Jacobs: Leica MP Microscope Protocol

Leica MP Microscope Protocol

Objectives

3 63X objectives. Two of which are immersion (1 for water for media, 1 oil for fixed tissue). The remaining one is a dry objective with a working distance of 2.5 mm.

Filters

• Red - EthD (live/dead)
• Green - FITC, GFP, Alex 488
• UV - DAPI

Procedure

Setup

1. There are three green switches on the bottom right side of the desk, (one for PC, one for Scanner, one for Laser), turn all three on and turn key on
2. Turn on Mercury Lamp (on floor next to desk)
3. Turn on control box for microscope (underneath microscope, must be on to start LAS AF hardware)
4. When using DAPI, turn on the 2-photon source (near back of the room, orange switch, this requires time to warm up - about 1 hr)
5. Open LAS AF on the desktop for imaging
   1. Select laser off for simulation to view images.
   2. Select laser on with or without photon for regular microscope use.
      • When using 2 photon click ‘configuration’ and select ‘MP-on’ at startup of software.
   3. Keep microscope stand at DMI 6000.
   4. When prompted to initialize stage, click no if you do not need to take sequential images or stage mapping feature are required. Also click no if you have already focused your sample. If you select ‘yes’, make sure to lower the objective first and the microscope is tilted back.

LAS AF

1. Configuration -> Laser to turn on lasers
   1. If using MP, turn MP on
   2. If using just 488, you only need to turn on Argon.
   3. If using other dyes, turn on full spectrum (HeNe 543, 494, 633).
   4. Set the Argon at 20-30% as anything higher will accelerate the bleaching.
   5. Acquire-> Acquisition to adjust resolution and rate of sampling
   6. For live (video), select 1024 x1024, 600Hz
      1. Smaller resolution will allow for faster scanning at higher frequencies. A 512x512 resolution is usually good enough.
      2. A slower scanning frequency leads to more information but also more exposure time and thus more bleaching.
   7. If using DAPI,
      1. select MP DAPI from the list of preselected beam path settings
      2. click ‘MP laser’: MP shutter should be at 800 nm
      3. On panel turn knobs to: Fw(25%), Gain (30%-50%), Offset (0%), MP1 (1)

Loading Sample

1. Change stage if needed (use provided allen key to remove screws)
2. If using an oil objective, put a small drop of oil on the objective. If using a water objective, put a small drop of water on the objective.
3. If not place sample in holder and slide together to grasp firmly
4. If imaging on a glass slide, place coverslip down as it is thinner.

Imaging

1. Control knobs: X, Y, and Z all have fine and coarse motions. MAKE SURE WHICH IS TURNED ON TO NOT DAMAGE OBJECTIVE.
   1. For imaging through a coverslip, after a fresh drop of oil distance from slip should read between 700nm and 900nm (approx). This number will decrease as you image as the oil will be spread across the slide as you move, thus decreasing the focal plane distance.
2. If imaging a Z-Stack, use knob on panel to set heights, not controller (knob has a feedback display allowing you to know your position).
   1. Always set the final position first, then the initial position as the Z stack will start from where the panel feedback shows, not what you actually set.
   2. Select if you want to determine Z stack by time, or number of slides.
   3. Click start to begin the imaging sequence
3. Adjustments
   1. Adjust the smart/detector offset until you see the green (zero signal)
   2. Adjust the smart/detector gain until you see the blue.
      1. If you are at max gain and still have not saturated, you can increase the argon.
   3. You can also use sequential scans to improve your image. Instead of simultaneous imaging, each excitation and emission is done separately.
4. For multi-photon imaging, the normal exciation with the confocol should also work.
   1. To turn on emission, you may need to hold the mouse down for a longer (click like you mean it.).
   2. Set the FW first. Then set the detector gain to about 800 and then adjust the EOM. Then adjust the detector settings.
   3. If you’re having problems make sure the photon mode is pulsed not CW (continuous wave)

Saving Images

1. Click on the experiments tab
2. Select file you wish to save as an image, right click it and select export as tiff
3. Name and save

Power Off

1. Bring all laser power down
2. Turn off all lasers (under the configuration-> lasers)
3. Close LAS AF program and turn off computer
4. Turn off everything except Laser power switch, turn key to off.
5. Shut off laser power ~15 min after turning the key off
6. Turn off microscope, Hg power and 2-photon source

Maintenance

• If the objective is blurry clean with lens paper and 70% EtOH.