CSU-W1 Spinning Disk/High Speed Widefield

FPBASE

Objectives

1. Plan Apo λ 10x / 0.45

2. Plan Apo λ 20x / 0.75

3. Apo LWD 40x / 1.15 WI

4. Plan Apo 60x / 1.27 WI

5. Plan Apo VC 100x / 1.4 Oil

6. Apo TIRF 100x / 1.49 Oil

(only 10x and 20x are installed on Microscope; others are in cabinet)

Filter Turret (top)

5. Photobleaching / photactivation cube (has an OD2 excitation filter and a TB 355-405-473 dichroic mirror)

Filter Turret (bottom)

1. Sedat Quad (Semrock)

2. CFP/YFP (Semrock)

3. Empty

4. Empty

5. Empty

6. Analyzer

Emission Wheel (for Widefield)

1. Open

2. 440/521/607/700 Multipass

3. 440/40

4. 525/30

5. 607/36

6. 684/34

7. 472/30

8. 542/27

9. 464/547 Multipass

Emission Wheel (for W1)

1. 447/60

2. 525/50

3. 607/36

4. 685/40

5. empty

6. empty

7. empty

8. empty

9. empty

10. empty

Dichroics in the W1

1. CSU-W1 Quad Dichroic 405/445/514/785
2. CSU-W1 Penta Dichroic 405/488/561/640/755

Hardware

• Andor Zyla 5.2 (Right Camera Port)

• Andor Zyla 4.2 (USB3, CSU-W1 left port)

• Andor iXon Ultra DU888 1k x 1k EMCCD (CSU-W1 back port)

• Nikon Ti with PFS3, Stage up with two turrets

• ASI XYZ stage (150 um travel piezo Z; COM8 Baud 115200)

• Lumencor Spectra-X (COM4, Baud 9600) with ESIO AOTF controller (COM5, Baud 57600)

• Sutter Emission Wheel and Lambda 10-B controller (COM9, Baud 128000)

• Sutter TLED, White

• Rapp Optoelectronic UGA-40 photobleaching system (COM6)

• Vortran 405 and 473 nm lasers for photoactivation and photobleaching

• Arduino (BF shutter/Zoom encoder; COM3)

• W1 Spinning Disk with Borealis upgrade (Left Camera Port; COM7, Baud 115200)

• ILE Laser Launch with 4 laser lines (405nm, 488nm, 561nm, 640nm) (COM2, Baud 19200)

Firmware / Software

• Ti firmware v5.0 (Combination N)

• Ti Driver v2.0.5.1

• TiControl v4.4.1

• Micro Manager 2.0 Gamma

PC

• Asus Prime X299-A

• Windows 10 64-bit

• Core i9 3.3GHz

• 64 GB RAM

• NVidia GeForce RTX2080

• Samsung 970 EVO Plus 2000Gb M.2 NVMe x 4 22x80 SSD (Fast Data)

• HGST 0F27452 DC HC510 ISE 10,000gb 7200rpm 256mb Cache SATA 6.0Gb/s (Data)

• Micron 5300 PRO 960gb SSD

Laser Powers from ILE Launch

Laser Power to Output 10x objective (mW)

Photobleaching

Open the Projector plugin in Micro-mananger to control the photobleaching system. Instructions on using the Projector plugin are here. To control the laser powers, open the Vortran control panel. To calibrate either laser, you can use an 0.01 mg/ml fluorescein solution, imaged in the FITC channel. A bottle of this solution is in the cabinet. Right now there is no way to save the calibration, so if you switch lasers you'll need to recalibrate.

For photoconverting mEos2, using the correct laser power is critical. If the laser power is too high, the protein will bleach instead of photoconverting. With the current OD2 filter, a 405 nm power of 50 mW works well (once we switch to an OD1 filter, use 5 mW). The Rapp unit ignores the spot dwell time parameter; to control how long the area is converted, adjust the loop parameter. For the laser power above, 10 loops gives good photoconversion and is still relatively rapid (~1s for areas of a few μm² at 100x). For bleaching GFP you'll need to use higher power.

Laser powers measured out of 10x objective, each laser at 50 mW power (no ND filter in)