Spreadsheet: 152605d1335450992-excel-2007-automatically-sizing-a-table-according-to-another-tables-size-maintenance-schedule-maintenance-summary-max-power-monthly-power
Sheet: Performance Maintenance Sheet

Confocal Performance Assessment |  |  | Time (h) = |  | update
Instrument: | LSM4
Serial Number: | 2102000144
Date of Inspection: | March 16/11
Conducted by: | Aleks
Test 1: Laser Power Test |  |  | Time (h) = | 1
Configuration:
Objective: [NAME] 10x/0.3NA |  | PMT settings: NA | Light path elements: NA
Frame Size: 512 x 512 | Zoom: 10 | Scan Speed: 9
Argon laser power: ~6 A | Single track | Averaging: none
Protocol:
1. Warm up lasers for at least 1 hour (refer to previous [NAME]) |  |  | 6. Choose appropriate laser power and dichroic
2. Focus the objective using a brightfield test sample |  |  | 7. Run continuous scan
3. Replace specimen with the power meter sensor |  |  | 8. Record measurement (mW) in Table 1 below
5. Set wavelength on power meter |  |  | 9. Repeat steps 5 to 8 for other configurations
6. Turn off lights if possible, hit Auto, then Zero
Table 1: Laser Power Measurements
Dichroic | HFT UV/488/543/633 | HFT 405/488/543 | HFT 405/514 | HFT 458 | HFT 458/514 | HFT UV/488/543/633 | HFT 405/488/543 | HFT 488 | HFT 488/543 | HFT 405/514 | HFT 458/514 | HFT UV/488/543/633 | HFT 405/488/543 | HFT 488/543 | HFT UV/488/543/633
Approx. Full Power | 25 | 25 | 25 | 3 | 3 | 16.8 | 16.8 | 16.8 | 16.8 | 10.2 | 10.2 | 1 | 1 | 1 | 1
Wavelength (nm) | 405 | 405 | 405 | 458 | 458 | 488 | 488 | 488 | 488 | 514 | 514 | 543 | 543 | 543 | 633
0.2
0.4
0.6
0.8
1
Test 2:  Laser/Stage/Detector Stability Test (LTST) |  |  | Time (h) = | 3
Configuration:
Objective: [NAME] 10x/0.3NA |  | Frame Size: 512 x 512 | Zoom: 1
Pinhole Size: ~2 AU | [NAME] Gain: 1 | Bit Depth: 8 Bit | A few line averages/frame can be used
Scan speed: 7 | Gain: ~625 | [NAME]: 1 | Sequential scan
Track 1 (405): HFT UV/488/543/633>Mirror>NFT490>LP490>Ch2 |  |  |  | how does 405 turn into something greater than 490????
Track 2 (488): HFT UV/488/543/633>Mirror>NFT490>BP505-530>Ch3 |  |  |  | isn't NFT490 too close to 488? I thought it was ~20nm difference after interacting with specimen???
Track 3 (543): HFT UV/488/543/633>Mirror>NFT545>LP560>Ch3 |  |  |  | ???
Track 4 (633): HFT UV/488/543/633>Mirror>NFT545>LP560>Ch3 |  |  |  | ???
Note: If using the same PMT for multiple LASER lines keep the PMT voltage constant and vary the LASER power,
however it is ideal to use a different PMT for each LASER line
Protocol:
1. Warm up lasers for at least 1 hour (refer to previous [NAME]) |  |  |  | 7. Turn off overhead lights
2. Focus the objective using a brightfield test sample |  |  |  | 8. Set laser powers and detector gains to read ~150 grey levels
3. Replace specimen with the red plastic Chroma slide |  |  |  | 9. Record laser power and channel gain settings in Table 2 below
4. Focus ~20 mm into specimen, limiting imaging specks and scratches |  |  |  | 10. Close room door and place a sign over the light switch
5. Insert primary dichroics  and filters as listed above |  |  |  | 11. Acquire 360 frames @ 30s intervals (~3h)
6. Activate ChD and remove polarizer/filters for halogen lamp |  |  |  | 12. Shift the slide ~1/2 the field of view and collect another image
Table 2: Laser/Detector Settings
 | Laser Power (%) | Confocal Ch Gain (PMT) | ChD Gain   (T-PMT)
405 nm
488 nm
543 nm
633 nm
Test 3: Short Term Stability Test (STST) |  |  | Time (h) = | 0.5
Configuration: |  |  |  |  |  |  |  |  | * separate field uniformity and stability test b/c field uniformity must be 512x512
Objective: [NAME] 10x/0.3NA |  | Frame Size: 64 x 64 | Zoom: 1
Pinhole Size: ~3 AU | [NAME] Gain: 1 | Bit Depth: 8 Bit | Sequential scan
Scan speed: maximum | Gain: ~625 | [NAME]: 1
Track 1 (405): HFT UV/488/543/633>Mirror>NFT490>LP490>Ch2 |  |  |  | how does 405 turn into something greater than 490????
Track 2 (488): HFT UV/488/543/633>Mirror>NFT490>BP505-530>Ch3 |  |  |  | isn't NFT490 too close to 488? I thought it was ~20nm difference after interacting with specimen???
Track 3 (543): HFT UV/488/543/633>Mirror>NFT545>LP560>Ch3 |  |  |  | ???
Track 4 (633): HFT UV/488/543/633>Mirror>NFT545>LP560>Ch3 |  |  |  | ???
Note: If using the same PMT for multiple LASER lines keep the PMT voltage constant and vary the LASER power,
however it is ideal to use a different PMT for each LASER line
Protocol:
1. Warm up lasers for at least 1 hour (refer to previous [NAME]) |  |  |  | 7. Turn off overhead lights
2. Focus the objective using a brightfield test sample |  |  |  | 8. Set laser powers and detector gains to read ~150 grey levels
3. Replace specimen with the blue plastic Chroma slide |  |  |  | 9. Record laser power and channel gain settings in Table 3 below
4. Focus ~20 mm into specimen, limiting imaging specks and scratches |  |  |  | 10. Close room door and place a sign over the light switch
5. Insert primary dichroics  and filters as listed above |  |  |  | 11. Acquire 600 frames @ 0.5s intervals (~5min)
6. Activate ChD and remove polarizer/filters for halogen lamp |  |  |  | 11. Repeat for green, orange and red Chroma slides separately
Table 3: Laser/Detector Settings
 | Laser Power (%) | Confocal Ch Gain (PMT) | ChD Gain   (T-PMT)
405 nm
488 nm
543 nm
633 nm
Test 5:  PMT Co-Registration Test |  |  | Time (h) = | 1
Configuration:
Objective: Plan Apochromat 63x/1.4 NA |  | Frame Size: 256 x 256 | Sequential scan
Pinhole Size: 1 AU | [NAME] Gain: 1 | Bit Depth: 8 Bit | Z interval: 0.1µm
Scan speed: maximum | Zoom: ~10 | [NAME]: 1 | [NAME]:  55-70 nm
Track 1 (405): HFT UV/488/543/633>Mirror>NFT490>LP490>Ch2
Track 2 (488): HFT UV/488/543/633>Mirror>NFT490>BP505-530>Ch3
Track 3 (543): HFT UV/488/543/633>Mirror>NFT545>LP560>Ch3
Track 4 (633): HFT UV/488/543/633>Mirror>NFT545>LP560>Ch3
Note: Collect such that the pixel size is ~1/2 the resolution
Protocol:
1. Warm up lasers for at least 1 hour (refer to previous [NAME]) |  |  |  |  | 6. Collect a Z series using sequential scans of 3+ PMTs
2. Focus on 4 µm Tetraspeck beads using BF illumination
3. Adjust laser powers and PMT gains to acquire quantitative images in all channels |  |  |  |  |  | do we want to record laser powers and gains
5. Position sample so that multiple beads are seen in the field of  view
Table 5: Zmax and COM
laser channel | [NAME] |  | Bead2 |  | [NAME]
 | Zmax | COM | Zmax | COM | Zmax | COM
405 nm |  |  |  |  |  |  | analysis table
488 nm
543 nm
633 nm
Test 6:  Point Spread Function (PSF) Test |  |  | Time (h) = | 0.5
Configurations: |  |  | Protocol:
Plan Apochromat 63x/1.4 NA objective |  |  | 1. warm up lasers for 60 min (as required)
256 x 256 format, zoom = 10, 8 12-bit acquisition |  |  | 2. insert place a 0.1 µm yellowish-green microsphere PSFspeck bead sample on the microscope
Scan speed = fastest possible, frame averaging = 4 |  |  | 3. focus on beads using widefield fluorescence (FITC cube) |  |  |  |  | Use MeteroloJ
Detector offset = 0, digital gain = 1 |  |  | 4. Adjust laser intensities and PMTs to acquire quantitative images in all channels - no pixels reading zero or saturating, use the range indicator LUT
pinholes (PH) = 0.85 1 and 5 Airy units |  |  | 5. if possible position sample so that multiple beads are seen in the field of view
Detector offsets = 0, digital gains = 1 |  |  | 6. acquire a z-series in all three channels
z step size = 0.1 µm |  |  | 7. determine the FWHMxy of multiple beads for each channel
Track 1 (488):HFT UV/488/543/633>Mirror>NFT490>BP505-530>Ch3 |  |  | 8. determine the FWHMz of multiple beads for each channel
 |  |  | 9. repeat for at least 5 beads with the pinhole at 1 Airy unit
Table 6: PSF |  |  | 10. repeat steps 4-9 with the pinhole at 5 Airy units
 | 1 Airy Unit
 | FWHMxy | FWHMz
Bead 1
Bead 2
Bead 3
Bead 4
Bead 5
 | 5 Airy Unit
 | FWHMxy | FWHMz
Bead 1
Bead 2
Bead 3
Bead 4
Bead 5
Test 5:  Flow Cell Test |  |  | Time (h) = | 0
Use the Plan Apochromat 63x/1.4 NA objective, zoom 2, image from the field illumination/uniformity test
Test 5:  Pinhole Alignment Test |  |  | Time (h) = | 1
don't need to warm up LASERs
Test 6:  Detector Noise Test |  |  | Time (h) = | 0.5