Microscopy Facility

Frequently Asked Questions

General Questions

Our services are available to all Penn State departments on all campuses and to researchers outside the University.

The fee schedule for university affiliated researchers is subsidized by the Huck Institute for Life Sciences, which allows for much lower prices than the industry standard.

Non-university researchers cannot take advantage of the subsidized fee schedule.

  1. Contact us to request an appointment.
  2. Your first appointment will be a planning session
    • Future appointments will be scheduled at that time.
    • Be sure to bring along background literature illustrating the type of information you intend to collect.
  3. Register for a User account and add a billing account in the Research Instrumentation Management System (RIMS).

Billing rates vary according to the instrumentation. There is no charge for consultation on techniques, experimental design or subsequent data analysis.

Bills are generated each month electronically.

Estimate cost of experiments

  • Have students routinely estimate the cost of experiments... animals, reagents, antibodies, bench time, and facility time.
  • It is a great learning experience and useful for grant writing and running a lab in the future.
  • Carelessness does cost money.

Choose appropriate microscope

  • Confocals cost $35-40 per hour.
  • Widefield digital microscopes cost $20-25 per hour.
  • For data collection you often do not need the perfect picture.

Think ahead

  • Visit with us about experimental design, controls and proper samples for setup.
  • The more prepared you are, the faster you can collect data.

It depends on what you want to do. We offer training on equipment use and sample preparation. Various University safety courses are also recommended or required.

Equipment use and sample preparation

We provide training on an individual basis. Training is usually divided into several 1-2 hour sessions each encompassing specimen preparation and basic equipment use.

Required safety training

If uranyl acetate is used in your protocol, you need to complete the Penn State radionucleotide safety training and exam.

Recommended safety training

All persons working in a laboratory should attend the chemical safety training seminar, a free course given by Environmental Health and Safety at Penn State.

It depends on the type and number of samples you want to analyze.

TEM experiments

Usually take several days to one week of specimen preparation and several hours of microscope time.SEM experimentsNormally take 1-3 days, with the vast majority of this time being devoted to sample preparation.Cell sorters

  • Setup of the instrument settings and sorting gates will take approximately 30 minutes for each session.
  • Cells can be run at a maximum of about 12,000 cells/second if the concentration is high enough. We find that cells at a concentration of 10-million/mL work well for most people, but the ideal concentration will depend on the cell type and at what concentration the cells aggregate.
  • The total time required will depend on how many cells you desire to collect and the efficiency of the sort. In general, DOUBLE the time that you calculate it would actually take to collect the number of cells you want, because sorting is not 100% efficient, and cells will be lost due to aggregation, filtering, etc.

Confocal and Widefield microscopes

  • Setup can take 30-60 minutes, depending on complexity of the sample.
  • A reasonably high Nyquist limit z-scan will take about 5 minutes.

Data collection / analysis

  • Fees are charged for the collection of data.
  • No additional charge to analyze the images and flow data on laboratory computers with the appropriate analysis software.
    • We have licensed copies of popular software for data analysis.

It's your data; you should take it with you and archive it in multiple places. The facility keeps temporary copies depending on whether its flow cytometry or microscopy data.

You are responsible for storing your data!

Microscopy

We do not archive imaging data. Users are expected to back up all images.

Typically, files are moved from the optical microscopes to one of the lab computers after one week. For your convenience, they reside on the lab computers for one month for easy image manipulation. Do not consider this archiving. These computers are heavily used and can fail wiping out all data on them. You should always have your own copy of data.

Images must be transferred to the user's PASS spaceUSB drives are not allowed on the TEM computer.

Most likely you will have many citations from your own literature search. Some of the sources of information that we have used to develop general protocols are listed below.

  • Bell, P.B., 1984, Scanning Electron Microscopy of Cells in Culture, SEM, Inc. AMF O’Hare, IL.
  • Bozzola, John J. and Lonnie D. Russell 1999, Electron Microscopy: Principles and Techniques for Biologists, 2nd Ed., Sudbury, Ma.
  • Dykstra, M. J., 1992, Biological Electron Microscopy: Theory, Techniques and Troubleshooting, Plenaum Press, New York, NY.
  • Cross, P.C. and K.L. Mercer, 1993, Cell and Tissue Ultrastructure: A Functional Perspective. W.H. Freeman and Company, New York, NY.
  • Giberson, R.T. and Richard S. Demaree, 2001, Microwave Techniques and Protocols, Humana Press, Totowa, NJ.
  • Goldstein, J.I., D.E. Newbury, P. Echlin, D.C. Joy, C. Fiori, and E. Lifshin, 1981. SEM and X-Ray Microanalysis: A Text for Biologists, Material Scientists and Geologists, Plenum Press, New York, NY.
  • Hall, J.L. and C. Hawes, 1991, Electron Microscopy of Plant Cells, Academic Press, San Diego.
  • Hayat, M.A., 1989, Principles and Techniques of Electron Microscopy: Biological Applications, 3rd Ed., CRC Press, Inc. Boca Raton, FL.
  • Kok, L.P. and E. Boon. 1992. Microwave Cookbook for Microscopists, 3rd Ed. Coulomb Press, Leyden, Leiden, Netherlands.
  • Lee, R.L., 1993, Scanning Electron Microscopy and X-Ray Microanalysis, PTR Prentice-Hall Inc, Englewood Cliffs, NJ.
  • Rochow, T.G. and P.A. Tucker, Introduction to Microscopy by Means of Light, Electrons, X-Rays, or Acoustics, 2nd Ed., Plenum Press, New York, NY.
  • Rodin, J.A.G., 1977, Histology: A Text and Atlas, University Press, New York, NY.
  • Severs, N.J. and D.M. Shotton, Ed. 1995 Rapid Freezing, Freeze Fracture and Deep Etching: Techniques in Modern Biomedical Microscopy, Wiley-Liss, New York, NY.
  • Venable, J.H. and Coggeshall, R. 1965, A Simplified Lead Citrate Stain for Use in EM. , J Cell Biol, 25: 407-408.

Safety issues

Many of the chemicals used in electron microscopy protocols are hazardous. If handled improperly they may cause serious health problems. The purpose of raising this issue is not to scare or discourage anyone, but rather to educate users to properly and safely handle these chemicals. The regulations should be considered non-negotiable.

There is a zero tolerance policy for unsafe or careless handling of chemicals or equipment in the facility.

Recommendations

It is recommended that all persons working in a laboratory attend the chemical safety training seminar, a free course given by Environmental Health and Safety at Penn State.

Policies

  • All fixatives, solvents and embedding media must be handled in a hood with caution.
  • Assume all chemicals are hazardous, regardless of label, handle with caution.
  • Do not pour anything down the drain unless specifically directed to do so by a staff member.

More Resources

  • Smithwick, EB (1985) Cautions, Common Sense, and Rationale for the Electron Microscopy Laboratory. Journal of Electron Microscopy Technique 2: 193-200.
  • Barber, VC (1984) Safety in the Scanning Electron Microscopy Laboratory - 1984 Update. Scanning Electron Microscopy IV: 1719 - 1722

Are any radioactive materials used in the Facility?

Uranyl acetate is used in some of the staining procedures for TEM.  Uranyl acetate is radioactive, but when handled properly, does not pose a danger to the user.

How should I handle it?

If uranyl acetate is used in your protocol, you must use it with caution and dispose of it properly.  When working with radioactive materials, you must be careful not to contaminate other parts of the lab. There is a lead cabinet in the EM Facility for disposal of radioactive waste.

Required Training

Environmental Health and Safety requires all personnel working with radioactive materials (including uranyl acetate) to complete the radionucleotide safety training and exam.

Links

Books

The facility also maintains an extensive library and data base of existing protocols. We provide short-term loan of books.

  • Shapiro, H.M. (2003) Practical Flow Cytometry, Fourth Ed., John Wiley and Sons, Hoboken, NJ.
  • Hayat, M.A. (2000) Principles and Techniques of Electron Microscopy: Biological Applications, Fourth Ed., Cambridge University Press, Cambridge, UK
  • Bozzola, J.J. and Russell L.D. (1999) Electron Microscopy: Principles and Techniques for Biologists, 2nd Ed. Jones and Bartlett Publishers, Boston, MA.
  • Hall, J.L. and C. Hawes (1991) Electron Microscopy of Plant Cells, Academic Press, San Diego
  • Dykstra, M. J. (1992) Biological Electron Microscopy: Theory, Techniques and Troubleshooting, Plenaum Press, New York, NY.
  • Severs, N.J. and D.M. Shotton, Ed. (1995) Rapid Freezing, Freeze Fracture and Deep Etching: Techniques in Modern Biomedical Microscopy, Wiley-Liss, New York, NY
  • Rochow, T.G. and P.A. Tucker, Introduction to Microscopy by Means of Light, Electrons, X-Rays, or AcousticsSecond Ed., Plenum Press, New York, NY
  • Lee, R.L. (1993) Scanning Electron Microscopy and X-Ray Microanalysis, PTR Prentice-Hall Inc, Englewood Cliffs, NJ
  • Bell, P.B. (1984) Scanning Electron Microscopy of Cells in Culture SEM, Inc. AMF O'Hare, IL
  • Goldstein, J.I., D.E. Newbury, P. Echlin, D.C. Joy, C. Fiori, E. Lifshin (1981) SEM and X-Ray Microanalysis: A Text for Biologists, Material Scientists and Geologists, Plenum Press, New York, NY
  • Rodin, J.A.G. (1977) Histology: A Text and Atlas, Oxford University Press, New York, NY
  • Cross, P.C. and K.L. Mercer (1993) Cell and Tissue Ultrastructure: A Functional Perspective. W.H. Freeman and Company, New York, NY

Other Questions

Here are four examples of preparation protocols: for TEM and SEM of animal tissue, and for TEM and SEM of plant tissue.

Transmission electron microscopy (TEM) sample preparation

Animal tissue TEM preparation
  1. Primary Fixation: Immerse tissue blocks (usually 1mm3) in 2.5% glutaraldehyde in 0.1M Cacodylate buffer, pH 7.4 for 2 hours
  2. Primary Wash: 3 washes (each 5 minute duration) in 0.1 M cacodylate buffer with 4% sucrose, pH 7.4
  3. Secondary Fixation: Place sample in 1% osmium tetroxide in 0.1M cacodylate buffer, pH 7.4 for 1 hour
  4. Secondary Wash: 3 washes (each 5 minute duration) in 0.1 M cacodylate buffer, pH 7.4
  5. En Block Staining (optional): Stain samples with 1% uranyl acetate in 25% ethanol or water for 1 hour
  6. Dehydration: 1 x 10 min. in 50% ethanol1 x 10 min. in 70% ethanol1 x 10 min. in 90% ethanol1 x 10 min. in 95% ethanol3 x 10 min. in 100% ethanol (EM grade)3 x 10 min. in acetonitrile
  7. Infiltration and Embedding: Place sample in 50/50 acetonitrile/spurr’s resin for 1 hourPlace sample in 100% spurr’s resin for 1.5 hours – repeat once. Orient samples into mold and add resin
  8. Polymerization: Place mold containing samples in 60°C oven for 24 hours.
Plant tissue TEM preparation
  1. Primary Fixation: Immerse sample in 2.5% glutaraldehyde in 0.1M cacodylate buffer for 24 hours at 4oC
  2. Primary Wash: 3 washes (each 5 minute duration) in 0.1 M cacodylate buffer, pH 7.4
  3. Secondary Fixation: Place sample in 1% osmium tetroxide in 0.1M cacodylate buffer, pH 7.4 for 1 hour
  4. Secondary Wash: 3 washes (each 5 minute duration) in 0.1 M cacodylate buffer, pH 7.4
  5. En Block Staining (optional): Stain samples with 2% aqueous uranyl acetate for 1 hour or more.
  6. Dehydration: 2 x 10 min. in 50% ethanol2 x 10 min. in 70% ethanol2 x 10 min. in 90% ethanol2 x 10 min. in 95% ethanol3 x 10 min. in 100% ethanol (EM grade)3 x 10 min. in acetonitrile
  7. Infiltration: Place sample in 50/50 acetonitrile/spurr’s resin for several hours or overnight Place sample in 75/25 acetonitrile/spurr’s resin for several hours or overnightPlace sample in 100% spurr’s resin for several hours or overnight, repeat once.
  8. Polymerization: Place mold containing samples in 60°C oven for 24 hours.

Scanning electron microscopy (SEM) sample preparation

Animal tissue SEM preparation
  1. Primary Fixation: Immerse sample in 2.5% glutaraldehyde in 0.1M Cacodylate buffer, pH 7.4 for 2 hours at room temperature or at 4° C (in refrigerator) overnight.
  2. Primary Wash: 3 washes (each 5 minute duration) in 0.1 M cacodylate buffer pH 7.4
  3. Secondary Fixation: Immerse sample in 1% osmium tetroxide (aqueous) pH 7.4 for 1 hour at room temperature and in a light tight container.
  4. Secondary Wash: 3 washes (each 5 minute duration) in 0.1 M cacodylate buffer pH 7.4
  5. Dehydration: 1 x 10 min. in 25% ethanol1 x 10 min. in 50% ethanol1 x 10 min. in 70% ethanol1 x 10 min. in 85% ethanol1 x 10 min. in 95% ethanol2 x 10 min. in 100% ethanol1 x 10 min. in 100% ethanol (EM grade)
  6. Critical Point Dry: This automated process takes approximately 40 minutes to complete.
  7. Mounting: Mount the sample, which is now completely dried, onto metal stub with double sided carbon tape.
  8. Sputter Coating: Finally, apply a thin layer of metals (gold and palladium) over the sample using an automated sputter coater.  This process takes about 10 minutes.
Plant tissue SEM preparation
  1. Primary Fixation: Immerse sample in 2.8% glutaraldehyde in 0.1M Hepes buffer, pH 7.2 (with 0.02% Triton X-100), for several hours at room temperature or overnight at 4°C.
  2. Primary Wash: 3 washes (each 5 to 10 minute duration) in 0.1 M Hepes buffer, pH 7.2
  3. Dehydration: 1 x 10 min. in 25% ethanol1 x 10 min. in 50% ethanol1 x 10 min. in 70% ethanol1 x 10 min. in 85% ethanol1 x 10 min. in 95% ethanol2 x 10 min. in 100% ethanol1 x 10 min. in 100% ethanol (EM grade)
  4. Critical Point Dry: This automated process takes approximately 40 minutes to complete.
  5. Mounting: Mount the sample onto metal stub with double sided carbon tape.
  6. Sputter Coating: Finally, apply a thin layer of metals (gold and palladium) over the sample using an automated sputter coater.  This process takes about 10 minutes.

Software for analyzing fluorescent microscopy images

Obtain a copy of FV viewer

  1. Stop by the lab and check-out an installation disk
  2. Install it on your computer
  3. Return the installation disk to the lab 

System Requirements

  • Windows XP, Vista, or 7
  • 2 megs of memory (RAM)
  • 100 megs of free disk space

Capabilities

  • FV Viewer is a lite version of the same software installed on the microscope.
  • This software will read the data captured by the FV1000/FV500/FV300.
  • Images saved with the FV file format (OIF, OIB, FV Multi-Tiff) can be read.
  • An export function exists to create copies of your images in common image file formats (BITMAP, JPEG, PNG, TIFF etc.).
  • Image capture, measurement, analysis, 3D function, etc. cannot be used by this version.