Multiphoton Quick Start

Owned by Ki Hng
Last updated: Mar 25, 2024
5 min read

Before you start

The key on the side of the keypad must always be left in the horizontal position (see image below), even when the system is powered down. However, occasionally I find that it has been turned to the vertical position, which means it is switched off. Make sure it is in the horizontal position, otherwise the lasers and anything else connected to the "Components" switch will not come on. 

If you get an error message saying "Unexpected error during hardware control task. An attempt to move the filter-set "ShutterArgon" to position 1 failed", it is because the key is in the wrong position. 

If this happens, you should quit Zen, turn on the key and then open Zen again.

Switching On

  • Turn on the "Main switch" on the keypad
  • Turn on "Systems/PC" on the keypad
  • Wait for computer to boot up and log in to LSM-User account
  • Turn on the "Components" switch on the keypad. 
  • Click the "Start System" button

  • Click the" Acquisition" tab and expand the "Laser" menu. Turn on the lasers you need.  (Some of the lasers might show a red background initially, which means it is not ready to be used.  This will go away after a couple of mintues when the laser is warmed up)


 














Locating your Specimen

  • Click the "Locate" tab and expand the "Microscope Control" drop-down menu
  • Select the objective lens you'd like to use - either via the menu in locate', or via the touchscreen controller next to the screen. 
  • Select a suitable fluorescence filter. Only the first three should be used; the rest are special positions for the confocal and multiphoton
  • Put your specimen on the microscope and open either the transmitted light shutter or the fluorescence shutter depending on which mode you want to use to find your sample
  • Turn on the lamp by clicking its icon and pressing the On button

  • Open the fluorescence shutter to let light through

  • When your specimen is in focus, close the shutter and click the "Acquisition" tab.


Imaging Setup

  • Expand Imaging Setup
  • Choose LSM and Channel
  • (Non Descanned is for multiphoton only)
  • (Lambda Mode and Online Fingerprinting are for spectral imaging)
  • Use Smart Setup if you want to easily set up your experimental settings (e.g. filter and detector combinations) depending on your dyes.

  • Pick dyes from the list and Smart Setup will arrange the light path to optimally capture those dyes with three combinations:
    • Fastest (simultaneous acquisition with a risk of crosstalk)
    • Best Signal (Sequential acquisition with the spectral windows optimised for each dye in turn to maximise signal, but may be slow)
    • Smartest (Line sequential with the spectral windows balanced to get the best signal with the best speed)

  • You can manually setup or adjust the light paths in imaging Setup

  • Fastest shows all three colours being excited and captured simultaneously in one Track (Track 1)
  • Sequential acquisitions would require more than one Track.
  • Best Signal creates three tracks, one for each channel so they are all captured separately but switching between the tracks takes time because the components (e.g. dichroic mirrors) move
  • Smartest creates two tracks, with spectrally separated dyes in the same track as each other. Also, the physical components don't move to save time
  • NB lasers are always set to 2% in Smart Setup - but you can change them afterwards
  • Click Live to get a live scan.

  • Expand Acquisition Mode. In here you can change a variety of parameters but the main ones are the scan settings:


    • Scan Mode: Whether the scan switches channels in Frame or Line mode (frame by frame or line by line)
    • Frame Size = Scan Format
    • Line Step: Increases scan speed but lowers the resolution in one dimension by skipping lines in the scan
    • Scan Speed: the line frequency. Expressed in pixel dwell time as opposed to in Hz
    • Averaging, including number of scans and whether it should be done line by line or frame by frame
    • Method: Mean = Average, Sum = Accumulate
    • Bit Depth: 8 bit is default
    • Direction: unidirectional or bidirectional (Stick to unidirectional)
    • Scan Area: Zoom and Scan Field Rotation
  • Expand Channels
  • In here you can determine which channels are actually scanned by unchecking the tracks and you can define laser powers and detector settings such as detector gain
  • For most purposes you will use Integration Mode, which is the normal mode of using a PMT, but photon counting can be used to quantify signals so that each intensity level corresponds to a detected photoelectron.
  • Range Indicator at the bottom of the screen will indicate saturation and black pixels

  • Single Channel allows you to see just a single channel, which is useful when adjsting the range. Note that the confocal is still scanning all the channels - it just isn't displaying them
  • Live scans at the maximum possible speed. If you click Continuous it will scan using the settings in Acquisition Mode
  • Save by clicking the floppy disk icon in Images on Documents (top right). Delete by clicking the X
  • Z-Stack optimise scetioning and step. Diagrams illustrate the degree of sampling, which is also shown in percentage form below. This changes when the step size is changed.

  • The function Match Pinhole sets the pinhole to keep this optimal interval and in addition sets the slice thickness for all detection channels approximately the same. This typically results in slightly thicker slices for channels detecting the longer wavelength range. In case the channels are assigned to different tracks and a Frame wise Multitracking scheme is applied, the pinhole diameter is set for each track such that the values of the resulting optical sections from the different channels are identical and have double the value of the optimal interval.