Microscopy report outline

• Follow the lab report general guidelines.
• Provide a thorough and accurate discussion of error sources and measurement uncertainty. An outstanding error discussion is an essential element of a top-notch report.
• 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, with a .pdf extension. Example: CrickFranklinWatson.pdf.

Week 1 report: Microscope construction and bright-field characterization

In addition to answering the questions shown in bold in Optical Microscopy Week 1: Brightfield Microscopy, do the following:

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.

Week 2 report: Microscope construction and fluorescence characterization

In addition to answering the questions shown in bold in Optical Microscopy Week 2: Fluorescence Microscopy, do the following:

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 3 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?

Week 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

• 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 40× 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 40× 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.

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

Week 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.

• 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 τ data in log-log plot form and comment.
  • Use your MSD vs τ data to calculate estimates of G' and G" as described in Cellular microrheology.
  • Compare your results to the literature provided and/or any other relevant literature.
• Comment on/quantify uncertainty. How can you/did you improve it?

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