Nikon N-STORM Bedienungsanleitung

The Nikon N-STORM User Guide

General Guidelines:

If you get stuck at any point, do not understand something or find the system does not

function properly ALWAYS warn the staff!!!

Microscope:

1. NEVER switch the microscope and stage off as the system as the heat generated by of the

motors, circuits and perfect focus system can generate excessive drift while heating up.

2. Do not forget to switch the 405 nm laser of as its switch is not as visible as for the others.

Objectives:

1. Objectives are very expensive always handle with extreme delicacy especially while changing

samples. Never touch or scratch the front lens directly.

2. When starting to work check for visible damage.

3. Always start imaging with the 10x objective and proceed to larger magnifications. This helps

to avoid crashing into the sample and damaging the objective.

4. When changing objectives always make sure the distance between the objective and stage is

appropriate. ESPECIALLY if using a different stage insert than the previous user.

5. When applying oil always check if the objective is indeed an oil immersion objective.

6. After using the 100x oil immersion objective NEVER forget to clean with lens cleaning paper:

1. Fold the lens cleaning paper three times to form a strip, 2. Hold it on the two ends on both

sides, 3. Gently wipe with left-right motion. Never press with your finger directly. 4. Repeat

previous step until you cannot see oil stain and repeat once after that one final time.

7. When finished ALWAYS turn to the 10x objective and lower the stage to a safe distance.

Sample Holder:

1. When using the universal sample holder (Ti-SH-U) never loosen the left slider!! This is the

fixed point and changing its position will prohibit users to find positions when reimaging slides.

Lasers:

1. Only switch on lasers that will be used for your experiments.

Computers:

1. Never save data on the local hard drives. Please use the network drives instead.

Booking:

1. When switching off always check the calendar. Users might delete their booking during your

time slot. If that happens to be in the afternoon the system might stay. This is potentially

dangerous and waists the lifetime of the lasers. If you delete your booking please make sure to

warn the previous user!!

2. When deleting a booking please send an e-mail to the previous/subsequent user and the institute

mailing list.

System Start up:

Step 1.: Switch on the control computer.

Step 2.: Power up the laser board with the power switch located on the back part of the

unit.

Step 3.: Power up the lasers needed for the current experiment. To prolong the lifetime of

the laser heads make sure to ONLY switch on the lasers that are really needed!

The 405nm (blue) has a single switch on the top rack shelf. The 488nm (green) and the

561nm (red) lasers have a separate controller with a main switch on the back and a key on

the front. After switching the main switch and before turning the key you have to wait 10-

15 seconds otherwise the laser will not turn on (even if the lights suggest otherwise). The

647nm (far red) laser is a diode pumped laser and has to be switched on in two stages:

1. First the main power has to be switched on the front of the MPL laser controller and its

key has to be turned. The pumping laser will switch on and the emission lamp will light up.

2. Finally the main laser has to be switched on using the GUI-VFL v3.0 software. Start the

program by clicking on the desktop icon. Start the emission with the on/off button. Set the

output power to 30mW, press the accept button and wait for the measured power to reach

the 30mW value. Finally reach the maximal output of 330mW in three 100mW steps

waiting at each step to wait the system to reach the appropriate output power.

Step 4.: Switch on the confocal controller that is situated to the left of the microscope. The

controller is on the lower shelf of the rack.

Step 5.(Optional): If you will use diascopic illumination switch the Halogen lamp on. The

controller is on the higher shelf of the rack by the microscope.

Step 6.: Log on to the network and start NIS elements. Please NEVER save data to the

local hard drives as they might be deleted!!

System Shut Down:

Step 1.: ONLY proceed if you are the last user or if the instrument is not booked for an

hour or more.

Step 2.: ALWAYS switch back to the 10X objective and distance the objective from the

stage. If the 100x oil immersion lens has been used NEVER forget to clean with lens

cleaning paper: 1. Fold the lens cleaning paper three times to form a strip, 2. Hold it on the

two ends on both sides, 3. Gently wipe with left-right motion. Never press with your finger

directly. 4. Repeat previous step until you cannot see oil stain and repeat once after that one

final time.

Step 3.: Stop NIS elements. If you proceed with the shutdown process without exiting NIS

Elements it will prompt you with error messages.

Step 4.: Switch the diascopic lamp OFF.

Step 5.: Switch OFF the confocal controller by pressing the button on the front panel.

Step 6.:

Power OFF the 405nm (blue) laser. Turn the key on the front panel to OFF and switch the

main power off on the controllers for the 561nm (red) and 488nm (green) lasers. For the

647nm (far red) laser:

1.Bring the output power down in 100mW steps to 30mW and switch off the laser using

the MPB Control software. 2. Switch the pumping laser and controller off using the key

and power switch on the front panel of the MPB controller on the top of the rack.

Step 7.: Shut down the laser board with the switch on the back part of the laser unit.

Step 8.: Switch off the computer and check if everything is off before leaving.

STORM Imaging:

Step 1.: Start the latest version of NIS Elements, enable the multicamera settings.

Step 2.: Right click on the NIS desktop and open the following windows: Ti Pad, C2plus

Compact GUI, C2plus scan area, LU4APad, N-STORM ans DU-897 Settings.

Step 3.: Check if the required lasers are on. This should include the 405nm laser as well

for activation!

1. STORM lens should be pushed in position (usually the 4x!)

2. The manual TIRF angle should be set to 45 degrees towards the back.

3. Illuminator slider positions should be as follows: ND filters OUT, Polarizers IN.

4. The motorized TIRF angle should be set to 3300 or higher!

Step 4.: Check the field stops

1. Field stop slider should be IN

2. Field stop should be set to the size of the ROI.

3. D Lens Should be IN!

Step 5.: Objective and slide should be clean and when approaching NEVER use the

escape/refocus button a since this case small bubbles can get trapped and cause unwanted

drift. If the objective has to be withdrawn from the surface clean both the slide and objective

from oil residue.

Step 6.: Check filter cubes: STORM, Dual STORM or the Quad cube

Step 7.: 6 Check Camera Settings: 30ms, EM gain 10 MHz, EM gain 300, Conversion gain

2.4

Step 8.: Check STORM Software settings:

1. 3D should be set to active.

2. Laser settings 405 10%, 647 100%.

1. Open the settings window by pressing on the Settings button of the N-STORM module

and select the "Sequence Panel"

2. Acquisition should be in Image Data Acquisition Priority.

3. Set activation to Continuous.

1. Press options on the Settings window.

2. Remove the Z-stack option tick.

3. Camera options: 256x256 image and keep recommended settings OUT.Set image

acquisition parameters: by pressing on the "Edit" button of the N-STORM window ->

405/647: 10000 frame.

Step 9.: Check C2 Confocal camera settings: Either reuse camera settings from previous

run or run the appropriate macro to set the confocal scan area (align the confocal channel

with EMCCD). The location of the macro is:

Z:\other\manuals\STORM\C2_Andor_registration\confmove_2017_01_25_STORM_cub

e_3D.mac (if more recent macro is available use that one).

Step 10.: Set Confocal settings and remember that for 2 channel imaging set channel series

OFF and for three channel acquisition channel series should be active with the following

order:(3->1,2).

Step 11.: Capture confocal stack before STORM imaging!!

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