microscopes-seqsr

Sequential Super-Resolution Microscope

The sequential super-resolution microscope is a custom-built multi-target super-resolution imaging system utilizing the sequential imaging strategy. This microscope is optimized for using photo-physical properties of the fluorophore ‘Alexa Fluor 647’ for each of the multiple targets being imaged, and images are recorded using a high quantum efficiency digital CMOS camera. This microscope is also equipped with automated re-finding of once registered cells and active image stabilization for nanometer scale drift correction while imaging. Computer control of the instrument is done using custom written MATLAB software.

MIC_SEQ_SRcollect();

MIC_SEQ_SRcollect is a super resolution data collection software. This class requires Matlab 2014b or higher and works with Matlab Instrument Control (MIC) classes matlab-instrument-control

For the complete operating guide — sample loading, cell selection, acquisition, sequential imaging, and troubleshooting — see the User Guide.

Equipment List

Microscope body

• Home built base.
• Thorlabs, MAX381/M, 3-Axis NanoMax stage, travel 4 mm with stepper motor and 20 $\mathrm{\mu m}$ with piezo.

Objective

• Olympus, UPLSAPO 100XS, silicone oil Immersion, NA 1.35, WD 0.2 mm, FN 22.

Camera

• Hamamatsu, C11440-22CU, sCMOS camera, 2048x2048 pixels, pixel size 6.5 $\mathrm{\mu m}$.

Illumination

• MPB Communications, 2RU-VFL-P-500-647-B1R, 647 nm laser, 500 mW.
• Thorlabs, DL5146-101S, 405 nm laser, 40 mW.
• Thorlabs, M660L3, 660 nm LED, 640 mW, for bright field illumination.

Other equipments

• National Instrument, USB-6008, DAQ card.

Filters

• Semrock, Di02-R635, dichroic mirror
• Semrock, FF01-708/75-25-D, band pass filter.

Sequential Super-Resolution Microscope Setup Guide

For setting up the sequential super-resolution microscope for data collection, Follow the steps in the order listed below:

Note:

To ensure proper initialization and safety, start with every individual component of the microscope turned off.

1. Turn ON the Power for Stepper Motor Controller

   • Turn ON the power switch of the three-channel bench top stepper motor controller. The switch is located at the back.

2. Power ON the Camera

   • Power ON the camera using the power switch on the rear face of the camera.

3. Power ON the 647nm Laser Control Module

   • Power ON the 647nm laser control module. The power switch is located on the front face.
   • Important: Do not turn the ‘key’ to ‘Laser ON’ at this stage. This will be done after loading the sample and closing the box.

4. Power ON the Extension Power Strip

   • Go to the back of the optical table, and turn on the extension power strip, ensuring that all piezo and strain-gauge controllers are powered ON.

5. Turn ON Laser Speckle Reducer

   • For uniform laser illumination, turn on the switch at the bottom of the speckle reducer.

6. Open MATLAB and Load Microscope Control Software

   • Open MATLAB, and execute the following command to load the sequential microscope control software:

     SEQ = MIC_SEQ_SRcollect();

   • The software will connect to the stepper motors and move the stage to a safe position.
   • If the stepper controller was power cycled, run SEQ.homeSteppers() after startup to re-home the motors. Do not home with a sample loaded.
   • If the software was previously closed abnormally (e.g. MATLAB crash) while a sample was loaded, answer "Yes" to the startup prompt. The software will safely raise the stage so the sample can be removed, then continue with normal setup.

7. Verify Initialization of Controllers

   • Confirm that all piezo/strain-gauge controllers have initialized correctly and are displaying the desired values.
   • If any controller is not initialized correctly, use the ‘Reconnect Piezos’ button on the control GUI to initialize them one-by-one. This may sometimes require power cycling the respective controllers.

8. Ready to Load Sample and Begin Imaging

   • Now microscope is ready for SR data collection.

Once you are finished using the microscope, please switch off the speckle reducer and execute the following code:

SEQ.delete();

Now, proceed to turn off every device except computer.

Citation:

David J. Schodt, Farzin Farzam, Sheng Liu, and Keith A. Lidke, "Automated multi-target super-resolution microscopy with trust regions," Biomed. Opt. Express 14, 429-440 (2023)