User:Kevinly

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This Wiki page is a comprehensive compilation of Kevin's personal memos for each Assignment and component of 20.309. The intended audience is just Kevin, but if others read it and happen to learn from it, then that's nice too. Perhaps in the future, when there is a new generation of 20.309 instructors, these logs and memos will be a valuable resource.

A cartoon centipede reads books and types on a laptop.
Kevin at the Royal Observatory Greenwich in the UK to see John Harrison's clocks. The exhibits were unfortunately closed the entire week of his visit.


This page is loosely organized by Assignments/Labs.

Assignments

Assignment 1: Optics boot camp

Overview
  1. Work on mystery negative lens on paper before going to the lens station
  2. Verify lens-maker formula
  3. Become familiar with MATLAB indexing and plotting of single/multiple pixels
Lab notes
  • The lens-maker-verifying component is already built and tucked away in the cubbies in the lab

Assignment 2: Brightfield transillumination microscope

Overview
Coding notes
Lab notes
  • Aligning the aluminum mirror

Assignment 3: Single-color epifluorescent microscope

Overview
Lab

Assignment 4: Resolution and particle tracking in glycerin


  • Make sure students know how to lock and unlock the stage
  • Teach them how to use ROI (Region of Interest)


Overview
  • Measure the resolution of their optical system (40x Nikon with 125mm tube lens and 590nm light); Expect resolution around 625nm
Lab notes
  • The PSF beads for measuring the resolution are particularly small
    • Set the gain to max and use a exposure of about 200,000 µs when focusing and searching for a good patch of beads
    • When you actually take the PSF image, you can lower the gain and expect an exposure of about 800,000 µs (with current around 0.5 A)
    • Some slides are better prepared than others (the older slides are photobleached)
  • Preparing the sample slide with particles diffusing in glycerin
    • Make a channel on a glass slide using two double-sided tape with a cover slip sandwiched on top. Then let the glycerin-bead mixture capillary action its way through before sealing each end of the channel with nail polish
    • You'll have to let the slide sit for a while because there may still be flow from the capillary action movement
      • Even if there is flow, you can account for it using the difference trajectory
    • Its better to do this with higher concentration of beads (10~30 in the FOV)

Assignment 5: Particle tracking in cells


Reminder or checklist to do before starting this week of lab!


Overview
  • Students measure fixed 0.84 µm beads to quantify the stability of their optical system

This Assignment is known for applying the particle tracking code from Assignment 4.

Diagram for Assignment 5 lab protocol.
Lab notes
  • Cells were already soaking with beads (of what size again?)
  • Try to find about 2-5 beads in a cell
  • Helpful to have a transillumination light source on (but very dim or angled away) while the fluorescence is on (exposure around 100,000 µs)
    • This allows you to ensure the found beads are actually in treated/untreated cells
Code notes
  • Bug: 3xN output of FindParticlesInMovies.m (input to Track.m) doesn't work sometimes (namely for fixed beads)
    • Error: Track.m: 'You are not going any further, check your params and data'; 'the code broke at line 1045'
    • Solution: Don't put the whole 3xN centroidWithFrames into Track.m input — just put as many frames in as possible before Track.m fails


Assignment 6: Equivalent circuits and voltage divider lab

Assignment 7: AC circuits, frequency sweep, and step response

Lab notes
  • When they export the frequency sweep data from Waveforms as a CSV, uncheck all boxes except for "Labels"
Uncheck all export settings except for "Labels" which is the horizontal axis information.
  • Students oftentimes use an insufficient time base when trying to see their signal on the scope
  • Remind them to always be mindful of the time scale of their filter and signal and choose the appropriate time base
Code notes

For MATLAB, the new A7 template should be good to go. The only thing they need to input/modify are as follows:

  • The name of their CSV files (correctly formatted columns)
  • The resistor and capacitor values they used
  • The correct transfer functions
  • The correct numerator and denominator forms

Assignment 8: Mystery circuits and adding second color

Lab notes
  • The white circuit is not like the other ones
  • The second-color should already be in their kits; just give them the blue excitation filter and Blue/Green dichroic mirror

Assignment 9: Build microfluidic system and controls


  • Reminder or checklist to do before starting this week of lab!
  • Assemble the microfluidics kit


Lab notes
  • When building the control circuit, all sub-circuits (signal and power) must share the same ground
    • You can connect the grounds of both LED power channels right at the power supply
      Note the black wire connecting the ground for both channels.
  • The circuit can be built on a breadboard. Here is an example implementation
    Very neat implementation by Steve. Take advantage of the plug-in ports at the top of the breadboard.


Assignment 10: Sacha osmotic shock

Lab Chore Tutorials

Cleaning camera sensors

With the new camera sensor wipers that Steve ordered (they're actually designed for DSLR camera sensors), this Lab Chore is much easier (and almost fun). The traditional method was to use lens cleaning tissue, but sometimes that left lint or deposited even more dirt. The key with cleaning lenses and sensors is to firmly wipe it once.

  • New cleaning method:
    1. Wet one side of the wiper with the cleaner fluid
  • Old cleaning method:
  • General cleaning tips:
    • Never have the exposed camera sensor angled upward; dirt and dust settles downwards!
    • Wipe once

Set screw guidelines

There are a handful of microscope parts that use set screws: C6W, CP02, and the cage rods.

Part Set Screw Length Additional Notes
CP02 2/16" In default position, set screws should peak out just a hair to allow cage rods to slide past through-holes
C6W 4/16" or 5/16" In default position, top of set screw should line up with the face C6W
ER025 3/16" For standardization purposes, all cage rods should have only one set screw tightly fastened with the appropriate length. This is to ensure that when the cage rod screws into another cage rod or cage cube, the set screw remains attached to its original rod after unscrewing.
ER2 4/16"
ER3 4/16"
ER4 4/16"

Microscope Kit Assembly

The microscope parts need to be pre-assembled into a kit. The kits do not include any mirrors or filters because these parts are more fragile — hand these components to students when they finish the rest of their build.

I divide the microscope build into 3 parts:

  1. Brightfield transillumination (Assignment 1)
  2. Two-color epifluorescent (Assignment 3 & 8)
  3. Microfluidic system (Assignment 9)

Here are the kit parts, counts, and other notes/recommendations:

Brightfield transillumination kit

Two-color epifluorescent kit

Students will use half the parts in this kit in Assignment 3 for the single-color epifluorescent microscope used in particle tracking. In Assignment 8, students build out the second-color to visualize the yeast response to an osmotic shock.

Two-color epifluorescent microscope kit with parts laid out.
Part Quantity Additional Notes
C6W 1 Holds the excitation dichroic mirror
B1C 1 Bottom of the C6W
CP02 2 Support for each of the excitation arms
SM1L05 2 Holds each of the condenser lenses for the excitation LEDs
ER025 4 Holds the microscope base C6W to the dichroic mirror C6W
ER3 6 3 for each arm of the excitation
PH2 2 For each excitation arm support. Can alternatively use a PH4
TR1 2 Connect to TR3 to make a 4" vertical support for each excitation arm
TR3 2 Connect to TR1
TR4* 2 Alternative to connecting TR1 to TR3
BA1 2 Base of each excitation arm support
75mm lens 1 Housed in the side of the microscope base C6W
20mm aspheric lens 2 Condenser lens for each excitation LED

Microfluidics Caddy

Part Quantity Additional Notes
C6W 1 Holds the excitation dichroic mirror
L-shaped black acrylic fluidics caddys 1 Holds the fluids and valve
50 mL conical tubes 2 Container to hold a 15 mL tube, which will actually contain fluid
Routing clamps 2 Secures the 50 mL conical tubes to the caddy
Two-line routing clamp 1 Holds the pinch valve (on the clamp closer to the P14 post
Pinch valve 1 Pinches the soft tubing
1/4-20 screw w/ wing nut 2 pairs Holds the routing clamps for the 50 mL tubes
8-32 screw w/ wing nut 1 pair Holds the two-line routing clamp
Diablotek power supply 1 12 volt power supply for the pinch valve.
Molex connector 1 Use with Diablotek

Cell Culturing Protocols

309PEPs

List of "309PEPs" inspired from Python Enhancement Protocols. I will probably make this its own Wiki Page as it grows.

Scaffolds for coding assignments

  • Type: Process
  • Status: Draft

The purpose of students coding is to gain the valuable skill of being able to program and to understand how data/information is manipulated, transformed, and computed.

  • Programming skills include reading documentation, understanding use cases of data types, understanding the inputs and outputs of functions, writing clean code.
  • Programming skills improve through revisions, reading/writing, and time.
  • Data analysis skills include …
  • Data analysis skills improve by actively translating math and physical concepts into data structures and manipulating, transforming, and computing them

Given how jam-packed 20.309 already is with content, if we had to choose between programming and data analysis skills, I’d say we should focus more on the data analysis. In practice, students should focus more on coding Helper Functions, which encapsulate very physical transformation and computations of data/information.

Goal: Make students focus more on the data analysis, math, and theory rather than getting muddled by managing end-points between functions. Most of the learning and coding should happen inside the Helper Functions

Administer: For each of the coding parts of assignments, package all the helper functions, tests, and templates into a ZIP file.

Code Templates: Bring all the Helper Functions together in a sequential and chronological way (manages end-points). The Template need not be modified; only if students are changing global parameters (Example: NUMBER_OF_PARTICLES, BEAD_DIAMETER).

Helper Functions: Most of the learning happensExisting examples include (DrawOneFluorescentMicrosphere.m, BlurAndAddNoiseToImage.m).

Assignment Submission: Students usually submit output figures that are labeled with a legend. They probably don’t need to submit the Template, just the Helper Functions and any figures.