DEPARTMENT OF PATHOLOGY CONFOCAL MICROSCOPY CORE

CONFOCAL NEWSLETTER
V OLUME 1, I SSUE 1 A UGUST 2008

O LYMPUS FV1000 MPE C ONFOCAL M ICROSCOPE

The Olympus FV1000 is a state-of-the-art multi-photon confocal laser scanning microscope with 5 fluorescent laser lines from 405 to 635 nm and with pinhole detectors for imaging up to 3 colors simultaneously as well as a detector for phase contrast type imaging. A unique feature of the FV1000 is that it operates in the 2-photon excitation mode which allows the fluorescent imaging of living cells, of explanted living tissues, and even of tissues and organs in living animals. The 2-photon functionality allows penetration depths of hundreds of microns into tissues which is achieved without significant photo-damage making time-lapse studies of living cells and tissues feasible. The FV1000 system has an excellent selection of objective lenses from 5x to 60x including

Confocal Microscopy Core Support Services

The Confocal Core is supported on a day-to-day basis by faculty and staff in the Department of Pathology. As Director of the Core, Dr. Ryerse oversees the management of the core and assists researchers in planning projects, obtaining and interpreting images and with the preparation of images for publication and presentations. The day-to-day maintenance, operation and new user training is the responsibility of Ms. Alice Rickard who is available on a 95, 5 day-a-week basis to assist users with questions and operational issues. Dr. Ryerse is in daily contact with Alice Rickard and will assist in user training and operational issues as required. We are also very fortunate to have the expertise of Mr. Bill Petersen from Olympus to help with questions about specific research projects and hardware and software issues. Bill lives in the area and will come to work with the confocal staff and/ or individual researchers.

The new state-of-the-art Olympus FV1000 2-photon confocal microscope is located in Doisy Research Center several immersion objectives which can be dipped into culture media or physiological saline. Optical zoom provides magnifications up to 100x and beyond to meet the customized needs of all users. The Olympus FV1000 MPE comes with a full complement of software for customizing and storing userspecific settings and a second work station for image processing and analysis.

R ESEARCH M ICROSCOPY S ERVICES F ACILITY

Researchers with microscopy projects might like to know that Jan Ryerse and Barbara Nagel have initiated a fee-for-service basic research microscopy facility in the Pathology Department. We guarantee rapid turnaround times for sample processing and will assist researchers in designing experiments, evaluating results, and in organizing and labeling micrographs for publications, seminars and grant proposals. Digital imaging is provided for all types of light and electron microscopy including bright field, phase and Nomarski LM, TEM, SEM, epi- and confocal immuno-fluorescence, EM immuno-gold labeling and negative staining. Contact Jan Ryerse (ryersejs@slu.edu) or Barb Nagel (nagelba@slu.edu) for a price schedule, a consultation or to submit samples.

A CONFOCAL WEBSITE is under construction which will include operating instructions, technical information, online sign-up calendars, helpful hints, confocal images and other information for confocal users. Stay tuned.

C ONFOCAL M ICROSCOPY S EMINAR

Dr. Ryerse will present a seminar on confocal microscopy. 12 Noon Monday September 22nd Learning Resource Center Room 113

Confocal Support Contact Information

Dr. Jan Ryerse Doisy Hall R514 (office) 314-497-8549 (cell 24/7) 314-977-7848 (office) ryersejs@slu.edu Alice Rickard 11th Floor Desloges Towers 314-268-5220 rickarda@slu.edu
USER FEES for trained users from 8am to 5pm weekdays is $30.00 per hour billed in half-hour increments and $15 per hour for evenings and weekends. The charge is $60.00 per hour when Jan Ryerse or Alice Rickard is present training users or working on projects.

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CONFOCAL NEWSLETTER

B IO R AD 1024 C ONFOCAL M ICROSCOPE ( CONT D

FROM

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The BioRad lacks a laser line for DAPI; however, propidium iodide staining and the far red detector can be used to image cell nuclei. Over the years many researchers have been trained on the BioRad confocal microscope and have successfully used it to obtain excellent images for their research publications, seminars and grant proposals. Although this is an older microscope it continues to provide good service and excellent images.

The BioRad confocal microscope is presently located in 1120 Desloges Towers but soon will be re-located to Room 107A

I MAGES FROM THE B IO R AD C ONFOCAL M ICROSCOPE

In the upper panel a protein (left side, green) is being synthesized by 2 transfected cells and transported into the cell nuclei. In the center image in the upper panel all of the cell nuclei in the field have been stained with propidium iodide which emits in the red wavelength. The yellow color in the merged image on the right shows that the protein has entered the cell nuclei. A mutant version of the protein which lacks a nuclear localization signal is synthesized in several transfected cells (lower panel left). The middle image shows all the nuclei in the field stained red. As shown in the merged image on the right the protein remains outside the nuclei in small vesicles in the cell cytoplasm which otherwise is not stained in these images.
Images courtesy of Dr. G. Chinnadurai and S. Vijayalingam

RESEARCH MICROSCOPY SERVICES FACILITY Researchers with microscopy projects might like to know that Jan Ryerse and Barbara Nagel have formalized a fee-for-service basic research microscopy facility in the Pathology Department. We guarantee rapid turn-around times for sample processing and we will assist researchers in designing experiments, evaluating results, and in organizing and labeling micrographs for publications, seminars and grant proposals. We provide digital imaging for all types of light and electron microscopy including bright field, phase and Nomarski light microscopy, transmission and scanning electron microscopy, epi-fluorescence and confocal immuno-fluorescence, EM immuno-gold labeling and negative staining.

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I MAGES FROM THE O LYMPUS FV1000 MPE C ONFOCAL

A B
Do two nuclear proteins co-localize? Using indirect immuno-fluorescence staining one protein was labeled with a red fluorophore (A) and the other with a green fluorophore (B). DAPI staining shows all of the nuclei in the field in C. Merging the A and B images (D and the enlarged image) indicates that the two proteins do not colocalize except for perhaps some minor association in the do-nut holes as indicated by yellow.
Images taken by Jan Ryerse using a sample provided by Dr W. Wold and Ann Tollefson in the Microbiology Department

I MAGING L IVING A NIMALS U SING THE M ULTI -P HOTON M ODE OF THE O LYMPUS FV1000 MPE C ONFOCAL M ICROSCOPE
The 2-photon mode of the Olympus FV1000 microscope allows the deep imaging of living cells and explanted living tissues and organs without photo-toxicity effects. It even permits fluorescence imaging in living animals. A good example is the Zebrafish which is widely used as a model system for studies in developmental biology. An adult zebrafish is shown in A and a zebrafish embryo is shown in B. Using zebrafish embryos which express enhanced green fluorescent protein in the sensory ganglia, Dr. Mark Voigt and PhD student Angie LaMora in the Pharmacology Department are using the FV1000 to study how outgrowing axons find their way from the CNS to their target organs. The box in B shows the region scanned. Voigt and LaMora collect a hundred or more 1 um thick image slices in the Z-axis which are merged into a single image (C). The pathways of the axons from the ganglia at the bottom of the image to their target tissues are readily apparent. By studying mutants with abnormal pathways they are beginning to understand the complex cellular signals which direct the growth of axons to their target tissues.

B A

100 um

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HELPFUL HINTS
Fluorescence Staining of Cell Nuclei with DAPI and Propidium Iodide
DAPI should be used as a general fluorescent stain for nuclei in all samples destined for the Olympus confocal microscope. One reason is that it makes it easy to find the correct focal plane by epi-fluorescence prior to switching to the confocal operational mode. This is especially helpful for monolayers of cultured cells grown in plastic dishes, on slides or on coverslips which are difficult to focus using bright field optics or if the labeling of the target is weak. Second, being able to see the nuclei of all cells in a field from an transfection experiment provides a measure of transfection efficiency of the fluorescent construct. Third, merging the DAPI image with the fluorescent image of the target protein allows one to determine the location and the morphology of the target protein in or on the cell. DAPI staining is also equally effective with fluorescently labeled paraffin sections. One can use a commercially available mounting medium such as Vecta-Shield DAPI which, as the name implies, contains DAPI, or alternatively, you can make your own DAPI mounting medium using my recipe given below. Commercially available mounting media generally contain an anti-fade reagent. I add n-propyl-gallate to my homemade mounting solution as an anti-fade reagent. An advantage of the Vecta-Shield is that over several days it hardens to form a permanent mounting medium. Fluorescently-stained slides should be stored in the dark in a refrigerator or in a freezer. The BioRad confocal microscope does not have a laser line or filter set for DAPI, however one can use propidium iodide, which emits in the far red, to image nuclei. A formula for making and using propidium iodide is given below. There are three detectors in the BioRad - for green, red and far red emission signals. One can still use two fluorescent colors in addition to the far red propidium iodide by pseudo-coloring either the red signal or the far red signal.

DAPI MOUNTING MEDIUM 4-6-diamidino-2-phenylindole Make 1 mg/ml stock in PBS or DW Add 1 ul stock to 1 ml mounting medium of 1:1 glycerol:PBS containing 1% n-propyl gallate

PROPIDIUM IODIDE Make a 1 mg/ml stock in PBS For use dilute 1:3000 with PBS Stain sections for 5 min Wash with PBS Mount in 1:1 glycerol:PBS

Use #1.5 (0.17 mm thick) Glass Coverslips

You may never have never noticed this but glass coverslips come in different thicknesses, for example #1, #1.5, #2. The optics in the Olympus FV1000 MPE confocal are designed for use with #1.5 glass coverslips. Use #1.5 (0.17 microns thick) coverslips to obtain the best possible confocal images.

Jan Ryerse (Newsletter Editor) and Alice Rickard at the Olympus FV1000 MPE Confocal Microscope DRC Room 254A Contact information on p1