Difference between revisions of "Microscopy report outline"

This lab requires 4 submissions, one for each part of the lab. 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 them all at 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:
   • 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.
   • Why not put in a nice snapshot of your microscope? (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.
   • Provide examples of images of beads captures with each of the three objectives (10×, 40× and 100×).
3. Analysis:
   • Clarify the approach and steps you chose to go from the raw images to the inferred magnification and field of view quantitative results for your brightfield microscope. Show an example calculation.
   • Establish how the uncertainty of your measurements will be reported ("± standard deviation", "± standard error", "± percentage of average") and what equation you followed to quantify this uncertainty.
4. Results:
   • 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×):
     • Include a table with the following values for the 10×, 40× and 100× objectives:
       • (Don't forget to indicate units!)
       • 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 (and justify the size of the microspheres chosen for each of the objectives).
5. Discussion:
   • 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 and characterize its source of error: Possibly in table format,
     • indicate which sources of error contribute to the compounded uncertainty reported,
     • whether each source of noise is systematic or random, user- vs. instrument-based,
     • how each source of noise impacts numerically your results,
     • how predominant or minor each source of noise is toward reaching your conclusions,
     • which sources of error can be compensated / improved on, and which are intrinsic to your microscope design.
   • Explain how multiple measurements improve (or not!) the accuracy of your calculations.
6. Conclusion / Further Work:
   • Suggest possible design choices that would improve microscope performance.

Part 2 Report: Microscope construction and fluorescence characterization

1. Apparatus:

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

2. Procedure:

• Document the critical operations and parameters of your instrument that would be necessary for another person to replicate your work. (Assume that person has taken 309 and has access to all course materials: wiki, Stellar, lectures, problem sets, etc).

Epi-fluorescence imaging capability of your enhanced microscope

3. Data:

• Include the fluorescent reference image you made 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 3.26 μm and 0.84 μm fluorescent bead samples. Compare and contrast these pictures before and after flat-field correction to counteract nonuniform illumination.
• Also provide log(y) scaled histograms of at least one original and corrected image pair.

4. Analysis:

• Describe your flat-field correction procedure, from recording the reference image through applying the correction.

5. Discussion:

• 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?

Measured microscope resolution with 40× objective

3. Data:

• Provide a sample of the images used for resolution estimation.
  • Include a figure showing how many PSF points ended up being selected to participate in the averaged resolution estimate.
  • Include an overlay of the Gaussian fit to the 2D Airy disc (see plotgaussfit command in Matlab).

4. Analysis:

• Provide a bullet point outline of your data analysis methodology.

5. Results:

• Report the measured resolution of the 40× objective, as approximated by the full width half maximum (FWHM) of the Gaussian fitting.

6. Discussion:

• Discuss error sources and uncertainty in the measurement.
• Comment on measured versus theoretical value.

7. Conclusions and Future Work:

• Broach on the relevance of your fluorescence imaging and resolution deductions toward upcoming tasks in the 20.309 lab.

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