Microscopy report outline
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Revision as of 03:27, 16 September 2012 by Steven Nagle (Talk | contribs)
General guidelines
- Submit one paper per group.
- Bulleted list or outline format is encouraged
- Report numerical results properly
- Indicate the units of measurement
- Include the sample size and an appropriate measure of variability, such as a range, standard deviation, or standard error
- Use the abbreviation "s.d." for standard deviation and "s.e.m." for standard error after the "±"
- For example: 1.21 ± 0.03 GW (±s.d., n=42)
- Explain how you analyzed the data
- Summarize the algorithm used for all calculations and analyses
- Provide an outline of each MATLAB (or other language) function or script you used
- Put the complete code in an appendix
- Indicate the source of any code you did not write yourself
- Discuss your results
- Compare your results to theoretical predictions, reported values from literature, or other students' results
- Explain any factors that may have affected your results
- Describe what you would do differently if you had the opportunity to do the experiment again.
- Provide a detailed, correct, and comprehensive discussion of error sources for each measurement
- Is the resulting error random or systematic?
- If the error is systematic, can you determine the sign of the effect and its approximate magnitude?
- How could the error be reduced?
- Present data properly
- Images should include a scale bar
- Plots should have a clear title; axes should be labeled with units; use a plot legend when appropriate
- See the Lab report general guidelines.
- The report should be in PDF format, submitted electronically to Stellar in advance of the deadline.
- The report title should contain the last name of each of your group members.
Part 1: Microscope construction and bright field characterization
Microscope documentation and design
- Microscope block diagram, including all optical elements and relevant distances. It is unnecessary to document the details of the mechanical construction.
- Design calculations and considerations
- Photograph of your setup (optional, but nice)
Microscope characterization
- Characterize the transmitted bright field performance of the microscope.
- Provide calculations of magnification, plus examples of images used for that purpose, and comment on the accuracy of your calculations as described below.
- After calibration, measure an appropriate size of microspheres for each of the objectives and comment on the mean and uncertainty of your measurements.
- Magnification and field of view
- Include a table with the following values for the 10X, 40X, and 100X objectives:
- Theoretical resolution
- Actual magnification by multiple measures (Air Force Target, Ronchi Ruling)1
- Actual field of view in the sample plane (FOV)1
- Comment on and quantify the uncertainty of these measurements. How can you/did you reduce uncertainty?
- Provide a few example transilluminated pictures from each objective (there is no need to provide all).
- Include a table with the following values for the 10X, 40X, and 100X objectives:
Part 2: Fluorescence microscopy characterization
- Characterize the fluorescent imaging performance of your microscope
- Provide your fluorescent reference image(s) (as an image or a surface plot (see surf command in Matlab) and provide a cross-section across the diagonal (see improfile command in Matlab)
- Correct all images for nonuniform illumination (the flat field correction). Compare to the uncorrected versions in two or three cases.
- Describe your flat-field correction procedure from recording the reference image through applying the correction.
- Also provide histograms of at least one original and corrected image pair
- Comment on your corrections and relate your results to your choices during beam expander design and construction
Part 3: Resolution, Brownian motion and stability test
Summary
- Image PSF beads and calculate resolution
- Track microspheres suspended in a solvent and measure microscope stability
- Estimate diffusion coefficients; calculate viscosities from those estimates
- Comment on/quantify uncertainty. How can you/did you improve it?
Details
- Provide a bullet point outline of image processing methodology
- Report measured resolution of 40X (or 100X) objective1
- Provide a sample of the images used for resolution estimation (overlay the fit – see plotgaussfit command)
- Provide a table with measured estimates of FWHM resolution by Gaussian fitting1 for the 40x objective
- Bullet point outline of data analysis methodology
- Comment on estimated versus theoretical value
- Stability
- Provide X-Y plots of sum and difference tracks for fixed particles.
- MSD versus time interval for sum and difference tracks1
- Bullet point outline of data analysis methodology
- Comments on observed vs. expected data trend
- Viscosity samples
- Estimate diffusion coefficient, viscosity and other mechanical properties for each sample
- Comment on results, specifically how they are influenced by microscope stability, resolution,
- Comment extensively on sources of error and approaches to minimize them, both utilized and proposed
- Bullet point outline of all calculation and data processing steps
Part 4: 3T3 experiments
- Report your findings on 3T3 actin visualization and cytoplasm microrheology
- Quantify your investigations of the actin structure.
- Quantify your investigations of the cytoplasm/cytoskeleton microrheology.
- Comment on/quantify uncertainty. How can you/did you improve it?
1Remember to include uncertainty and a discussion of error sources for all numerical results.