Instituto de Biomedicina y Biotecnología de Cantabria Servicio de Microscopía Óptica Avanzada

Time Lapse Experiment with the SP5 Laser Scanning Microscope

1. Turn on the Microscope, LASER, Temperature, Arc Lamp and CO2 controls, etc. at least 1 hour before starting the experiment to allow the system to stabilize. Set the Argon/Krypton LASER to 10% instead of the habitual 20%. 2. Initialize the microscope Stage: Say YES to “Initialize Stage: DM16000 stage”. 3. CAUTION: Initialization of the stage can damage the microscope stage and/or lenses. Be sure that the condenser turret is lift and that the objective is well bellow the stage path. 4. Pre-warm the immersion oil, incubation camera, media, etc. at 37ºC. 5. Place a cover with cells in the incubation camera and focus with the x40/1.3 objective (it is slightly brighter than the x63/1.4). Let the cells to stabilize in the incubation camera for at least 30 minutes. You can go ahead and start adjusting the microscope setting during this time. 6. Microscope Settings • In Acquisition mode select XYZt, Sequential and Multiposition (Mark and Find Panel) • In Sequential Scan load you pre-defined setting For example for CFP, GFP, RFP and DIC Scan 3: Laser 458nm – Emission filter from 465 to 490 - Dectector 1 Scan 2: Laser 488nm – Emission filter from 500 to 530 Dectector 2 and 4 Scan 2: Laser 543nm – Emission filter from 569 to 623 Dectector 3 • Select Scan Between Lines • In XY select: Format: 512x512 pixels Speed: 700Hz Pinhole: Between 2 and 3 Frame Average: Between 2 and 3 7. Adjusting exposure First, set PMT Voltage between 900 and 1000 for detectors 1, 2 and 3 and around 400 for detector 4. Then, keeping the zoom at the minimun value (=1.7) to minimize phototoxicity, adjust the LASER Power trying to keep as low as possible. Set the appropriate Offset.

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Instituto de Biomedicina y Biotecnología de Cantabria Servicio de Microscopía Óptica Avanzada

8. Selecting positions Avoid using cells that were used to adjust the exposure. Once you find a cell that you like try to align it in the middle of the field of view with the ocular and memorize the Stage Position (Mark Position in Mark and Find Panel). Repeat for 3 – 5 cells It is not necessary to scan the cells, but you might perform a quick scan to check them on the computer screen before adding the position. 9. Adjusting the Z-Stack Set Zoom to 5 (Image Size: 77.5µm – Pixel Size: 151nm) Select scan Between Frames and, if possible, select the red channel (red ligh is less toxic) to stablish the Z-stack. Go back to Position 1 using the Mark and Find panel. Quickly set the Z-Stack Begin and Z-Stack End. Number of Steps <10 Z-Step Size = 1µm If necessary, re-center and Redefine Position (Mark and Find panel). Now, go to the other positions and check that Z-Stack is OK (Go to Begin, Go to Focus Plane and Go to End). If necessary, expand the Z-stack and re-center and Redefine each position (Mark and Find panel). 10. Starting the Experiment Set the Time Interval and Frame Number. Time Interval: 2 minutes Frame Number: 20 Frames Select again Scan Between Lines

Star Experiment and keep the fingers cross

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