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Experimental & Basic Sciences Platform

The Experimental and Basic Sciences core of SERI comprises of all the research laboratory space and core equipment contained therein that facilitate and support the translational and basic ocular disease research within SERI. This platform provides the core facilities available for all SERI researchers engaged in wet or bench laboratory research. The Experimental and Basic Sciences Platform is further divided into five separate facilities.


1. Experimental Molecular and Cell Biology


Head: Dr Seet Li Fong  


2. Ocular Proteomics


Head: Dr Zhou Lei


Head: Assoc Prof Eranga Vithana


4. Experimental Microscopy and Imaging


Head: Dr Gary Yam


5. Translational Ophthalmic Pathology


Head: Dr Anita Chan


(1) The Experimental Molecular and Cell Biology Facility includes all the tissue culture and -80 degree freezer rooms, laboratories dedicated to electrophysiology, chemistry and microbiology, as well as 33 general lab benches. Both specialised cell biology and general core lab equipment are part of this facility. This platform is supported by two laboratory managers who oversee daily laboratory operations, procure equipment, monitor utilisation of space, update risk assessments and ensure that biological and general workplace safety are in compliance with the rules and regulations of the SingHealth Institutional Biosafety Committee (SHSIBC).

(2) The Ocular Proteomics Facility, housed within a micro-analytical laboratory, is equipped with 2 HPLCs (nano- and high-flow) and 2 mass spectrometers (high-resolution TripleTOF and triple quadrupole) for proteomics, metabolomics and drug analyses. This facility provides 25 types of services, including gel protein identification, affinity purification of complex samples, relative/absolute quantitative proteomic analysis, identification/quantification of post-translational modifications, biomarker discovery/ validation, untargeted/targeted metabolomics, and pharmacokinetics/drug quantitation. New proteomics/metabolomics technologies to access the frontier “Omics” technologies are being developed.

(3) The Genomics Facility comprises of one tissue-processing laboratory for DNA and RNA extraction and includes the instrumentation for real-time qPCR and chip based gene expression analysis. The genomics facility facilitates genetic analyses of small- to medium-sized sample cohorts or samples derived from animal models. We are also setting up ophthalmic tissue processing to enable technologies such as RNAseq.

(4) The Experimental Microscopy/ Imaging Facility provides instrumentation for high resolution microscopic imaging and morphometric analysis of biological specimens. It is equipped with instruments for brightfield (standard/ phase-contrast/ differential interference contrast), fluorescent (standard/ confocal) and stereo microscopies, including a Carl Zeiss AXIOVERT 200 Motorized Microscope, a Carl Zeiss AXIOPLAN 2 Imaging Microscope, a Nikon C2 Confocal Laser Point Scanning System, a Nikon Eclipse TS100-F Stereomicroscopes, a BioRad ZOE Fluorescent Live Cell Imager and an Essen Bioscience Incucyte Zoom. Digital imaging is available for immediate capture and processing of images for publications, presentations and grants. The facility provides advisory assistance in experiment design, choice of microscopies, optimal sample processing, visualization and data collection.

(5) The Translational Ophthalmic Pathology Facility works in synergy with the Experimental Microscopy and Imaging Facility to provide specialised non-routine histopathology services involving ophthalmic tissues. This facility is led by a trained ophthalmic pathologist, Dr Anita Chan. Non-routine histological services include but are not limited to specialised techniques for tissue processing for transmission electron microscopy, multiplexed immunohistochemistry and preparation of ocular microvasculature and retinal tissue whole mounts.

The Translational Ophthalmic Pathology as well as The Experimental Microscopy and Imaging Facilities interact with the Department of Anatomical Pathology of the Singapore General Hospital and the Advanced Bio-imaging Core. They provide SERI researchers with access to a variety of advanced imaging equipment, as well as specialized services that help keep them abreast with advancing technology in this area.


Proteomics


Overview

The Ocular Proteomics Laboratory is a core platform in SERI and provides expertise and instrumentation in cutting-edge proteomics and metabolomics research. Established in 2004, the Ocular Proteomics Laboratory focuses on the application of proteomics to clinical samples from the eye to find new biomarkers and understand the disease. Additionally, we collaborate with other research groups within SERI and SNEC. We offer a broad range of proteomics/metabolomics and biological mass spectrometry (MS) services, including protein identification, quantitative proteomics (iTRAQ, high resolution – MRM, SWATH, etc.), characterisation of post-translational modifications (PTMs), MS-based non-targeted and targeted metabolomics and drug analysis. Recently, we have been asked to participate in the Human Proteome Organisation (HUPO) Eye Proteome project, which aims to establish a human eye proteome database.

Facilities

  • AB SCIEX TripleTOF 5600 Mass Spectrometer

  • Dionex UltiMate 3000 RSLCnano system

  • AB SCIEX API 2000 MS/MS system

  • Waters 2695 HPLC system

  • Waters Acquity UPLC I-Class system

Capabilities

  • Protein identification (identify proteins from a gel band, or whole proteome)

  • Quantitative proteomics and proteomic profiling (iTRAQ, mTRAQ, high-resolution MRM, SWATH, etc.)

  • Post-translational modification discovery (glycosylation, phosphorylation, acetylation, methylation, etc.)

  • MS-based metabolomics

  • Small molecule quantitation (drug level, targeted metabolomics, etc.)


Projects

One of our focuses is on tear fluid proteomics and metabolomics. We believe that tear fluid is a useful source for discovering biomarkers associated with the various components of the lacrimal functional unit, because of the close relationship of tears to the disease sites. Recent studies by our group showed that more than 1500 proteins and 60 small molecule metabolites were identified in healthy human tears using the latest proteomics and metabolomics technology. This comprehensive tear composition repertoire can be served as a background for future biomarker research of ocular diseases.

The current biomarker research projects which use tears as the source are:

  • Validation study of tear biomarkers for dry eye

  • Epidemiologic studies of tear proteomics in normal Chinese, Malay and Indian population

  • Tear proteomic profiles in post-LASIK (Laser in-situ keratomileusis) dry eye

  • Effect of glaucoma medication on tear protein profiles and use them as biomarkers to predict the risk of glaucoma surgical failure

  • Tear biomarkers in lacrimal gland tumor

  • Tear proteomic profiles in thyroid eye disease

  • Tear proteomic profiles in keratoconus

Other important proteomics/metabolomics projects include:

  • Integrated “Omics” in ocular important drug-resistance pathogens

  • Circulating biomarkers in diabetic retinopathy using a metabolomics approach

  • Molecular mechanism of myopia and atropine treatment in a mouse model using quantitative proteomics

  • Vitreous proteomic profile in diabetic retinopathy

  • To identify blood biomarker for responder vs non-responder of AMD patients towards anti-VEGF therapy

  • To identify novel aqueous humor biomarker for AMD/PCV

  • Inflammation markers in aqueous humor from PAS, PAC, and PACG


Publications

  1. Chen L, Gao Y, Wang LZ, Ning Cheung N, Tan GSW, Cheung CMG, Beuerman RW, Wong TY, Chan ECY, Zhou L*. Recent Advances in the Applications of Metabolomics in Eye Research. Analytica Chimica Acta. 2018, accepted. (IF = 5.123). * Corresponding author.

  2. Chen G#, Walmsley S#, Cheung GCM, Chen L, Cheng CY, Beuerman RW, Wong TY, Zhou L*, Choi HW*. Customized consensus spectral library building for untargeted quantitative metabolomics analysis with data independent acquisition mass spectrometry and MetaboDIA workflow. Analytical Chemistry. 2017, 89(9):4897-4906. (IF = 6.320). * Corresponding author.

  3. Tong L, Zhou L, Beuerman RW, Simonyi S, Hollander DA, Stern ME. Effects of punctal occlusion on global tear proteins in patients with dry eye. Ocular Surface. 2017, S1542-0124(16)30285-3. (IF = 5.530).

  4. Zhou L*, Beuerman RW. The power of tears: how tear proteomics research could revolutionize the clinic (Editorial).  Expert Review of Proteomics. 2017, 14(3):189-191. (IF = 3.849). * Corresponding author.

  5. Chen L, Cheng CY, Choi HW, Ikram MK, Sabanayagam C, Tan GS, Tian DC, Zhang L, Venkatesan G, Tai ES, Wang JJ, Mitchell P, Cheung CM, Beuerman RW, Zhou L*, Chan ECY*, Wong TY*. Plasma Metabonomic Profiling of Diabetic Retinopathy. Diabetes. 2016, Apr;65(4):1099-108. (IF = 8.474). * Corresponding author.

  6. Chen L, Li J, Guo T, Ghosh S, Koh SK, Tian D, Zhang L, Jia D, Beuerman RW, Aebersold R, Chan EC, Zhou L*. Global Metabonomic and Proteomic Analysis of Human Conjunctival Epithelial Cells (IOBA-NHC) in Response to Hyperosmotic Stress. Journal of Proteome Research. 2015, 14, 3982-3995. (IF = 5.001). *Corresponding author.

  7. Tong L, Zhou XY, Jylha A, Aapola U, Liu DN, Koh SK, Tian D, Quah J, Uusitalo H, Beuerman RW, Zhou L*. Quantitation of 47 Human Tear Proteins using High Resolution Multiple Reaction Monitoring (HR-MRM) Based-Mass Spectrometry. Journal of Proteomics. 2015, 115, 36-48. (IF =5.080). *Corresponding author.

  8. Barathi VA, Chaurasia SS, Poidinger M, Koh SK, Tian D, Ho C, Iuvone MP, Beuerman RW, Zhou L*. Involvement of GABA transporters in Atropine-treated Myopic Retina as revealed by iTRAQ Quantitative Proteomics. Journal of Proteome Research. 2014, Nov. 7; 13(11): 4647-4658. (IF = 5.001). *Corresponding author.

  9. Zhou L*, Wei Ruihua, Zhao Ping, Koh Siew Kwan, Beuerman Roger W, Ding Chuanqing, Proteomic Analysis Revealed the Altered Tear Protein Profile in a Rabbit Model of Sjögren’s Syndrome-Associated Dry Eye. Proteomics, 2013, 13(16), 2469-2481. *Corresponding author.

  10. Zhou L, Beuerman RW. Tear analysis in ocular surface diseases. Progress in Retinal and Eye Research, 2012, 31(6), 527-550. (IF = 11.653).

  11. Zhou L*, Zhao SZ, Koh SK, Chen L, Vaz C, Tanavde V, Li XR, Beuerman RW. In-depth analysis of the human tear proteome. Journal of Proteomics, 2012, 75(13), 3877-3885. (IF = 5.080). *Corresponding author.

  12. Chen L, Zhou L*, Chan E, Neo J, Beuerman, RW, Characterization of The Human Tear Metabolome by LC-MS/MS. Journal of Proteome Research. 2011, Oct 7;10(10):4876-82. (IF = 5.617). *Corresponding author.

  13. Wong TT, Zhou L, Li J, Tong L, Zhao SZ, Li XR, Yu SJ, Koh SK, Beuerman RW, Proteomic profiling of inflammatory signaling molecules in the tears of patients on chronic glaucoma medication, Invest. Ophthalmol. Vis. Sci. 2011, Sep 22;52(10):7385-91. (IF = 3.933).

  14. Zhou L, Liu SP, Li J, Ong LB, Guo L, Wohland T, Tang C, Lakshminarayanan R, Mavinahalli J, Verma C, Beuerman RW. The structural parameters to antimicrobial activity, human epithelia cell cytotoxicity and killing mechanism of 10-AA synthetic analogues and covalent dimer derived from hBD3 C-terminal. Amino Acids. 2011 Jan;40(1):123-33. (IF = 4.106).

  15. Zhou L, Beuerman RW, Chew AP, Koh SK, Cafaro TA, Urrets-Zavalia EA, Urrets-Zavalia JA, Li SFY, Serra HM. Quantitative Analysis of N-linked Glycoproteins in Tear Fluid of Climatic Droplet Keratopathy by Glycopeptide Capture and iTRAQ. Journal of Proteome Research, 2009, 8(4), 1992-2003. (IF = 5.737).

  16. Zhou L, Beuerman RW, Chan CM, Zhao SZ, Li XR, Yang H, Tong L, Liu SP, Stern ME, Tan DTH. Identification of Tear Fluid Biomarkers in Dry Eye Syndrome Using iTRAQ Quantitative Proteomics. Journal of Proteome Research, 2009, 8(11), 4889-4905. (IF = 5.737).


Head

Dr Zhou Lei


Contact us

Dr Zhou Lei at zhou.lei@seri.com.sg


Experimental Microscopy


Overview


The Experimental Microscopy Support platform (EMSP) provides collaborative and advisory assistance for scientists in Singapore Eye Research Institute/Singapore National Eye Centre to use high resolution imaging for their research programs. To conduct fundamental and advanced research relating studies of morphological and expression characteristics to biological and material properties, EMSP is equipped with “state of the art” light, stereo and confocal microscopes.

Facilities

  • Carl Zeiss AXIOVERT 200 Motorized Microscope (AxioVision 4.8)

  • Carl Zeiss AXIOPLAN 2 Imaging Microscope (AxioVision 4.8)

  • Carl Zeiss AXIO Imager System with ApoTome (AxioVision 4.7.1)

  • Nikon C2 Confocal Laser Point Scanning System (NISElementsAr-4)

  • BioRad ZOE Fluorescence Cell Imager

  • Nikon SMZ1500 Stereomicroscope

  • Nikon Eclipse TS100-F Stereomicroscopes (NIS-Elements D)

Most of them are outfitted with digital imaging to allow an investigator to quickly use the images for manuscripts, presentations and grants.


Capabilities

  • Live cell and time-lapse imaging (cellular dynamics for biological functions)

  • Fluorescence microscopy (target protein expression in sub-cellular structures and single molecule tracking and quantification)

  • Phase contrast / differential interference contrast DIC microscopy (contrast-enhanced topographic imaging)

  • Confocal microscopy (high-resolution point scanning imaging for 3D image reconstruction)

  • Polarized light microscopy (anisotropy, birefringence composition)

  • Stereomicroscopy (for close work and micro-dissection)


Manpower

This platform has a team supported by scientists, research officers and laboratory executives, who are committed to ensuring a high standard of research excellence and facility support.


Collaborators

  • Advanced Bio-imaging Core Platform, Academia, SingHealth

  • Electron Microscopy Unit, National University of Singapore (transmission and scanning electron microscopy, atomic force microscopy, cryosectioning and immunoelectron microscopy)

  • NanoCore, National University of Singapore (helium ion microscopy)

  • Institute of Molecular and Cell Biology, A*STAR Singapore (laser capture micro-dissection)


Projects

Morphological visualization accompanied with the dynamic biological processes is the backbone to most biological research. The EMSP supports image-related research, offering advices and technology support on various microscopies and image analysis resources.

We participate in different research projects involving:

  • Ocular tissue imaging (eg. corneal stromal collagen fibrillation, corneal endothelial cells)

  • Ultrastructural morphology and morphometric analysis

  • Protein expression on cells and tissues; co-localization analysis & quantification; 3D imaging

  • Live cell imaging and time-lapse protein expression and trafficking

  • Tissue dissection


Publications

  1. Zhang T, et al. The effect of amniotic membrane de-epithelialization method on its biological properties and ability to promote limbal epithelial cell culture. Invest Ophthalmol Vis Sci. 54(4):3072-81 (2013).

  2. Lee MC, et al. Expression of the primary angle closure glaucoma susceptibility gene PLEKHA7 in endothelial and epithelial cell junctions in the eye. Invest Ophthalmol Vis Sci. 55(6):3833-41 (2014).

  3. Yam GH, et al. Ex vivo propagation of human corneal stromal “activated keratocytes” for tissue engineering. Cell Transplantation. 24(9):1845-61 (2015).

  4. Yam GH, et al. Decellularization of human stromal refractive lenticules for corneal tissue engineering. Sci Rep. 6:26339 (2016).

  5. Ho H, et al. Altered anterior segment biometric parameters in mice deficient in SPARC. Invest Ophthalmol Vis Sci. 58(1):383-93 (2017).

  6. Riau AK, et al. Retreatment strategies following SMILE: in vivo tissue responses. PLoS One. 12(7):e180941 (2017).

  7. Li Z, et al. Optimization of spark plasma sintered titania for potential application as a keratoprosthesis skirt. J Biomed Mater Res Part A. 105(12):3502-13 (2017).

  8. Damgaard I, et al. Reshaping and customization of SMILE-derived biological lenticules for intrastromal implantation. Invest Ophthalmol Vis Sci. 59(6):2555-63 (2018).

  9. Yam GH, et al. Periodontal ligament as a novel adult stem cell source for regenerative corneal cell therapy. J Cell Mol Med. 22(6):3119-3132 (2018).

  
Head

Dr Gary Yam


Contact us

Dr Gary Yam at gary.yam.h.f@seri.com.sg


Experimental Molecular & Cell Biology

Overview

The Experimental Molecular and Cell Biology Platform provides the infrastructure and a range of essential and sophisticated core equipment to facilitate researchers in Singapore Eye Research Institute/Singapore National Eye Centre in their basic to advanced molecular and cell biology studies.

Facilities

Our laboratory space occupies two floors, on the 11th and 12th floors of The Academia. There are 33 general laboratory benches as well as rooms dedicated to the following:

  • Chemistry

  • Microbiology

  • Gel electrophoresis

  • Dark room/ X-ray processor

  • Tissue culture

Core equipment includes:

  • absorbance/ fluorescence/ luminescence plate readers

  • autoclaves

  • Bio-Plex 200 system

  • BD FACSVerse™ flow cytometer

  • ChemiDoc Imager 

  • circular dichroism spectroscopy

  • Essen IncuCyte zoom system

  • Guava Easycyte flow cytometer

  • Instron universal material testing system

  • real-time PCR instruments

  • droplet digital PCR system

  • medical X-Ray processor

  • microvolume spectrophotometers      

  • water filter system

  • xCELLigence RTCA systems

Manpower

This platform is managed by the SERI laboratory administration team and supported by scientists, research officers and laboratory executives to ensure optimal performance of all facilities and equipment. We comply with the rules and regulations of the SingHealth Institutional Biosafety Committee (SHSIBC).

Projects

Molecular and cellular experimentation is fundamental to understanding the mechanisms of eye diseases, as well as drugs for therapy. The projects facilitated by this platform are multidisciplinary, diverse and vary in both breadth and depth. Besides supporting experimental research, this platform also offers assistance for assessing other resources within the SingHealth campus and beyond. We collaborate extensively with other academic institutions within Singapore. We also welcome collaboration with industry partners interested in eye-related research or the exploration of innovative molecular and cell technologies. Our industry collaborators include AYOXXA Biosystems (Cologne, Germany) and Santen Pharmaceutical Co., Ltd. (Osaka, Japan).

Examples of ongoing research areas include:

  • Corneal endothelial cultures for ocular transplantation

  • Functional genomics

  • Therapeutics and drug delivery systems for dry eye, ocular surgery, etc

  • Anti-microbial therapeutics and biomaterial

  • Stem cell therapy for retinal diseases

  • Epidemiology and biomarkers of retinal diseases

 
Head

Dr Seet Li Fong


Contact us

Dr Seet Li Fong at seet.li.fong@seri.com.sg

 
Translational Opthalmic Pathology (TOP)

Overview

The TOP platform is a core platform that specializes in molecular pathology for the study of the pathology of ocular disorders. In our laboratory we have the latest DEPArray NXt © machine which has the capacity to sort single cells from fresh and fixed tissues, as well as paraffin embedded tissues for further downstream analysis. Tissues may be liquid based or solid tissue. This technology is superior to routine flow cytometry as it is able to handle single cells. The major disadvantage in working with ocular tissues is the small specimen size and limited cellular quantity. This technology is crucial for advancing molecular capabilities in our ophthalmic related research.

The platform is led by Dr Anita Chan who is a practicing ophthalmologist, as well as a trained ophthalmic pathologist. In our team, we have expertise in analyzing human and animal related histology for clinical and research studies respectively.

In addition to molecular pathology, we also offer advance histological services including:

  • Multiplex immunofluorescence imaging

  • Immunofluorescence imaging

  • Immunohistochemistry

  • Electron microscopy

Our platform works closely with the other core units in SERI (ocular genetics, proteomics, preclinical translational platform, as well as the microscopy imaging unit) to provide a comprehensive approach for ophthalmic research studies.

Projects

  • We currently have several industry research collaborations ongoing, including work with Santen Pharmaceutical and Menarini Biomarkers.

  • Other studies include the use of DEPARRay to study iris tissue in exfoliation syndrome.

 
Head

Dr Anita Chan (PI)

Team members

  • Mona Wang

  • Myoe Naing Lynn


Collaborators

  • Dr Lim Tong Seng (Co-PI, MBS collaboration)

  • Dr Tan Wei Jian (Post doc, MBS collaboration)


Contact us

Dr Anita Chan at anita.chan.s.y@singhealth.com.sg