User:Kevinly
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.
This page is loosely organized by Assignments/Labs.
Assignments
Assignment 1: Optics boot camp
- Overview
- Work on mystery negative lens on paper before going to the lens station
- Verify lens-maker formula
- 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
|
- 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.
- 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
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:
- 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 | 3/16" | In default position, set screws should peak out just a hair to allow cage rods to slide past through-holes |
C6W | 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:
- Brightfield transillumination (Assignment 1)
- Two-color epifluorescent (Assignment 3 & 8)
- 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.
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 |