Microscopy report outline

This lab requires 4 submissions, one for each part of the lab. Use the outline below. The report should begin with a section that documents the apparatus you built. This should be followed by one section for each measurement you make. The measurement sections should include subsections that detail the procedure you used, data you gathered, analysis you did, and results you obtained. You may revise any part of your report until the final deadline. Update the apparatus section to reflect any changes you make during the course of the lab.

Keep your lab report short.

In this lab, you will make dozens of images. Don't include all of them in your report. Where images are required, include one or a few that convey the character of the dataset. Size images appropriately. Few images or plots contain sufficient stunning and relevant detail that they merit half a page. A good trick for reducing plot size while maintaining clarity is to adjust the font size and line values to ensure that the plot remains clear even when it is small. To preserve the allure of your images and plots, be certain to save images in an uncompressed format; import and resize them in a way that retains the full quality; and create your final PDF file with settings that do not compress plots and pictures.

• Follow the lab report general guidelines.
• An outstanding error discussion is an essential element of a top-notch report.
• On each due date, one member of your group should submit a single PDF file to Stellar in advance of the deadline. The filename should consist of the last names of all group members, CamelCased, in alphabetical order, followed by a hyphen, the name of the assignment, with a .pdf extension. Example: CrickFranklinWatson-MicroscopyPart1.pdf.

Part 1

1. Apparatus:
   • Include a block diagram of your microscope, including all optical elements and relevant distances. It is not necessary to document the details of the mechanical construction.
   • Describe your design calculations and considerations.
   • Why not put in a nice snapshot of your ‘scope? (optional, but certainly a cherished memory in the making)
2. Magnification
   1. Procedure
      • Document the samples you used and how you captured images (camera settings, software used, etc…)
   2. Data
      • Include example images.
   3. Analysis and Results
      • Report the nominal and actual magnifications and fields of view you measured for the three objectives in a table. Report the length and width of the FOV (in distance units), not its area (in distance units squared).
      • Document the method you used to find magnification.
      • List the error sources that contributed significantly to systematic error and uncertainty in your measurement. To the degree possible, quantify the type and magnitude of the error.
   4. Discussion (optional for magnification measurement)
      • Explain any challenges you faced in the magnification measurement.
3. Particle diameter measurement
   1. Procedure
      • Document the samples you used and how you captured images (camera settings, software used, etc…)
   2. Data
      • Include example images.
   3. Analysis and Results
      • Report the average size of the microspheres in each sample and a measure of variation.
      • Describe how you measured the microspheres.
      • List the error sources that contributed significantly to systematic error and uncertainty in your measurement. To the degree possible, quantify the type and magnitude of the error.
   4. Discussion
      • Explain any challenges you faced measuring the size of silica microspheres.
      • How did your measurements differ from the manufacturer's specified values? What factors contributed to the difference?

Part 2

• Update the apparatus section of your report to reflect the changes you made in part 2.

4. Fluorescence imaging and flat-field correction
   1. Procedure
      • Document the samples you used and how you captured images (camera settings, software used, etc…)
   2. Data
   • Include raw images of the stained cell sample, reference image, and dark image.
   • It may be clearer to show the reference image as a surface plot.
   3. Analysis and Results
   • Plot intensity profiles of the reference, dark, and raw images on a single set of axes. (Use the improfile command in MATLAB.)
   • Use the reference and dark images to correct the raw image for nonuniform illumination and include the corrected image.
   • Include histograms of the sample, reference, dark, and corrected images on a single plot of log(count) vs intensity.
   • Document the steps you used to process the image.
   4. Discussion
   • How did your beam expander design affect your images?
5. Resolution
   1. Procedure
      • Document the samples you used and how you captured images (camera settings, software used, etc…)
   2. Data
   • Include an image of the PSF sample with the beads used for resolution measurement indicated.
   3. Analysis and Results
   • Report the resolution you measured. Make sure to include N and a measure of uncertainty.
   • Show sample Gaussian fits.
   • Include a thorough discussion of error sources.
   4. Discussion
   • Include a thorough discussion of error sources.
   • Compare the measured value to the theoretical value.

Part 3 report: Resolution, Brownian motion and stability test

In addition to answering the questions shown in bold in Optical Microscopy Week 3: Particle Tracking, do the following:

Summary

• 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

• 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.
    • You will likely find that a semilogy plot best shows the benefit of using the difference virtual particle.
  • Provide a bullet point outline of your data analysis methodology.
  • Comment on observed vs. expected data trends.

• 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.
    • Categorize the sources of error as systematic, random, or just mistakes (so-called "illegitimate" errors).
  • Provide a bullet point outline of all calculations and data processing steps.

¹Remember to include uncertainty and a discussion of error sources for all numerical results.

Part 4 report: Experiments in fibroblast cells

In addition to answering the questions shown in bold in Optical Microscopy Week 4: Microrheology Measurements in Fibroblast Cells, do the following: Report your findings on NIH 3T3 actin visualization and cytoplasm microrheology.

• Include images for + and - CytoD.
• Plot MSD vs time interval (τ) data on log-log axes.
• Use your MSD vs τ data to calculate estimates of G' and G" as described in Cellular microrheology.
• Discuss the results. Compare your results to classmates and values from literature.
• Comment on/quantify uncertainty.

Optical microscopy lab

Code examples and simulations

• Converting Gaussian fit to Rayleigh resolution
• MATLAB: Estimating resolution from a PSF slide image
• Matlab: Scalebars
• Calculating MSD and Diffusion Coefficients

Background reading

• Geometrical optics and ray tracing
• Physical optics and resolution
• Optical aberrations
• Aperture and field stops
• Optical detectors, noise, and the limit of detection
• Manta G032 camera measurements
• Understanding log plots