eye bank LASIK corneas) are correlated with histology, immunohistochemical, and ultrastructural findings obtained from the corneas in the L.F. Montgomery Laboratory and Dobbs Electron Microscopy Suite.  The in vivo confocal microscope is used for imaging mouse, rat, rabbit, and ex vivo human corneas obtained from various U.S. Eye Banks.   

Electron Microscopy

The Dobbs Electron Microscopy Suite contains a JEOL JEM CX transmission electron microscope in a separate room. The ultramicrotomy room contains a Leica Ultracut UCT ultra microtome and AO phase contrast microscope.  This room also contains equipment, much recently updated by the Emory Eye Center, to provide imaging and electronic transmission of digitized images to investigators. The EM now has a high resolution digital camera and digital images are transmitted via the internet to investigators. We anticipate that there will be more extensive use of the EM during the next cycle of the core grant. A critical point drier and Dento Vacuum Desk II sputter-coater are located in one room to prepare occasional specimens for scanning electron microscopy, which is performed in the Department of Chemistry on campus.

Experimental animal surgery imagery

A small animal experimental surgery unit is located in room BT427 adjacent to the L.F. Montgomery Laboratory and Dobbs Electron Microscopy Suite (Appendix xx).  This unit has been developed with departmental funds showing institutional support for the Core Grant and AVRC.  The unit includes a Wild Leitz M691 stereo dissection microscope, Panasonic DMR E95H DVD recorder, Olympus BX41 microscope with DP12 digital camera, and Dell Pentium 4 computer with an Ethernet connection.  The unit also has an ridex 810nm diode laser, Coherent photoactivator PDT laser with Zeiss slit lamp. All this equipment was purchased with departmental funds. The unit is supervised by Dr. Grossniklaus.  Principal investigators and their staff are trained by Dr. Grossniklaus and his research team on usage of the equipment. This is used for small animal surgery when imaging is required. Dr. Grossniklaus is available for assistance with use of these lasers by PIs at no cost to the Core Grant. This unit also has a 10-MHz B-scan ultrasound (Compact Touch, Quantel  Medical, Bozeman, MT) that is available for imaging intraocular structures. Additionally, a VisualSonics Vevo 2100 (Toronto, CA) ultrasound with blood flow imaging and microbubble contrast capabilities is available to Dr. Grossniklaus and the Core Grant through the Department of Pediatrics. 

Supplemental/ Backup facilities

Emory University and the School of Medicine support several “university-level” Core facilities that offer highly specialized, new, and state-of-the-art if not bleeding edge microscopy and imaging facilities including PALM, STED, STORM 4PI microscopes and live imaging microscopy facilities that the present Core Director Dr. Grossniklaus can use. As a member of the Winship Cancer Institute, he as access to a Zeiss 710 NLO multiphoton confocal scanning laser microscope which is on the 3rd floor of Building B, just across the atrium form the EEC. This allows for intravital (live animal) imaging with high magnification and resolution. This microscope is equipped with a Chameleon Vision S tunable multiphoton laser (680 nm to 1050 nm) that can penetrate farther into tissue than a traditional confocal laser. This system is mounted on an upright Zeiss Axioexaminer microscope, together with a flexible large area stage for imaging live mouse single cells, thick tissue sections, and cell lines. The confocal optics of the 710 house spectral detectors coupled with six lasers lines (405, 458, 488, 514, 543, and 633nm) for flexible acquisitions.  Two non-descanned detectors (NDDs) provide ultrasensitive detection of multiphoton signals. Additionally, the Emory University Apkarian Integrated Electron Microscopy Core offers cryo TEM and cryo SEM.  Dr. Grossnikalus serves as a facilitator when AVRC PIs need to use these microscopy techniques that are beyond the normal capabilities of any single PI. These same university Core labs also offer confocal and Electron Microscope services, should any of our own Core microscopes be out of service temporarily or be so overburdened with samples due to transient spikes in demand as we get close to major international meetings such as ARVO. 

Core B: FG&P

DNA/RNA section

The DNA/RNA section provides PCR, RT-PCR, and droplet digital PCR (ddPCR), SNP analysis, gene array analysis, linkage and candidate gene analysis, transgenic/knockout mouse analysis, and oligonucleotide synthesis using equipment and personnel of the Core. The Core has several PCR thermocyclers (see “Equipment”). The ddPCR service uses a Bio-Rad QX200 droplet digital PCR emulsifier/cycler for absolute quantification of nucleic acids and rare allele detection. This is currently a workhorse for CRISPR/Cas9 gene editing studies and disease model creation. It is also used it to quantify transcripts from laser capture microscopy (LCM) and individual cells from various mouse strains. In an innovative collaboration with the Bioanalysis Core, a complete PCR primer service was implemented that includes automated primer design, ordering, optimization, and archiving. An Agilent 2100 Bioanalyzer System is used to confirm single RT-PCR product identity and quality (RNA Integrity Number; RIN). A Bio-Rad Qiacube is used for semi-automated purification of DNA, RNA, and protein samples. This increases efficiency, repeatability, and productivity for the above procedures. The FG&P core also facilitates AVRC use of the Emory Integrated Genomics Core for microarray analyses using Illumina microarrays and Affymetrix chips.

Protein/small molecule Analysis

Protein/Small Molecule Analysis section provides peptide synthesis, HPLC/FPLC analysis, protein separation using 1- and 2-D gel electrophoresis, amino acid sequence analysis, and mass spectrometric analyses of proteins. Support for immunohistochemistry (IHC) and immunoblotting (IB) is provided in the form of a suite of Core-supplied antibodies and associated protocols commonly used by several AVRC laboratories. Additionally, Biotek Synergy multimode plate reader/washer systems are used for ELISAs and chemiluminescent and other fluorescence-based assays, BioRad Transblot Turbos are used for rapid western immunoblotting, and Bio-Rad ChemiDoc MP imaging systems are used for multiplex fluorescent western blotting, chemiluminescence detection, and general gel documentation. The EEC and VAMC have these same systems. This section also expanded with comprehensive HPLC assay development for protein and small molecule identification, providing complete development-through-analysis service for AVRC faculty previously inexperienced with these approaches. The HPLC system with fluorescence, UV/Vis, coulometric, and amperometric detectors, autoinjectors, and Agilent Chemstation is located on the 5th floor of the EEC. This area contains most of the shared molecular analytical equipment (Overall App. 4). Finally, this section facilitates AVRC use of the Emory Integrated Proteomics Core, which has an LTQ ion trap-Orbitrap Hybrid Mass Spectrometer coupled with an autosampler and HPLC system for automated capillary LC-MS/MS, a Quadrupole-Orbitrap Hybrid Mass Spectrometer coupled to an autosampler and nano-Acquity UPLC system, and an Orbitrap Fusion Tribrid Mass Spectrometer with Electron Transfer Dissociation. 

Cell isolation and culture section

The Cell Isolation and Culture section maintains the common tissue culture equipment (hoods, incubators, and microscopes) located on the EEC 5th floor (Overall App. 4). This section also provides flow cytometry, laser capture microdissection (LCM), and tissue culture capabilities, which includes assistance with sample preparation and processing of LCM material and cell line cryostorage. The LCM facility consists of an ArcturusXT LCM workstation, Leica 1850 cryostat, and sample preparation tools and instruments. 

Ocular Functional & Structural Phenotype section

Ocular Functional and Structural Phenotype section provides phenotypic analysis of retinal and visual function, ocular biometry, and in vivo ocular photography. Most of the assessments in this section are non-invasive and allow for longitudinal follow-up of ocular measures related to treatments, development, or degeneration. All in vivo and post-mortem analyses are conducted at the EEC and the VAMC using similar or identical equipment (see “Facilities and Resources” and “Equipment” for details). Retinal function is measured with electroretinography (ERG; various LKC systems). Visual function (visual acuity and contrast sensitivity) is recorded via optomotor response (OMR; CerebralMechanics OptoMotry systems). Structural phenotyping is via optical coherence tomography (SD-OCT) imaging systems (Heidelberg SLO/SD-OCT, Bioptigen 4300 series, and Phoenix Micron IV systems). We have three ERG/SD-OCT suites that allow rodent retinal function and structure to be sequentially assessed in the same rodent in vivo in the same anesthetic session. Refractive error of rodent eyes is measured with two custom-built infrared automated photorefractors. Axial length and other ocular dimensions and shape are also assessed ex vivo with Keyence high-resolution laser micrometers and 3-dimensional scanners. Rodent intraocular pressure is measured with tonometers. Fundus photography is performed with a RetCamII fundus camera, the Heidelberg SLO, and Micron IV retinal imaging systems. The section recently made available a high-resolution adaptive optics flood illuminated fundus camera (Rtx-1; Imagine Eyes, Paris, France) in support of AVRC laboratories that are initiating human subject studies and recently began testing adaptive lenses for the camera to allow use in animal models. This section also purchased and supports two new ERG systems (Celeris Diagnosys LLC) with full-field and pattern stimulation capabilities. These units are portable and are routinely used across AVRC laboratories. 

Rodent Ocular Microsurgery

The FG&P Core provides expertise in Rodent Ocular Microsurgery, principally at dedicated workstations in the EEC and VAMC, but also at other AVRC laboratories as needed (Ms. Sellers travels to the various AVRC laboratories). This service was created during the current funding period to assist AVRC investigators with optic nerve crush, subretinal, intravitreal, or other intraocular and transscleral drug and DNA/RNA construct delivery. The Core provides personnel (Ms. Sellers) and three surgical suites comprised of microscopes (with video capability), various manual and semi-automated injectors, and surgical instruments. The techniques developed by this section have resulted in several stand-alone methods publications [1-7] in addition to providing support that resulted in primary research publications. 

Mouse Colony Service

This Core also provides a Mouse Colony Service (breeding, genotyping, maintenance, and tracking) that currently includes 68 strains in 275-330 breeder cages and 1800 maintenance/experiment litter cages. The Department of Animal Resources has provided us with a dedicated, mite-free colony room (565 sq. ft.) on the third floor of the EEC for Core mouse breeding/housing. We genotype all of our breeders that have a mutation or transgene to ensure we are propagating the correct alleles. We also genotype experimental mice at various loci as needed.  All CRE driver mice are genotyped for presence of CRE.  Experimental mice that are produced from a het x het cross are all genotyped.  Floxed alleles are also genotyped when we are using CRE drivers that are leaky (i.e. RPE-CRE and Best1-CRE).Typically mice are genotyped for loci where the alleles are of interest or in question. For example, if an AVRC laboratory crosses two strains, progeny will be genotyped until homozygosity is established. A subset of all of our strains have been genotyped for rd8 to ensure it does not exist in the colony. If found, mice are backcrossed to C57BL/6J to establish new breeder lines without rd8. This section also provides genotyping and tracking services for AVRC members who use the VAMC vivarium (20 strains in 60 breeding and 200 experimental cages; see “Facilities & Resources” for details of VAMC animal services). Overall, 200-300 mice are genotyped per month. 

Core C:

For statistical analysis our core facilitates data collection, data storage and statistical analysis.

  • Provides PIs with aid developing protocols, data acquisition and analysis.
  • Aid investigators in data storage either on our local server or on the cloud through Amazon Web Services (AWS).
  • Facilitates image processing and Image analysis through training or for small jobs conducting the analysis.
  • Provide statistical analysis for basic and translational research.

We also provide bioinformatic analysis for high through put DNA and RNA sequencing.

  • Develops innovative advanced computational and bioinformatics resources to examine and manage large volumes of biological data as applied to vision science.
  • Provide integrative state-of-the-art software tools for genomics, proteomics, data mining, and understanding genetic interactions and visual systems biology.
  • Applies specific bioinformatics to vision science and visual systems biology.
  • Aid in large capacity computing and data storage on AWW.