Difference between revisions of "Microscopy report outline"

General guidelines

• 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.

• 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

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).

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 log(y) scaled 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. How do these choices relate to your intended experiments in weeks three and beyond?

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 reduce it?

Details

• Report measured resolution of 40X 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.
  • Provide a 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.
  • Provide plots of MSD versus time interval for sum and difference tracks¹.
  • Provide a bullet point outline of your data analysis methodology.
  • Comment on observed vs. expected data trend.

• Viscosity samples
  • Estimate diffusion coefficient, viscosity for each sample.
  • Comment on results, specifically how they are influenced by microscope stability and resolution.
  • Comment extensively on sources of error and approaches to minimize them, both utilized and proposed.
  • Provide a 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.
  • Use an application such as ImageJ to measure observed min/max separation between centers of the actin strands.
  • Measure min/max observed lengths of actin strands.
  • Comment on the effect of actin density as it relates to bead size in the microrheology portion.
  • If you wish, reference your favorite article to compare your images.
• Quantify your investigations of the cytoplasm/cytoskeleton microrheology.
  • Present your MSD vs sampling time (tau) data in log-log form and comment.
  • Use your MSD vs tau data to calculate estimates of G' and G" as described in Microrheology Measurements via Particle Tracking.
  • Compare your results to the literature provided and/or any other relevant literature.
• 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.