Integrated Microscopy and Imaging Laboratory

TAMHSC Arrows COM Arrows Basic Sciences Arrows SBTM Arrows Integrated Microscopy and Imaging Laboratory

Integrated Microscopy and Imaging Laboratory (IMIL)

The IMIL includes four imaging rooms, one for each of the large, shared imaging instruments, one preparation room, one cell culture room, one histology room, and an image processing room.

The facility was designed to dampen noise and vibration by isolation from the connecting building and exterior and by laminar air flow in all imaging rooms. It will be a biosafety level 2 research facility within a year.

This facility is located on the TAMHSC College of Medicine’s Temple campus and is connected to the Medical Research Building (MRB).

CORE EQUIPMENT

Confocal microscopy allows optical sectioning of samples to determine 3-D information using fluorescence microscopy. This offers great utility for imaging discrete planes in thick specimens.

The IMIL contains the following core equipment:

SHARED EQUIPMENT

The IMIL contains the following shared equipment:

LINKS

Meridian Ultima-Z Laser-Scanning Confocal Microscope System
Olympus BX51 Fluorescence Microscope
Simultaneous Calcium and Force/Sarcomere-Length Measurement System
Flow Cytometer
Cell Culture Prep Lab
Tissue Prep Lab
Histology Lab

Meridian Ultima-Z Laser-Scanning Confocal Microscope System

This microscope is outfitted with visible and UV lasers and is used for ratio imaging.

Capabilities:

sorting adherent cells
monitoring fluorescence recovery after photobleaching (FRAP)
releasing caged signaling molecules

Use of this microscope must be arranged with the facility director.

Olympus BX51 Fluorescence Microscope

Capabilities:

Fluorescence imaging of DAPI, FITC, and TRITC (or other similar fluorophores) individually and simultaneously
High signal-to-noise objectives for capturing weak fluorescence emission
Eliminates stray light that could increase background noise in images

Filter Sets:

DAPI/Hoechst - excitation: 320 - 390 nm, emission: 430 - 490 nm
FITC/GFP - excitation: 455 - 500 nm, emission: 570 - 560 nm
TRITC/Rhodamine - excitation: 505 - 560 nm, emission 575 - 655 nm
DAPI/FITC/TRITC - excitation: 380 - 400 nm, 415 - 430 nm, 475 - 495 nm, 545 - 570 nm, emission: 445 - 465 nm, 500 - 535 nm, 580 - 630 nm

Use of this microscope must be arranged with Dr. Xu Peng (xpeng@medicine.tamhsc.edu).

Simultaneous Calcium and Force/Sarcomere-Length Measurement System

This system includes:

Olympus IX70 microscope
Xenon lamp
Ionoptix hyper switch that operates at 200 Hz to allow quick switching between 2 different light pathways for excitation
CCD camera after long wavelength low pass filter for imaging
Photo-multiplier tube with narrow band pass filter at fluorescence wavelength for total intensity measurements
Aurora Scientific suite consisted of perfusion chamber, force transducer, position motor, temperature control, and electrical pacing

The Aurora portion of the system performs force measurements. The Ionoptix portion performs [Ca²⁺]_i and sarcomere length measurements. One can use this system to perform a variety of experiments such as intact cardiac myocytes, intact mouse papillary muscle, and engineered heart tissue. Currently, the imaging system is tailored for using Fura-2 as the [Ca2+]i detection dye.

Capabilities:

Simultaneous measurement of intracellular calcium [Ca²⁺]_iand force within intact heart tissue
Simultaneous measurement of intracellular calcium [Ca²⁺]_i and sarcomere length within living cardiac myocytes

Use of this microscope must be arranged with Dr. Carl Tong (ctong@medicine.tamhsc.edu).

Flow cytometer

- Beckman Coulter EPICS XL-MCL

Tissue Prep Lab

This lab contains:

Zeiss Stemi SV11 dissection microscope
Zeiss upright microscope
water heaters
NuAire Class II Biology Safety Cabinet
Thermo Scientific Forma 900 Series 23 cu. ft-80°C freezer
Kenmore refrigerator

Cell Culture Prep Lab

This lab contains:

Nikon phase microscope
NuAire Autoflow 4750 Water Jacket CO₂incubators
Thermo Scientific Forma 900 Series 23 cu. ft-80°C freezer
Thermo Scientific Revco 2-Door 4°C Refrigerator
NuAire Class II Biology Safety Cabinet
water bath
centrifuge
Mirus Nanopump 2.0 syringe pump for cell analysis under shear flow

Histology Lab

This lab contains:

Hamilton SafeAire II fume hood
NuAire Class II Biology Safety Cabinet
Thermo Scientific Revco 2-Door 4°C Refrigerator
cryostat
Thermo Scientific HM 355S Automatic Microtome
Boekel BB-600 Boiling Water Bath

RATES

The rates per hour for use of the core equipment are:

Fast NanoFluor II Integrated Microscope System - $____

Leica AOBS SP2 Confocal-Multiphoton Microscope System - $54

Olympus Confocal Microscope - $20

Image Processing - $___

Facility technician's operation of any of the above - $22

RESERVATIONS

The core instrumentation can be reserved online as follows:

Fast NanoFluor II Integrated Microscope System

Olympus Confocal Microscope

Leica AOBS SP2 Confocal-Multiphoton Microscope

Scheduling can be done up to but not exceeding 6 weeks in advance. Researchers should not schedule more time than needed. Unscheduled time and unused scheduled time will be used on a first-come first-served basis and are subject to normal fees. Schedules are subject to rearrangement by the facility directors and the facility technician.

After-hours and weekend scheduling is allowed for experienced users at the facility director's discretion.

To cancel reservations, email Anna Webb (annawebb@tamu.edu).

TRAINING

The procedure for initiating the use of IMIL core instrumentation is as follows:

Demonstrations can be organized by the facility director on as-needed basis in order to illustrate the capabilities and potential applications of the core instrumentation.
Interested investigators can then talk further with the facility director regarding the feasibility of their proposed experiments.
After appropriate training, the experiments can then be conducted by the investigator or members of his/her research group.

Trainings on each core instrument are offered quarterly. To sign up for a training, contact Anna Webb (annawebb@tamu.edu).

Before users can complete instrumentation training, they must complete a laser safety training and a fire suppression safety training. These trainings are offered by the HSC safety officer, Peter Gilmore (pgilmore@tamhsc.edu or 254-742-7024).

All users are required to complete a competency demonstration on each instrument before solo operation is allowed.

PUBLICATIONS

Gashev A. "Basic mechanisms controlling lymph transport in the mesenteric lymphatic net." Annals of the New York Academy of Sciences. 1207(S1) (2010): E16–E20.

Hai B, Zhenhua Y, Millar S, Yeon SC, Makoto MT, Andras N, Liu F. "Wnt/b-Catenin Signaling Regulates Postnatal Development and Regeneration of the Salivary Gland." Stem Cells and Development. 19(11) (2010): 1793-80.

Uddin MN, Horvat D, Childs EW, Puschett JB. "Marinobufagenin causes endothelial cell monolayer hyperpermeability by altering apoptotic signaling." American Journal of Physiology - Regulatory, Integrative and Comparative Physiology. 296 (2009): 1726-1734.

Stratman AN, Saunders WB, Sacharidou A, Koh W, Fisher KE, Zawieja DC, Davis MJ, Davis GE. "Endothelial cell lumen and vascular guidance tunnel formation requires MT1-MMP-dependent proteolysis in 3-dimensional collagen matrices." Blood. 114(2) (2009): 237-247.

Wang W, Nepiyushchikh Z, Zawieja DC, Chakraborty S, Zawieja S, Gashev A, Davis MJ, Muthuchamy M. "Inhibition of myosin light chain phosphorylation decreases rat mesenteric lymphatic contractile activity." American Journal of Physiology - Heart and Circulatory Physiology. 297(2) (2009): H726-734.

Bohlen HG, Wang W, Gashev A, Gasheva O, Zawieja DC. "Phasic contractions of rat mesenteric lymphatics increase basal and phasic nitric oxide generation in vivo." American Journal of Physiology - Heart and Circulatory Physiology. 297(4) (2009): H1319-1328.

Gashev AA, Davis MJ, Gasheva OY, Nepiushchikh ZV, Wang W, Dougherty P, Kelly KA, Cai S, von der Weid PY, Muthuchamy M, Meininger CJ, Zawieja DC. "Methods for lymphatic vessel culture and gene transfection." Microcirculation. 16(7) (2009): 615-628.

Francis H, Onori P, Gaudio E, Franchitto A, DeMorrow S, Venter J, Kopriva S, Carpino G, Mancinelli R, White M, Meng F, Vetuschi A, Sferra R, Alpini G. "H3 histamine receptor-mediated activation of protein kinase C-alpha inhibits the growth of cholangiocarcinoma in vitro and in vivo." Molecular Cancer Research. 7(10) (2009): 1704-1713.

Onori P, DeMorrow S, Gaudio E, Franchitto A, Mancinelli R, Venter J, Kopriva S, Ueno Y, Alvaro D, Savage J, Alpini G, Francis H. "Caffeic acid phenethyl ester decreases cholangiocarcinoma growth by inhibition of NF-kappaB and induction of apoptosis." International Journal of Cancer. 125(3) (2009): 565-576.

S Glaser, E Gaudio, A Rao, L Pierce, P Onori, A Franchitto, H Francis, D Dostal, JVenter, S DeMorrow, R Mancinelli, G Carpino, D Alvaro, S Kopriva, J Savage, and G Alpini. "Morphological and functional heterogeneity of the mouse intrahepatic biliary epithelium." Laboratory Investigation. 89 (2009): 456-469.

Alfano RW, Leppla SH, Liu S, Bugge TH, Meininger CJ, Lairmore TC, Mulne AF, Davis SH, Duesbery NS, Frankel AE. "Matrix metalloproteinase-activated anthrax lethal toxin inhibits endothelial invasion and neovasculature formation during in vitro morphogenesis." Molecular Cancer Research. 7(4) (2009): 452-461.

Singh VP, Le B, Khode R, Baker KM, Kumar R. "Intracellular angiotensin II production in diabetic rats is correlated with cardiomyocyte apoptosis, oxidative stress, and cardiac fibrosis." Diabetes. 57(12) (2009): 3297-3306.

Singh VP, Baker KM, Kumar R. "Activation of the intracellular renin-angiotensin system in cardiac fibroblasts by high glucose: role in extracellular matrix production." American Journal of Physiology - Heart and Circulatory Physiology. 294(4) (2009): H1675-H1684.

S DeMorrow, H Francis, E Gaudio, Y Ueno, J Venter, P Onori, A Franchitto, B Vaculin, S Vaculin, G Alpini. "Anandamide inhibits cholangiocyte hyperplastic proliferation via activation of thioredoxin 1/redox factor 1 and AP-1 activation." American Journal of Physiology. 294 (2008): G506-G519.

Fava G, Alpini G, Rychlicki C, Saccomanno S, DeMorrow S, Trozzi L, Candelaresi C, Venter J, Di Sario A, Marzioni M, Bearzi I, Glaser S, Alvaro D, Marucci L, Francis H, Svegliati-Baroni G, Benedetti A. "Leptin enhances cholangiocarcinoma cell growth." Cancer Research. 68(16) (2008): 6752-6761.

DeMorrow S, Francis H, Gaudio E, Venter J, Franchitto A, Kopriva S, Onori P, Mancinelli R, Frampton G, Coufal M, Mitchell BM, Vaculin B, Alpini G. "The endocannabinoid anandamide inhibits cholangiocarcinoma growth via activation of the noncanonical Wnt signaling pathway." American Journal of Physiology - Gastrointestinal and Liver Physiology. 295(6) (2008): G1150-G1158.

Glaser S, DeMorrow S, Francis H, Ueno Y, Gaudio E, Vaculin S, Venter J, Franchitto A, Onori P, Vaculin B, Marzioni M, Wise C, Pilanthananond M, Savage J, Alpini G. "Progesterone stimulates the proliferation of female and male cholangiocytes by an autocrine/paracrine mechanisms." American Journal of Physiology - Gastrointestinal and Liver Physiology. 295(1) (2008): G124-G136.

Childs EW, Tharakan B, Byrge N, Tinsley JH, Hunter FA, Smythe WR. "Angiopoietin-1 inhibits intrinsic apoptotic signaling and vascular hyperpermeability following hemorrhagic shock." American Journal of Physiology - Heart and Circulatory Physiology. 294(5) (2008): H2285-H2295.