examples of images acquired

Confocal Microscopy (Zeiss LSM 710, inverted microscope)

A Zeiss Axio Observer Z1 inverted microscope is connected to a Zeiss LSM710 confocal. The available laser lines are 405, 458, 488, 514, 561 and 633nm. With the availability of a near UV laser this confocal can also visualize DAPI. In addition to the confocal images a DIC image can be acquired. Stage and objective heaters are available to aid live cell imaging.
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The most common reason to use a confocal is to obtain optical sections that have much less out-of-focus blur than images from widefield instruments. In addition, one can acquire a 3-D data set for volume determination or 3-D reconstruction. 
Multi-tracking provides considerable improvement in the separation of similar dyes over that of a widefield microscope. Therefore, even if you do not need the optical sectioning ability of the confocal microscope, you may want to use it instead of a widefield microscope in order to ensure separation of dye pairs like FITC and rhodamine. Another reason to chose a confocal over a widefield microscope is to have precision in the overlay of images that are taken with different filters.  

Zeiss

Widefield Light Microscopy (multiple stations, upright & inverted platforms)

A Zeiss Axioplan Imaging 2e infinity-corrected upright scope with DIC and epifluorescence. Uses either a Color Zeiss Axiocam (for brightfield imaging) or a B&W Zeiss Axiocam for fluorescence. Filter cubes are available for DAPI, FITC, rhodamine, Texas Red and Cy5-like dyes. 

Zeiss

Axiovert 100 TV inverted microscope

This inverted scope is equipped for phase, brightfield, and fluorescence. The filter cubes are suitable for fluoresceine (GFP), rhodamine, and DAPI like dyes. The available objectives are 1.25x, 5x, 10x and a 32x long working distance objective. Additional objectives are available upon request. The filters and optics on this scope are not as advanced as the orcaerzeiss but it is useful for e.g. checking on transfection efficiency. A Zeiss Axiocam MRm is connected connected to the microscope and provides as resolution of up to 1388 x 1040 pixels. The camera is suitable for fluorescence imaging and provides only B&W images.

Zeiss

Stereo Microscope

An Olympus SZX12 stereo microscope serves for low magnification microscopy needs. It is equipped with a Q-imaging color camera and uses Q-Capture software to acquire images. 

Olympus

Incucyte Zoom

IncuCyte ZOOM® consists of a microscope inside a standard cell incubator for environmental stability, and a networked external controller hard drive that collects and processes image data. The instrument has three microscope objectives (4x, 10x, 20x) that can be interchanged by the facility manager upon request.
It houses up to six microtiter plates which can be run simultaneously. Available imaging modes are phase contrast, red and green fluorescent. Imaging can be for extended periods of time.
Hardware and software tools for a scratch wound migration assay in 96-well plates is available.
Software can be remotely accessed by any networked computer (IP number 10.170.3.126). Ask Birgit for a download location of the program.

Essen BioScience/Satorius

Nikon SIMe

A Nikon SIMe (structured illumination microscopy) superresolution microscope is available on a motorized inverted Nikon Eclipse Ti stand. Available laser lines are 488nm, 561nm and 640nm for acquiring 3D-SIM images. A DAPI channel can be added as a fluorescent widefield channel. The system provides lateral resolution of up to 115nm and axial resolution of up to 269nm. 
The system is equipped with a Hamamatsu Orca Flash 4.0 camera with high sensitivity and low readout noise.

Publications which relied on the equipment: 
Chen et al. (2019). Nanoscale monitoring of mitochondria and lysosome interactions for drug screening and discovery. Nano Research 12(5):1009–1015.
Khamo et al. (2019). Optogenetic Delineation of Receptor Tyrosine Kinase Subcircuits in PC12 Cell Differentiation. Cell Chemical Biology  26(3): 400-410.e3
Chen et al. (2018) Super-Resolution Tracking of Mitochondrial Dynamics with An Iridium(III) Luminophore. Small 14 (41): 1802166

Flow Cytometer (BD LSRFortessa)

Flow Cytometry is a technology that analyzes multiple physical and/or chemical characteristics of single particles, usually cells, as they flow in a fluid stream through a beam of light. The properties measured include a particle’s relative size, relative granularity or internal complexity, and relative fluorescence intensity.

The Fortessa is equipped with four lasers (405nm, 488nm, 561nm and 640nm) and detects 2 light scatter parameters (forward and side scatter) and up to 15 fluorescence parameters. The Fortessa only accepts 5mL polystyrene tubes. Sheath fluid (NERL Diluent 2) is provided. 

BD

Fluorescent Activated Cell Sorter (FACS)

BD Aria III.
Cell sorting is a method to purify cell populations based on the presence or absence of specific characteristics, most often the absence or presence of fluorescent labels. The BD Aria Cell sorter uses in-air droplets to sort. As a collection system we can sort either into two tubes (15ml or 5ml receiving tube with medium) or four tubes (5ml tubes with medium). We also have the option to sort onto tissue culture plates. Due to generation of droplets, the instrument is enclosed in a biosafety cabinet. Sheath is NERL blood bank saline and is provided. 
The available lasers are 407nm, 488nm, 561 nm, and 633nm, and in addition to forward and side scatter up to 13 fluorescent parameters can get acquired. 

Where is the core located?

The Advanced Cell Analysis Service Center is located in the Vontz Center. Most instruments are located on the 3rd floor. Please contact Birgit Ehmer if you need further directions. 

What is the configuration of the Flow Cytometer (BD LSRFortessa)

Cytometer: LSRFortessa

The first number for an emission filter gives the peak transmission, the second number the width of the window which transmits light. A 530/30 emission filter will therefore let light from 515nm to 535nm through. In brackets are common fluorophores which are suitable for that detector. 

Excitation: 405 nm
Emission:
450/50 (Alexa Fluor 405; BV421; CFP; DAPI; Hoechst 33258; Pacific Blue)
610/20 (BV605)
710/50 (BV711)
525/50 (Alexa Fluor 430; BV510; Pacific Orange;  V450; V500; BFP)
670/30 (BV650)
780/60 (BV786)
 
Excitation: 488 nm
Emission:
488/0 (side scatter SSC)
530/30 (Alexa Fluor 488; FITC; GFP; CFSE, mBanana, YFP)
710/50 (PerCP-Cy5-5)
 
Excitation: 561 nm
Emission:
585/15 (DsRed; PE; AF555; Cy3; dTomato; mOrange)
610/20 (PE-Texas Red; mCherry; mRaspberry; mStrawberry; Propidium Iodide)
670/30 (7-AAD; PE-Cy5; mPlum; Nile Red)
710/50 (PE-Cy5.5)
780/60 (PE-Cy7)
Excitation: 640 nm
Emission:
670/14 (Alexa Fluor 647; APC; APC-Cy5-5)
730/45 (Alexa Fluor 700)
780/60 (APC-Cy7; APC-H7; APC-AF750)
 
 

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Yang, YG; Leonard, M; Zhang, YJ; Zhao, D; Mahmoud, C; Khan, S; Wang, J; Lower, EE; Zhang, XT (2018). HER2-Driven Breast Tumorigenesis Relies upon Interactions of the Estrogen Receptor with Coactivator MED1. CANCER RESEARCH 78(2): 422-435

Thomas, HE; Zhang, Y; Stefely, JA; Veiga, SR; Thomas, G; Kozma, SC; Mercer, CA (2018). Mitochondrial Complex I Activity Is Required for Maximal Autophagy. CELL REPORTS 24(9): 2404-+

Xuyang F, Hsu SJ, Bhattacharjee A, Wang YY, Diao JJ and Price CM (2018). CTC1-STN1 terminates telomerase while STN1-TEN1 enables C-strand synthesis during telomere replication in colon cancer cells. Nature Communications (9) Article number: 2827

Qiu, KQ; Ke, LB; Zhang, XP; Liu, YK; Rees, TW; Ji, LN ; Diao, JJ ; Chao, H (Chao, Hui). (2018). Tracking mitochondrial pH fluctuation during cell apoptosis with two-photon phosphorescent iridium(III) complexes. CHEMICAL COMMUNICATIONS 54(19): 2421-2424.

Sasha J. Ruiz-Torres, Nancy M. Benight, Rebekah A. Karns, Elyse E. Lower, Jun-Lin Guan, and Susan E. Waltz (2017). HGFL-mediated RON signaling supports breast cancer stem cell phenotypes via activation of non-canonical ß-catenin signaling. Oncotarget. Aug 29; 8(35): 58918–58933.

Zhang, YJ; Leonard, M; Shu, Y; Yang, YG ; Shu, D; Guo, PX; Zhang, XT (Zhang, Xiaoting) (2017). Overcoming Tamoxifen Resistance of Human Breast Cancer by Targeted Gene Silencing Using Multifunctional pRNA Nanoparticles. ACS NANO 11(1): 335-346

Jiang, X; Hu, C; Ferchen, K; Nie, J (Nie, Ji); Cui, XL; Chen, CH; Cheng, LT; Zuo, ZX; Seibel, W; He, CJ; Tang, YX; Skibbe, JR; Wunderlich, M; Reinhold, WC; Dong, L; Shen, C ; Arnovitz, S; Ulrich, B; Lu, JW; Weng, HY; Su, R; Huang, HL; Wang, YG; Li, CY; Qin, X; Mulloy, J; Zheng, Y; Diao, JJ; Jin, J; Li, C; Liu, PP; He, C; Chen, Y; Chen, JJ. (2017). Targeted inhibition of STAT/TET1 axis as a therapeutic strategy for acute myeloid leukemia. NATURE COMMUNICATIONS 8 Article Number: 2099

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Chen Q, Jin C, Shao X, Guan R, Tian Z, Wang C, Liu F. Ling P, Guan JL, Ji L, Wang F, Chao H Diao J (2018) Super-Resolution Tracking of Mitochondrial Dynamics with An Iridium(III) Luminophore. Small 14 (41): 1802166

Khamo JS, Krishnamurthy VV, Chen Q, Diao J, Zhang K (2019). Optogenetic Delineation of Receptor Tyrosine Kinase Subcircuits in PC12 Cell Differentiation. Cell Chemical Biology 26(3): 400-410.e3

Chen Q, Shao X, Tian Z, Chen Y, Mondal P, Liu F, Wang F, Ling P, He W, Zhang K, Guo Z, Diao J. (2019). Nanoscale monitoring of mitochondria and lysosome interactions for drug screening and discovery. Nano Research 12(5):1009–1015.

Alam P, Haile B, Arif M, Pandey R, Rokvic M, Nieman M, Maliken BD, Paul A, Wang YG, Sadayappan S, Ahmed RPH, Kanisicak O. 2019. Inhibition of Senescence-Associated Genes Rb1 and Meis2 in Adult Cardiomyocytes Results in Cell Cycle Reentry and Cardiac Repair Post–Myocardial Infarction. Journal of the American Heart Association 8:e012089.

Bruzas I, Brinson BE, Gorunmez Z, Lum W, Ringe E, Sagle L. 2019. Surface-Enhanced Raman Spectroscopy of Fluid-Supported Lipid Bilayers. ACS Appl. Mater. Interfaces 2019, 11, 36, 33442–33451.

Che L, Yang XY, Ge C, El-Amouri SS, Wang QE, Pan D, Herzog TJ, Du CY. 2019. Loss of BRUCE reduces cellular energy level and induces autophagy by driving activation of the AMPK-ULK1 autophagic initiating axis. PLoS One: 14(5):e0216553.

Chowdhury D, Alrefai H, Landero Figueroa JA, Candor K. Porollo A, Fecher R, Divanovic S, Deepe GS, Vignesh KS. 2019. Metallothionein 3 Controls the Phenotype and Metabolic Programming of Alternatively Activated Macrophages. Cell Reports 27(13): 3873-3886.

Conley R. Surprising similarities in photoreceptor membrane shedding between vertebrates and the beetle; Thermonectus marmoratus. 2019. Master Thesis, University of Cincinnati, Cincinnati, USA).

Fang H, Yao S, Chen Q, Liu C, Cai Y, Geng S, Bai Y, Tian Z, Zacharias AL, Takebe T, Chen Y, Guo Z, He W, Diao JJ. 2019. De novo-designed near-infrared nanoaggregates for super-resolution monitoring of lysosomes in cells, in whole organoids, and in vivo. ACS Nano 13, 14426.

Fang H, Yao S, Chen Q, Liu C, Cai Y, Geng S, Bai Y, Tian Z, Zacharias AL, Takebe T, Chen Y, Guo Z, He W, Diao JJ. 2019. De novo-designed near-infrared nanoaggregates for super-resolution monitoring of lysosomes in cells, in whole organoids, and in vivo. ACS Nano 13, 14426.

Ge C, Vilfranc CL, Che LX, Pandita RK, Hambarde S, Andreassen PR, Niu L, Olowokure O, Shah S, Waltz SE, Zou L, Wang J, Pandita TK, Du CY. 2019. The BRUCE-ATR signaling axis is required for accurate DNA replication and suppression of liver cancer development. Hepatology: 69(6): 2608–2622.

Shekhar H, Kleven RT, Peng T, Palaniappan A, Karani KB, Huang SL, McPherson DD, Holland CK. 2019. In vitro characterization of sonothrombolysis and echocontrast agents to treat ischemic stroke. SCIENTIFIC REPORTS 9: 9902

Wang, Chenran; Haas, Michael A; Yang, Fuchun; Yeo, Syn; Okamoto, Takako; Chen, Song; Wen, Jian; Sarma, Pranjal; Plas, David R; Guan, Jun-Lin 2019. Autophagic lipid metabolism sustains mTORC1 activity in TSC-deficient neural stem cells. Nature metabolism, 1 11, 1127-1140.

Che Lixiao, Kris G Alavattam, Peter J Stambrook, Satoshi H Namekawa, Chunying Du. 2020. BRUCE preserves genomic stability in the male germline of mice. Cell Death Diffe: 27(8):2402-2416.

Chen, Qixin; Shao, Xintian; Hao, Mingang; Fang, Hongbao; Guan, Ruilin; Tian, Zhiqi; Li, Miaoling; Wang, Chenran; Ji, Liangnian; Chao, Hui; Guan, Jun-Lin; Diao, Jiajie. 2020. Quantitative analysis of interactive behavior of mitochondria and lysosomes using structured illumination microscopy. Biomaterials, 250 , 120059

Deng, Shan; Essandoh, Kobina; Wang, Xiaohong; Li, Yutian; Huang, Wei; Chen, Jing; Peng, Jiangtong; Jiang, Ding-Sheng; Mu, Xingjiang; Wang, Chenran; Peng, Tianqing; Guan, Jun-Lin; Wang, Yigang; Jegga, Anil; Huang, Kai; Fan, Guo-Chang. 2020. Tsg101 positively regulates P62-Keap1-Nrf2 pathway to protect hearts against oxidative damage. Redox biology, 31, 101453

Lafond M, Shekhar H, Panmanee W, Collins SD, Palaniappan A, McDaniel CT, Hassett DJ, Holland CK. 2020. Bactericidal Activity of Lipid-Shelled Nitric Oxide-Loaded Microbubbles. FRONTIERS IN PHARMACOLOGY 10, 1540.

Qiu, Kangqiang; Du, Yang; Liu, Jiyan; Guan, Jun-Lin; Chao, Hui; Diao, Jiajie. 2020. Super-resolution observation of lysosomal dynamics with fluorescent gold nanoparticles. Theranostics, 10 13, 6072-6081.

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Wu, Hsin-Jung; Hao, Mingang; Yeo, Syn Kok; Guan, Jun-Lin. 2020. FAK signaling in cancer-associated fibroblasts promotes breast cancer cell migration and metastasis by exosomal miRNAs-mediated intercellular communication. Oncogene 39(12): 2539–2549.

Yang, Fuchun; Sun, Shaogang; Wang, Chenran; Haas, Michael; Yeo, Syn; Guan, Jun-Lin 2020. Targeted therapy for mTORC1-driven tumours through HDAC inhibition by exploiting innate vulnerability of mTORC1 hyper-activation. British journal of Cancer, 122, 1791–1802.

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Yeo, Syn Kok; Guan, Jun-Lin. 2020. Regulation of immune checkpoint blockade efficacy in breast cancer by FIP200: A canonical-autophagy-independent function. Cell stress, 4 8, 216-217

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Tang, Xin; Angst, Gabrielle; Haas, Michael; Yang, Fuchun; Wang, Chenran 2021. The Characterization of a Subependymal Giant Astrocytoma-Like Cell Line from Murine Astrocyte with mTORC1 Hyperactivation. Int J mol sciences, 22 8,

Vilfranc CL, Che LX, Patra KC, Niu L, Olowokure O, Wang J, Shah SA, Du CY. BIR repeat-containing ubiquitin conjugating enzyme (BRUCE) regulation of ß-catenin signaling in the progression of drug-induced hepatic fibrosis and carcinogenesis. World J Hepatol 2021; 13(3): 343-361

Wang C, Haas MA, Yeo SK, Paul R, Yang F, Vallabhapurapu S, Qi XY, Plas DR, Guan JL. 2020. Autophagy mediated lipid catabolism facilitates glioma progression to overcome bioenergetic crisis. British Journal of Cancer: 124, 1711–1723 (2021)

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Vilfranc CL, Che LX, Patra KC, Niu L, Olowokure O, Wang J, Shah SA, Du CY. BIR repeat-containing ubiquitin conjugating enzyme (BRUCE) regulation of ß-catenin signaling in the progression of drug-induced hepatic fibrosis and carcinogenesis. World J Hepatol 2021; 13(3): 343-361 [PMID: 33815677 DOI: 10.4254/wjh.v13.i3.343]

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Qixin Chen, Liu-Yi Liu, Zhiqi Tian, Zhou Fang, Kang-Nan Wang, Xintian Shao, Chengying Zhang, Weiwei Zou, Fiona Rowan, Kangqiang Qiu, Baohua Ji, Jun-Lin Guan, Dechang Li, Zong-Wan Mao, Jiajie Diao. (2023) Mitochondrial nucleoid condensates drive peripheral fission through high membrane curvature. Cell Reports 42:12, 113472

Mingang Hao, Peixin Lu, Sarah Sotropa, Kanakaraju Manupati, Syn Kok Yeo, Jun-Lin Guan. (2024) In vivo CRISPR knockout screen identifies p47 as a suppressor of HER2+ breast cancer metastasis by regulating NEMO trafficking and autophagy flux. Cell Reports, Vol 43(2),113780.

Tang, X., Walter, E., Wohleb, E., Fan, Y., & Wang, C. (2023). ATG5 (autophagy related 5) in microglia controls hippocampal neurogenesis in Alzheimer disease. Autophagy, 20(4), 847–862.

Yeo, S. K., Haas, M., Manupati, K., Hao, M., Yang, F., Chen, S., & Guan, J. L. (2023). AZI2 mediates TBK1 activation at unresolved selective autophagy cargo receptor complexes with implications for CD8 T-cell infiltration in breast cancer. Autophagy, 20(3), 525–540.