Difference between revisions of "Microscopy report outline"

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==General guidelines==
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This lab requires 3 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.
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* See the [http://measure.mit.edu/~20.309/wiki/index.php?title=20.309:Lab_Report_Guidelines#Lab_report_guidelines Lab report general guidelines].
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*'''The report should be in PDF format, submitted electronically  to Stellar in advance of the deadline.'''
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*'''The report title should contain the last name of each of your group members.'''
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* Submit one paper per group.
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Keep your lab report short.  
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* Bulleted list or outline format is encouraged.
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* Report numerical results properly:
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** Indicate the units of measurement.
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** Include the sample size and an appropriate measure of variability, such as a range, standard deviation, or standard error.
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** Use the abbreviation "s.d." for standard deviation and "s.e.m." for standard error after the "±".
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** For example: 1.21 ± 0.03 GW (±s.d., n=42)
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* Explain how you analyzed the data:
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** Summarize the algorithm used for all calculations and analyses.
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** Provide an outline of each MATLAB (or other language) function or script you used.
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** Put the complete code in an appendix.
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** Indicate the source of any code you did not write yourself.
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* Discuss your results:
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** Compare your results to theoretical predictions, reported values from literature, or other students' results.
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** Explain any factors that may have affected your results.
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** Describe what you would do differently if you had the opportunity to do the experiment again.
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* '''Provide a detailed, correct, and comprehensive discussion of error sources for each measurement''':
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** Is the resulting error random or systematic?
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** If the error is systematic, can you determine the sign of the effect and its approximate magnitude?
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** How could the error be reduced?
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* Present data properly:
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** Images should include a scale bar.
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** Plots should have a clear title; axes should be labeled with units; use a plot legend when appropriate.
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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.
  
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==Week 1 report: Microscope construction and bright-field characterization==
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* Follow the [[20.309:Lab Report Guidelines|lab report general guidelines]].
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* An outstanding error discussion is an essential element of a top-notch report.
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* Include any MATLAB code you developed in an appendix at the end of your report.
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* 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: <code>CrickFranklinWatson-MicroscopyPart1.pdf</code>.
  
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In addition to answering the questions shown '''in bold''' in [[Optical Microscopy Week 1: Brightfield Microscopy]], do the following:
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==Part 1==
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{{:Optical Microscopy: Part 1 Report Outline}}
  
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===Microscope design===
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==Part 2==
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{{:Optical Microscopy: Part 2 Report Outline}}
  
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* 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.
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==Part 3 report: Resolution, stability, and particle tracking==
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* Describe your design calculations and considerations.
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{{:Part 3 Combined Report Outline}}
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* Illustrate your apparatus with a photograph (optional, but nice).
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===Microscope characterization===
 
  
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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×):
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{{:Optical microscopy lab wiki pages}}
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* 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.
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* Include a table with the following values for the 10×, 40× and 100× objectives:
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** Theoretical resolution
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** Actual magnification by multiple measures (Air Force Target, Ronchi Ruling)
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** Actual field of view in the sample plane (FOV)
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* Quantify the uncertainty on your measurements (with an appropriate number of significant decimal points!).
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* 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.
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===Discussion on uncertainty===
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* Explain why some samples could not be well imaged with some objective(s), and whether the results differed from your expectations.
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* 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. 
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* 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.
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==Week 2 report: Microscope construction and fluorescence characterization==
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In addition to answering the questions shown '''in bold''' in [[Optical Microscopy Week 2: Fluorescence Microscopy]], do the following:
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===Microscope design===
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* Update the block diagram description of your microscope, its photograph, and annotation of pertinent optical components and distances.
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===Fluorescence imaging characterization===
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Demonstrate the epi-fluorescence imaging capability of your enhanced microscope:
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* 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).
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* 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.
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* Describe your flat-field correction procedure, from recording the reference image through applying the correction.
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* Also provide log(y) scaled histograms of at least one original and corrected image pair.
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===Discussion on design and image quality===
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* Comment on your corrections and relate your results to your choices during beam expander design and construction.
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* How do these choices relate to your intended experiments in weeks three and beyond?
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==Week 3 report: Resolution, Brownian motion and stability test==
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====Summary====
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* Image PSF beads and calculate resolution.
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* Track microspheres suspended in a solvent and measure microscope stability.
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* Estimate diffusion coefficients; calculate viscosities from those estimates.
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* Comment on/quantify uncertainty. How can you/did you reduce it?
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====Details====
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* Measured resolution of 40× objective<sup>1</sup>
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** Provide a sample of the images used for resolution estimation (overlay the fit – see plotgaussfit command).
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** Provide a table with measured estimates of FWHM resolution by Gaussian fitting<sup>1</sup> for the 40× objective.
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** Provide a bullet point outline of data analysis methodology.
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** Comment on estimated versus theoretical value.
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* Stability
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** Provide X-Y plots of sum and difference tracks for fixed particles.
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** Provide plots of MSD versus time interval for sum and difference tracks<sup>1</sup>.
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** Provide a bullet point outline of your data analysis methodology.
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** Comment on observed vs. expected data trend.
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*Viscosity
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** Estimate diffusion coefficient, viscosity for each water-glycerin mixture sample.
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** Comment on results, specifically how they are influenced by microscope stability and resolution.
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** Comment extensively on sources of error and approaches to minimize them, both utilized and proposed.
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** Provide a bullet point outline of all calculations and data processing steps.
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==Week 4 report: Experiments in fibroblast cells==
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Report your findings on NIH 3T3 actin visualization and cytoplasm microrheology.
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* Quantify your investigations of the actin structure.
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** Use an application such as ImageJ to measure observed min/max separation between centers of the actin strands.
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** Measure min/max observed lengths of actin strands.
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** Comment on the effect of actin density as it relates to bead size in the microrheology portion.
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** If you wish, reference your favorite article to compare your images.
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* Quantify your investigations of the cytoplasm/cytoskeleton microrheology.
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** Present your MSD vs sampling time &tau; data in log-log plot form and comment.
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** 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]].
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** Compare your results to the literature provided and/or any other relevant literature.
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* Comment on/quantify uncertainty. How can you/did you improve it?
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<sup>1</sup>Remember to include uncertainty and a discussion of error sources for all  numerical results.
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==Lab manual homepage==
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[[Lab Manual: Optical Microscopy]]
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{{Template:20.309 bottom}}
 
{{Template:20.309 bottom}}

Latest revision as of 17:04, 11 January 2017

20.309: Biological Instrumentation and Measurement

ImageBar 774.jpg


This lab requires 3 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.
  • Include any MATLAB code you developed in an appendix at the end of your 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

  1. Microscope documentation
    1. Include an updated block diagram of your microscope.
  2. Images
    1. Include a figure with an images of the 3.26 μm fluorescent microsphere samples, and the stained cell samples with and without Cyto-D.
      • For each sample, create 1 figure with 5 panels.
      • The panels of the figure should be: A) unprocessed image; B) reference image; C) dark image; D) flat-field corrected image; and E) histogram.
      • In the caption, specify the exposure and gain settings. Each image should have a scale bar. State the dimension of the scale bar in the caption.
      • For panel E, plot histograms of the unprocessed, dark, reference, and corrected image on the same set of axes. Plot log10( count ) on the vertical axis and intensity on the horizontal axis. Use a line plot instead of a bar chart for the histogram.
    2. Image profile
      • For one reference, dark and cell image set, plot an intensity profile across the same diagonal. You may also use a bead image, along with it's unique reference and dark images. The intensity of your three images should be on the same scale, i.e., 0 to 65,535 or 0 to 1. Place all three profiles on a single set of axes for comparison. (Use the improfile command in MATLAB.)
  3. Discussion
    1. How did your beam expander design affect your images?
    2. What differences did you observe between the cells with and without CytoD?

Part 3 report: Resolution, stability, and particle tracking

  1. 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 indicating which beads were used for resolution measurement..
      • Include the intensity histogram of your image (created by imhist)
    3. Analysis and Results
      • Report the resolution you measured. Make sure to include N and a measure of uncertainty.
      • Show sample Gaussian fits.
      • Explain the Matlab algorithm used for data analysis.
    4. Discussion
      • Compare the measured value to the theoretical value.
      • Include a thorough discussion of error sources. Do not comment on insignificant sources of error. To determine which error sources are significant, and which are not, you must think carefully about the uncertainty related to each error source and estimate its magnitude and sign. Include these estimates in your report along with your estimate of the combined, total uncertainty. It may be helpful to list out the error sources in a table, including a category for the error source, type of error (random, systematic, fundamental, technical, etc.), the magnitude of the error, and a description and way to minimize each one.
  2. Stability
    1. Procedure
      • Document the samples you used and how you captured images (camera settings, frame rate, total number of frames, exposure, software used, etc…)
    2. Data
      • Show an example frame from the stability movie.
      • Include the MSD vs. tau plot from your stability video, containing at least 2 sum trajectories and 2 difference trajectories on a log-log scale
    3. Discussion
      • What are the benefits and drawbacks of differential tracking?
      • Include a thorough discussion of error sources.
  1. Viscosity
    1. Procedure
      • Document the samples you prepared and used and how you captured images (camera settings including frame acquisition rate, number of frames, number of particles in the region of interest, choice of sample plane, etc)
    2. Data
      • Include a snapshot of the 0.84 μm fluorescent beads monitored.
      • Plot two or more example bead trajectories for each of the glycerin samples. (Hint: If you subtract the initial position from each trajectory, then you can plot multiple trajectories on a single set of axes.)
    3. Analysis and Results
      • Plot the average MSD vs τ results for all glycerin samples (A, B, C, and D); use log-log axes. Use the minimum number of axes that can convey your results clearly.
      • Include a table of the diffusion coefficient, viscosity and glycerin/water ratio for each of the samples (A, B, C, and D).
      • Provide a bullet point outline of all calculations and data processing steps.
    4. Discussion
      • How do your viscosity calculations compare to your expectations? (This chart is a useful reference.)
      • Include a thorough discussion of error sources and the approaches to minimize them. It may be helpful to list out the error sources in a table, including a category for the error source, type of error (random, systematic, fundamental, technical, etc.), the magnitude of the error, and a description and way to minimize each one.
  1. Particle Tracking in Cells
    1. Procedure
      • Document the samples you prepared and used and how you captured images (camera settings including frame acquisition rate, number of frames, number of particles in the region of interest, choice of sample plane, etc)
    2. Data
      • Include a snapshot of the 0.84 μm fluorescent beads monitored.
      • Plot two or more example bead trajectories for each of the samples. (Hint: If you subtract the initial position from each trajectory, then you can plot multiple trajectories on a single set of axes.)
    3. Analysis and Results
      • Combine your data with others from the class to increase your sample size.
      • Plot the average MSD (from the difference trajectories) for untreated and Cyto D treated cells on a single set of log-log axes.
    4. Discussion
      • What kind of motion do you see described by your MSD vs τ results?
      • What differences do you see between the untreated and Cyto D treated MSD curves?
      • Please suggest an interpretation of the behavior of your cells based on your data.
      • Include a discussion of your error sources.


Optical microscopy lab

Code examples and simulations

Background reading

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