Weekly Updates
Contents
Week 03/14 – 03/18
This Week
- Defined a modified equipment list with the help of Uli and Jeff at the Danuser lab (simplified design without TIRF and a single laser)
- Research was made on the best choices for lasers, cameras and objectives
- The microscope at station 3 was approved for use with the e-STORM project, with minor modifications depending on the specific parts acquired
Next Week
- A final equipment list will be compiled and quotes will be obtained
- Ideally, the equipment will be ordered
- Work will start with the image analysis programming, including the analysis of code obtained from the Danuser lab plus literature reading
Risks and Issues
- The adoption of the microscope frame at station 3 might be premature depending on the final parts and design we acquire
- There might be a fair amount of experimentation with the setup since we are using the most minimalist approach possible
- There is a delay on the planned time for part ordering, which has to be addressed next week or shipping and processing time might interfere with the project execution
Week 03/21 – 03/25
This Week
- Additional reading was done on the part selection software
- The options for lasers were evaluated. In particular, OEM seems to be the best option based on their price to quality relation. The particular wavelength will depend on the dye selection
- Research was done into the image processing software, and the best option seems to be adapting the QuickPALM code available in open source to our needs
- Jeff and Uli gave more feedback on the project, clarifying that the image processing can probably be done with a simple Gaussian fit, so research was put into that
Next Week
- Try and finish the research for part selection
- Get the best photon budget possible for part selection
- Continue work in image processing, in particular adapting the QuickPALM to our needs
Risks and Issues
- The part selection process is taking a lot longer than expected, which could result in delays in the schedule
- The reading required for the photon budget seems to be considerable, so again that may affect the proposed schedule
- Reverse engineering and adapting the QuickPALM code might be more complicated than just programming our own code--JAVA does not have an open source code for nonlinear least squares regression for multidimensional fits.
Week 03/28 – 04/01
This Week
- Continued research on parts and photon budget, taking a lot of information from the Olympus resource website. The light path from laser to sample is complete
- Research was put into dSTORM, and determining if it is a viable option for our project, both in terms of equipment and software
- Quotes were obtained for the Andor and Hamamatsu cameras. The latter seems to be the best option (Orca Flash 2.8)
Next Week
- Again attempt to finish the photon budget, addressing the sources of noise and the path from sample to camera
- Determine if dSTORM is viable, so that one laser can be used
Risks and Issues
- We are falling behind schedule in both the part selection and the image processing, the amount of information necessary keeps increasing
- If dSTORM is selected, there is a risk of not having enough photons to perform the experiments, even at long integration times
- The performance of the Orca Flash 2.8 is a lot lower than the EM-CCD and Neo-sCMOS cameras, so there is a chance there is no way of neglecting the high-price cameras
Week 04/04 – 04/08
This Week
- Finished the sample to camera photon budget, tailored for single particle detection based on Gaussian fits
- The dye selection seems to point to ATTO 680, since it is an order of magnitude faster than Cy5 for dSTORM, even when Cy5 is complexed
- Evaluated different camera and dye options using the photon budget calculations
Next Week
- Order the laser and setup the demo for the camera
- Fully understand the QuickPALM code
- Adapt the existing microscope frame for addition of the new laser and the new camera
Risks and Issues
- The election of dSTORM is a risky choice, since there might not be enough photons to collect and the system might be too slow
- The availability of the ATTO 680 dye might be less ideal than Cy5
Week 04/11– 04/15
This Week
- The laser was finally ordered. This is a Dragon Lasers 655 nm Diode Laser, 500 mW with analog modulation. It should come within the next couple of weeks.
- The filter set to use was also ordered (Chroma Technologies, one dichroic mirror and one barrier filter). We decided to use ATTO 655, since it perfectly fits the spectrum of the laser we have.
-The demo was set with the Hamamatsu representative. A date is to be determined
- Received code from Uli for Gaussian fit, particle detection, and example data sets. This code is in C.
- Slowly but surely sifted through QuickPalm code to understand methods for particle detection and position, stdev calculations.
Next Week
- Adapt the microscope removing some unnecessary features, since it wasn’t done this week
- Determine where we are going to get the sample
- Acquire some sort of enclosure for the system
Risks and Issues
- The laser we bought has to come from China, so there might be some delays
- There is still no actual date for the demo of the camera
- Sample preparation is very uncertain at this point
Week 04/18– 04/22
This Week
- The demo date was set for May 2 at 10 AM, and we’ll have the camera for a week
- The filters came in, but they won’t be installed until the new laser arrives
- The microscope was finally adapted to the required specs, including the removal of an extra mirror and the optimization of the laser and camera paths
Next Week
- Characterize the response of the Manta camera to a simulated STORM. For that we’ll need to measure the photon to electron response.
- Hopefully receive and install the laser along with the filters.
- Install the enclosure for the setup
Risks and Issues
- The sample keeps being an unsolved issue in the process
- The laser has not been confirmed yet
- The image processing code has not made considerable advances yet
Week 04/25– 04/29
This Week
- We set for ATTO655-Phalloidin, and microtubules as the system target. Therefore, the dye was ordered, and we started looking at ways to obtain the related reagents for sample preparation.
- The microscope was aligned and power measurements were performed. At maximum gain, we are looking at 17 photons per count with the Manta camera after background subtraction
- We received all the pieces of the enclosure, but there is a low profile screw missing so assembly cannot be completed
Next Week
- Get through the camera demo and install it. Also, characterize it using the same method used for the Manta
- Prepare samples with cells from Brian to be ready to obtain data
- Set up the completed system, including laser and enclosure
Risks and Issues
- The laser was sent just this week, so no delays are desired
- There is no enclosure for the camera demo
- The sample preparation reagents are still partially uncertain
Week 04/30 - 5/7
This Week
- Thawed cells for sample preparation. Two cell lines were thawed. The first is MDA-MB-231, an established human breast cancer cell line. The other is J1 Mouse Embryonic Stem Cells.
- Cells split every 3 days
- Identified and ordered reagents necessary for sample preparation: (beta-mercaptoethylamine, paraformaldehyde, mounting medium with anti-fade agent (MOWIOL with DABCO)).
Next Week
- Attempt to create sample preparations
Risks and Issues
Week 05/08-05/15
-Obtained samples from Zhuang lab
-Discovered that laser was off spec--does not get filtered by dichroic
-Reworked configuration to replace green laser to take images of beads
-Obtained 632.8nm laser from Junior Lab (Physics Department)
Next week
- Take images of beads
- Start taking images of real samples
- Write report
Issues and Risks
- New laser is much lower power (~5mW). Power is needed to switch the fluorophore to its dark state.
- Will images of real samples be bright enough?
- The new laser's beam diverges and splits--> more loss of power
Week 05/16-05/19
- Installed new laser
- Took images of PS Speck Invitrogen Deep Red beads
- Took image stacks of cell samples and ran data
Future Directions
- Continue working to incorporate non-linear Gaussian fit into QuickPalm Code
- Try to get STORM to work with Manta camera.
- Work to finalize sample preparation protocol with ATTP655, including cell fixation and staining.
- Devise a method to determine the approximate resolution of our STORM images.
