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IMAGING SPECIALIST POSITION AT UCSF

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Hilary Beggs

IMAGING SPECIALIST POSITION AT UCSF

The Beckman Vision Research Center at the University of California, San Francisco is
seeking a qualified and motivated candidate to act as a dedicated expert in confocal
microscopy and digital imaging for our NEI-funded Core facility.

The Imaging Specialist will provide training to users on Zeiss Pascal laser scanning
microscopes, as well as assistance with image acquisition and analysis, sample preparation,
and troubleshooting. The candidate will also update and optimize imaging protocols, and
oversee maintenance of several confocal microscopes as well as standard microscopes
equipped with CCD cameras. Opportunities for individual projects and authorship on
publications are possible for motivated individuals. The candidate will also serve as an
interface between our Core facility and other imaging facilities at UCSF.

The variety of projects in the Vision Core will require the Imaging Specialist to be creative,
versatile and up to date on a wide variety of imaging techniques and analysis methods.
Minimum requirements include a Bachelors or Masters degree in biological sciences,
laboratory experience, meticulous technique, and strong communication and people skills.
Preference will be given to candidates with experience in live cell, tissue slice or whole
mount imaging, as well as experience with image analysis and quantitation (Image J,
Imaris/Bitplane, MatLab, Metamorph).

Please send CV, cover letter, and names of 3 references by email or mail to:

Hilary Beggs, Ph.D.
University of California, San Francisco (UCSF)
Beckman Vision Center
10 Koret Way, Rm K127
San Francisco, CA 94143-0730
Email: [hidden email]

Salary is commensurate with experience.

Jerry Sedgewick-2

Re: SHG of Collagen Gels

I have been tearing the little hair left out of my head trying to figure
out why I can get great images of forward scattered SHG of collagen from
cow's knees, but give me a collagen gel and I can't see a thing. Both
are type 1 collagen, what should work. I've tried wavelengths from
870nm (where I get the best signal) to 920nm, used both half- and
quarter-waveplates individually and together, and so on.

I have asked about the gel preparation, and, at first, these were given
to me after they matured overnight, but now we are trying gels that have
matured for a week. I'm not doing the prep, and so I'm in the dark
about the procedure.

This is on a custom built device channeled through an Olympus FV300
head, an Olympus BX61WI and an Olympus 60x 0.9 N.A. water immersion lens.

Any ideas?

Jerry

--
Jerry (Gerald) Sedgewick
Program Director, Biomedical Image Processing Lab (BIPL)
Department of Neuroscience, University of Minnesota
312 Church St. SE, 1-205 Hasselmo Hall
Minneapolis, MN 55455
(612) 624-6607
[hidden email]
http://www.bipl.umn.edu
Author: "Scientific Imaging with Photoshop: Methods, Measurement and Output."

Rawlight.com (dba Sedgewick Initiatives)
965 Cromwell Avenue
Saint Paul, MN 55114
[hidden email]
(651) 308-1466
http://www.quickphotoshop.com
http://www.heartFROMstone.com
http://www.rawlight.com

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Charras, Guillaume

Re: SHG of Collagen Gels

I would try reflection. With the FV-1000 you can do that quite easily with
the light path builder.

Peter Friedl has done some nice imaging of cells moving in collagen gels
and all of his early images of collagen gels are using reflection.

Cheers,
Guillaume

On Nov 8 2008, Jerry Sedgewick wrote:

> I have been tearing the little hair left out of my head trying to figure
> out why I can get great images of forward scattered SHG of collagen from
> cow's knees, but give me a collagen gel and I can't see a thing. Both
> are type 1 collagen, what should work. I've tried wavelengths from
> 870nm (where I get the best signal) to 920nm, used both half- and
> quarter-waveplates individually and together, and so on.
>
> I have asked about the gel preparation, and, at first, these were given
> to me after they matured overnight, but now we are trying gels that have
> matured for a week. I'm not doing the prep, and so I'm in the dark
> about the procedure.
>
> This is on a custom built device channeled through an Olympus FV300
> head, an Olympus BX61WI and an Olympus 60x 0.9 N.A. water immersion lens.
>
> Any ideas?
>
> Jerry
>
>

--
___________________________________
Guillaume Charras
Royal Society University Research Fellow
London Centre for Nanotechnology and Department of Physiology
University College London
17-19 Gordon street
London, WC1H 0AH
UK

Guenter Giese

Re: SHG of Collagen Gels

In reply to this post by Jerry Sedgewick-2

Jerry,

could you please provide us with some more details concerning your equipment (multiphoton laser, filters /mirrors in the excitation as well as in the transmission path etc.)

We made good experiences with the configuration described in:
Han, M., Giese, G., and Bille, J. F. (2005). Second harmonic generation imaging of collagen fibrils in cornea and sclera. Optics Express 13, 5791-5797.

Briefly:

ZEISS LSM510 NLO
Axioskop 2 FS MOT
COHERENT Chameleon XR at 800 nm
40x/0.8 or 63x/1.0 ZEISS water dip lens

Transmission path (forward SHG):
ZEISS NA1.4 oil condensor
Two IR beam block filters (Zeiss KP685)
bandpass filter (400/10 nm)

Epifluorescence path (backward SHG):
Two IR beam block filters (Zeiss KP685)
bandpass filter (400/10 nm)

I experienced huge differences in multiphoton performance of objectives (differing by a factor of up to 10 at the same NA / immersion type). Do you have access to comparable objectives (ZEISS objectices should fit, and the ZEISS 63x/1.0 water dip lens, e.g., performs very well in SHG imaging)

Did you check for possible effects of some polarizing optics in your beam path?

Best,

Guenter

Dr. Guenter Giese
Light Microscopy Facility
Dept. of Biomedical Optics
MPI fuer Medizinische Forschung
D-69120 Heidelberg, Germany
phone (xx49 or 0)-6221-486-360 (fax -325)
e-mail: [hidden email]

----- your message -----
from: Jerry Sedgewick <[hidden email]>
date: Sat., 8. November 2008, 2:00
cf: Re: SHG of Collagen Gels
to: [hidden email]

> I have been tearing the little hair left out of my head trying
> to figure
> out why I can get great images of forward scattered SHG of
> collagen from
> cow's knees, but give me a collagen gel and I can't see a
> thing. Both
> are type 1 collagen, what should work. I've tried
> wavelengths from
> 870nm (where I get the best signal) to 920nm, used both half-
> and
> quarter-waveplates individually and together, and so on.
>
> I have asked about the gel preparation, and, at first, these
> were given
> to me after they matured overnight, but now we are trying gels
> that have
> matured for a week. I'm not doing the prep, and so I'm in
> the dark
> about the procedure.
>
> This is on a custom built device channeled through an Olympus
> FV300
> head, an Olympus BX61WI and an Olympus 60x 0.9 N.A. water
> immersion lens.
>
> Any ideas?
>
> Jerry
>
> --
> Jerry (Gerald) Sedgewick
> Program Director, Biomedical Image Processing Lab (BIPL)
> Department of Neuroscience, University of Minnesota
> 312 Church St. SE, 1-205 Hasselmo Hall
> Minneapolis, MN 55455
> (612) 624-6607
> [hidden email]
> http://www.bipl.umn.edu
> Author: "Scientific Imaging with Photoshop: Methods, Measurement
> and Output."
>
> Rawlight.com (dba Sedgewick Initiatives)
> 965 Cromwell Avenue
> Saint Paul, MN 55114
> [hidden email]
> (651) 308-1466
> http://www.quickphotoshop.com
> http://www.heartFROMstone.com
> http://www.rawlight.com
>
>
>
>
> --- Get FREE High Speed Internet from USFamily.Net! --
> http://www.usfamily.net/mkt-freepromo.html ---

----- Ursprüngliche Nachricht -----
Von: Jerry Sedgewick <[hidden email]>
Datum: Samstag, 8. November 2008, 2:00
Betreff: Re: SHG of Collagen Gels
An: [hidden email]

J. Paul Robinson

Re: SHG of Collagen Gels

In reply to this post by Jerry Sedgewick-2

A long time ago (1999-2004) we did a whole lot of work on collagen and
collagen gels...using reflection confocal - i think it was some of the
earlier work in the field...we have some really fantastic images, and 3d
reconstructs, videos, etc...I think most of the papers below are
available for download on our website see below....all done I might add
on our Biorad 1024 (UV/viz) - frankly one of the best confocals ever
built! Pity it was killed off!

There are also a bunch of videos associated with the first paper below
showing real time fibrilogenesis
http://www.cyto.purdue.edu/flowcyt/research/imaging/tlrecm.htm

regards

Paul Robinson
Purdue

http://www.cyto.purdue.edu/flowcyt/research/pub1.htm

Title: Time-lapse confocal reflection microscopy of collagen
fibrillogenesis and extracellular matrix assembly in vitro
Author: A. O. Brightman, B. P. Rajwa, J. E. Sturgis, M. E. McCallister,
J. Paul Robinson, S. L. Voytik-Harbin
Publication: Biopolymers, Vol. 54, 222-234, 2000

Title: Three-dimensional imaging of extracellular matrix and
extracellular matrix-cell interactions.
Author:Voytik-Harbin SL, Rajwa B, Robinson JP.
Publication:Methods Cell Biol. ;63:583-97, 2001

Title: Tensile mechanical properties of three-dimensional type I
collagen extracellular matrices with varied microstructure.
Author: Roeder, B.A., Kokini, K., Sturgis, J.E., Robinson, J.P., and
Voytik-Harbin, S.L.
Publication: J Biomech Eng 124:214-222, 2002.

Title: Analysis of Orientations of Collagen Fibers by Novel
Fiber-Tracking Software.
Author: Jun Wu, Bartlomiej Rajwa, David L. Filmer, Christoph M.
Hoffmann, Bo Yuan, Ching-Shoei Chiang, Jennie Sturgis, and J. Paul Robinson
Publication: Microscopy and Microanalysis 9, 574–580, 2003

Title: Simultaneous mechanical loading and confocal reflection
microscopy for 3D micro-biomechanical analysis of biomaterials and
tissue constructs.
Author: Voytik-Harbin, S.L., Roeder, B.A., Sturgis, J.E., Kokini, K.,
and Robinson, J.P.
Publication: Microscopy and Microanalysis 9(1):74-85. 2003.

Title: Local, three-dimensional strain measurements within largely
deformed extracellular matrix constructs.
Author Roeder BA, Kokini K, Robinson JP, Voytik-Harbin SL.
Publication: J Biomech Eng. 2004 Dec;126(6):699-708.

Roeder BA, Kokini K, Sturgis JE, Robinson JP, Voytik-Harbin SL.
Abstract
Tensile mechanical properties of three-dimensional type I collagen
extracellular matrices with varied microstructure.
J Biomech Eng. 2002 Apr;124(2):214-22.

Jerry Sedgewick wrote:

> I have been tearing the little hair left out of my head trying to figure
> out why I can get great images of forward scattered SHG of collagen from
> cow's knees, but give me a collagen gel and I can't see a thing. Both
> are type 1 collagen, what should work. I've tried wavelengths from
> 870nm (where I get the best signal) to 920nm, used both half- and
> quarter-waveplates individually and together, and so on.
> I have asked about the gel preparation, and, at first, these were given
> to me after they matured overnight, but now we are trying gels that have
> matured for a week. I'm not doing the prep, and so I'm in the dark
> about the procedure.
> This is on a custom built device channeled through an Olympus FV300
> head, an Olympus BX61WI and an Olympus 60x 0.9 N.A. water immersion lens.
>
> Any ideas?
>
> Jerry
>

--
J. Paul Robinson
SVM Professor of Cytomics
Professor of Immunopharmacology & Biomedical Engineering
Director, Purdue University Cytometry Laboratories
Past-President, International Society for Analytical Cytology
(now International Society for Advancement of Cytometry)

Purdue University Cytometry Laboratories
Bindley Bioscience Center
1203 West State Street
Discovery Park, Purdue University
West Lafayette, IN 47907-2057
Ph (765) 494 0757; Fax (765) 494 0517
email: [hidden email]
www.cyto.purdue.edu

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