Introduction to Cell Culture and the Nano-imaging Facility at the Nano Bio-systems and Bio-mimetics Lab

Transcription

1 Introduction to Cell Culture and the Nano-imaging Facility at the Nano Bio-systems and Bio-mimetics Lab Dr. Scott Lenaghan (Post-doc) March 2009

2 Nano Bio-systems and Bio- Housed in Dougherty 206 mimetics Lab Self-sustained research lab with cell culture and nano-scale imaging capabilities Consists of Cell Culture, Imaging, and Office facilities Primary purpose: to experimentally explore the potential for bio-inspired nanoscale research

3 Offices for Laboratory Personnel

4 Cell Culture Facility Purpose: maintain experimental cell lines for nanoscale study and imaging Types of cells: mammalian cell lines, plant cell cultures, nonairborne pathogens Approved for BL-2 organisms,

5 Cell Culture Facility

6 Cell Culture Facility (Equipment) Self-sustained lab with all equipment necessary for cell culture Two CO 2 incubators for anaerobic cell culture Forma Scientific Water Jacketed Incubator Thermo Scientific Direct heat w/ Sterilization Cycle Incubator

7 Cell Culture Facility (Equipment) Thermo Scientific Revco, Flammable and explosion proof refrigerator and freezer VWR International., Gravity Convection Oven, 32 0 c C

8 Cell Culture Facility (Equipment) Shel Lab Hybridization Chamber o C in 7 min, precise to C w/ rotisserie VWR International water Jacketed CO 2 Incubator

9 Cell Culture Facility (Equipment) Thermo Scientific 1300 Series A2 Biological Safety Cabinet, with attached vacuum pump and trap

10 Cell Culture Facility (Sample Storage) Liquid Nitrogen storage for long term storage of cells Thermo scientific 8036, cane storage and transport dewar Thermolyne Locator 8, box type dewar

11 Cell Culture Facility (Sample Storage) Terra Universal, industrial grade desiccator cabinet

12 Cell Culture Facility (Benchtop Equipment) Eppendorf 5702RH, refrigerated centrifuge with swinging bucket rotor 15 and 50 ml conical tubes, max 3000xg IEC HN-S2 Centrifuge, swinging bucket rotor for 15 and 50ml conical tubes, max ~9000xg

13 Cell Culture Facility (Benchtop Equipment) Drummond Pipet Aid Denver Instrument SI-234, Electronic Analytical balance, accurate to g, max load 230g Thermo Scientific Finnpipette II, pipets from µl

14 Imaging Facility

15 Imaging Facility Olympus Fluoview 1000 Confocal Laser Scanning Biological Microscope on Olympus IX81 inverted base Agilent technologies State-of-the-art AFM

16 For reduction of vibration crucial for AFM, the system is housed in a Herzan cabinet with a Herz DT- A series vibration dampening table Imaging Facility

17 Olympus Fluoview FV1000 Lasers: Multi-line Argon (457nm, 488nm, 515nm) HeNe gas (543nm) Violet diode (405mn) Diode (635nm) Our system does not have IR laser or UV Ar (770nm, 351nm)

18 Olympus Fluoview FV1000 (Probes) Cos-7 Cells Orange = nucleus Blue = mitochondria Green = actin Cos-7 Cells Orange = nucleus Green = actin Yellow = mitochondria (Olympus Confocal Image Gallery)

19 Olympus Fluoview FV1000 (Autofluorescence) Cherry bud blossom Rubber Tree Leaf (Olympus Confocal Image Gallery)

20 Olympus Fluoview FV1000 Strengths Allows visualization of internal structures within cells Can view live and fixed cells With proper label can specifically label structures Can take time-lapse over long time scale to track intracellular movements Z-sectioning allows for 3-D sectioning

21 Olympus Fluoview FV1000 Limitations Most biological specimen need to be labeled with a probe Bleaching often occurs Autofluorescence can complicate results

22 Olympus Fluoview FV1000 Applications for Bio Inspired Nano technology Visualization of drug delivery Tracking of particles within cells (bio machines) Potential visualization of nanoparticles (~70nm and greater)

23 Agilent Technologies AFM How does AFM work, the theory behind AFM

24 Agilent Technologies AFM What is unique about our system? Increased Z-depth Advanced laser alignment and calibration Increased resolution due to superior design and manufacturing Integration with a confocal system for simultaneous imaging of surface and internal structure of specimen

25 Agilent Technologies AFM Imaging with AFM generates surface topography DNA Breast Cancer Tissue Pacific Nanotechnology, Inc.

26 Agilent Technologies AFM AFM can also be used for force measurements and determining adhesive strength

27 Agilent Technologies AFM Potential applications for imaging and force determination in biological systems Imaging of membrane interactions with foreign particles Indentation of membranes to determine membrane strength Introduction of wounds with tip, and observation of repair

28 Functional AFM Attaching a ligand, nanoparticle, receptor, etc. to a tip and then measuring adhesive forces of this functionalized tip Can be used for receptor binding Nanoparticle interaction with surfaces Bonding strength of chemicals, and specific functional groups

29 Agilent Technologies AFM Strengths of AFM Nano resolution for imaging of particles Can measure nano-scale force measurements for interactions including weak forces such as van der Waals forces Functional AFM can measure binding kinetics of any attached particle

30 Agilent Technologies AFM Weaknesses of AFM Cannot see internal cellular structure, only topographical mapping Samples must be strongly adhered to a surface (can be difficult for biologicals) Probe can damage live cells and destory membranes

31 Unique Nature of Nano Bio- systems and Bio-mimetics Lab One of the only systems that combines simultaneous imaging of both confocal and AFM State-of-the-Art platform for looking at the nano-scale interior and exterior of a cell Diverse group with experience in aspects of engineering, biology, and chemistry

32 Questions?