Preliminary

Protocol

1. turn on:
   • microscope power supply (Fig.1.3),
   • lamp (Fluoarc) (Fig.1.2),
   • computer: login: Z1imager: psw: zeiss;
   
   NB! if you turn off the lamp, you should wait at least 20min before to turn it on again
2. start program AxioVision Rel. 4.5;
3. open Microscope Window (MW) from the main menu: "Microscope" —> "microscope";




4. set the lamp intensity to 20% (Fig.1.1);
5. set regulator position (Fig.2.2) to see image on the screen;
6. select the 20x objective;
7. load the FlowCell (FC):

   Fig. 3.  FC-position.

   • click "Load position" on the touchscreen (Fig.2.5),
   • clean the microscope table (Fig.1.7) with optic cleaning pad (Fig.1.4),
   • load FC to the "right-back" corner of the table (Fig.3),
   
   NB! FC sits loosely in the standard glass holder
   • click "Set work position" on the touchscreen (Fig.2.5),
   • set coordinates: X= ##, Y= ##; Z= 0 (in the bookmark "Stage" of the MW),
   • click on the icon "Live" in the main menu,
   • select the bookmark "transmitted" in the MW,
   • open the "transmitted light" shutter, adjust focus distance (Fig.2.4) to see the edge of the channel,
   • move to the center of the 8-th channel,
   • close the "transmitted light" shutter;
   • write Y-positions for all channels:

     Channel Position Cluster density Y(ch8) (curr. posit.)           Y(ch7) (+ 1500)   Y(ch6) (+ 2x1500)   Y(ch5)     Y(ch4)     Y(ch3)     Y(ch2)     Y(ch1)

8. ajust focus distance and analyse clusters:
   • select the bookmark "reflected" in the MW: 38HEgreen filter,
   • open shutter,
   • adjust focus distance (Fig.2.4) (clusters are on both internal surfaces of the FC. The shift to the bottom surface is +75µm)
   
   NB! UV is not good for the clusters, be quick!
   • check cluster densities in all 8 channels;
   • close the shutter;
9. finish:
   • move table to the "load" position,
   • put FC back in Petry dish,
   • close the AxioVision program,
   • transfer files on your computer;
10. if you are the last user:
    • turn off the Power Supply,
    • turn off the FluoArc lamp,
    • logout.

Notes

cluster pictures

• open Multidimensional Acquisition Window (MAW): select from the main menu "Acquisition" —> "multidimensional acquisition",
• select the bookmark "C", leave one channel (you can delete/add channels right-clicking on their numbers) and chose the eGFP dye,
• go to "extended parameters" and set:

  enable	Channel 1
  Name	eGFP
  Channel color	green
  Exposure time	506
  Exposure mode	Channel settings
  Max. pixel intensity	230
  Shading correction	Use camera setting
  Shading correction name	---
  Setting during acquisition	User:HE_EGFP_VIS
  Setting after acquisition	Shutter_closed.zvhs
  Snap channel	Snap
  Dye	eGFP
  Display settings	System default
  Focussing	Current focus position
  User interaction	---
  Z Stack mode	ZStack

• photo: press "start", activate screen with image, "###", "Save",