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 report: Microscope construction and bright-field characterization

Microscope design

• Draw a block diagram of your microscope, including all optical elements and relevant distances. It is unnecessary to document the details of the mechanical construction.
• Describe your design calculations and considerations.
• Illustrate your apparatus with a photograph (optional, but nice).

Microscope characterization

Describe the transmitted bright-field performance of your microscope by calculating its magnification and field of view, with each of the three objectives (10×, 40× and 100×):

• Provide examples of transilluminated images used to calculate the magnification of the microscope. It is not necessary to show an exhaustive set of all images.
• Include a table with the following values for the 10×, 40× and 100× objectives:
  • Theoretical resolution
  • Actual magnification by multiple measures (Air Force Target, Ronchi Ruling)
  • Actual field of view in the sample plane (FOV).
• Quantify the uncertainty on your measurements (with an appropriate number of significant decimal points!).
• After calibration, measure the size of microspheres (justify the size of the microspheres chosen for each of the objectives), and comment on the mean and uncertainty of your measurements.

Discussion on uncertainty

• Explain why some samples could not be well imaged with some objective(s), and whether the results differed from your expectations.
• Identify the limits of accuracy of your microscope. Indicate which sources of error contribute to the compounded uncertainty reported ("± s.d." or "± s.e.m."), which dominate, which can be compensated / improved on, and which are intrinsic to your microscope design.
• Recognize user- vs. instrument-based errors. Explain how multiple measurements improve (or not!) the accuracy of your calculations. Suggest possible design choices that would improve microscope performance.

Part 2 report: Microscope construction and fluorescence characterization

Microscope design

• Update the block diagram description of your microscope, its photograph, and annotation of pertinent optical components and distances.

Fluorescence imaging characterization

Demonstrate the epi-fluorescence imaging capability of your enhanced microscope:

• Provide the fluorescent reference image you collected with both the 40× and 100× objectives, as either an image (see imshow command in Matlab) or a surface plot (see surf command in Matlab), as well as a cross-section of signal intensity across its diagonal (see improfile command in Matlab).
• Exhibit your images of the 4 μm and 1 μm fluorescent bead samples, with both the 40× and 100× objectives. Compare and contrast these pictures before and after flat-field correction to counteract nonuniform illumination.
• 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.

Discussion on design and image quality

• 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 3a report: 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

• 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
  • Estimate diffusion coefficient, viscosity for each water-glycerin mixture 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 calculations and data processing steps.

Part 3b report: Experiments in fibroblast cells

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 plot form and comment.
  • Use your MSD vs tau data to calculate estimates of G' and G" as described in Optical Microscopy Part 3b: Microrheology Measurements in Fibroblast Cells.
  • 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.