Difference between revisions of "Microscopy report outline"

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

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, 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
    • Calculate the actual field of view (FOV)¹
  • Comment on and quantify the uncertainty of these measurements. How can you/did you improve it?
  • Provide a few example transilluminated pictures from each objective (there is no need to provide all)

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

Parts 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

• Bullet point outline of image processing methodology
• Measured resolution of 40X (or 100X) objective¹
  • Images used for resolution estimation (preferably with fit – see plotgaussfit command)
  • Estimate of FWHM by Gaussian fitting¹
  • Bullet point outline of data analysis methodology
  • Comments on estimated versus theoretical value
• Stability
  • 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
• X-Y plots of tracks for all samples
• MSD versus time interval plots for measured samples
• Estimate of diffusion coefficient, viscosity and other mechanical properties for each sample
• Comments on results, specifically 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.