DIVE start-up procedure

Owned by Andrew Vaughan
Last updated: Nov 02, 2023

New Chamber

The microscope chamber has been changed. See image below. The blacked-out front panel now slides aside rather than up and down (A). There is a clear panel under the blacked out one that also slides aside (B). The entire front can be opened using the handle (C). There a switch to change between white and red LEDs inside the chamber (E).

The instructions below assume that the standard six-position nosepiece is fitted to the DM6 microscope with three standard M25 thread lenses installed: 20x, 40x and 63x. The instructions also assume that the SuperZ Galvo stage is fitted. If an M32 thread lens or an alternative stage is installed, then different procedures will apply.

Turn on the PC and Supply Unit

It doesn't matter which one you turn on first. Press the power button on the front of the PC and switch on the Supply Unit by turning on the Power and Laser switches on the side of the Supply Unit and turning the key 1/4 turn clockwise to the vertical position. The PC can be turned on without switching on the rest of the system so you can analyse or copy data.

Check for obstructions that could damage the microscope

Make sure there is no sample on the stage or anything on or around the microscope that could cause damage during microscope initialisation.

To check the stage, slide down the hatch on the front of the microscope chamber. If it is hard to see, turn on the LED by rotating the white dial on top of the chamber clockwise. You will hear a click when the switch turns on, after which the dial increases the brightness of the light. The LED can also be switched on and off using the foot switch. If you turn the dial on and see no light, try pressing down the foot switch.

Raise the microscope nosepiece and lower the condenser

To avoid the possibility of damage to the objective lens during stage initialisation, raise the focus so the objective lens is far away from the stage. There are no focus knobs on the microscope stand itself but there are coarse and fine focus knobs on the STP8000 controller. The inner of the two focus knobs is the coarse focus.

To prevent damage to the substage condenser as the stage initialises, lower the condenser by rotating its focus knob anticlockwise until the front lens is clear of the bottom of the stage.

The DM6 is a fixed stage microscope, which means that the stage doesn’t move when the focus is changed. Instead, it’s the objective lens nosepiece that moves up and down. Fixed stage microscopes are used for experiments where the sample must be kept very stable, such as electrophysiological experiments where there may be probes inserted into the specimen that could be moved or damaged if the stage moves up or down. The fixed stage configuration also allows the user to change stage types and raise or lower the stage to accommodate different sized specimens.

Start the software and select a configuration

Double-click the LASX icon on the desktop. A splash screen will appear with two drop-down menus for selecting the Configuration and Microscope.

The microscope should always be DM6, but you will need to select one of the following configurations depending on the hardware features you need:

  • machine_MP-Laser_OFF.xlhw - which is for confocal imaging without using the multiphoton laser
  • machine_MP-Laser_OFF_climate_ON.xlhw - which is for confocal imaging of live specimens requiring warm incubation
  • machine_MP-Laser_ON.xlhw - which is for multiphoton or confocal imaging
  • Machine_MP-Laser_ON_climate_ON.xlhw - which is for multiphoton or confocal imaging of live specimens requiring warm incubation

A window will appear asking whether you want to initialise the stage. You should initialise the stage if you need to tile a specimen or capture multiple positions using LASX Navigator. Note that the option to use LASX Navigator will not appear in the software if the stage is not initialised.

Note that the focus should automatically raise the objective lens to the safe Home position when the stage is initialised, but it is wise to manually move the objective lens to a safe position before initialising (see above).

Click Configuration at the top of the LASX user interface. Click the Laser Config button and turn on the lasers you need. The Coherent Discovery laser has a tunable line and a fixed 1040 nm line. MP1 is the fixed 1040 nm line and MP2 is the tunable line. Click MP2, MP1 will automatically come on.