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Notes for confocal training session I
Also completed dossier for import-export of human tissues for exchange with English colleagues. Put in an envelope to the INSERM tomorrow.
For one single color: can do live imaging also.
Blue notebook is a detailed tutorial (also online); small binder with white rings is more useful for day to day.
Software LSM510. Start Expert Mode. Oil from 40x and up.
Work in menu “acquire” (buttons). First four are most useful.
Lasers – Helium-Neon or Argon. Only turn on when need. But can leave on standby if will need during session.
Microscope: Objective choose and let automatic motor do its work. Then reflector – only for the fluorescence mercury lamp, in order to choose one’s field, and is necessary. Shutter on the right in front – slide up and down to get more or less intense. FL is the button for on/off Hg lamp (will have had to turn on the generator).
Was on the menu in mode Vis (will work for Nomarski – buttons on right are labeled “HAL” (for halogen; potentiometer in front on slider) then change over to LSM which will be for the confocal. Suggests focus in fluo anyhow.
Hit the Config button – how many and which colors will have. Single vs multi-track. NT 80/20 is the dichroic filters. Click on diskette for pre-saved config gfiles. Eg. red t, green, Qdot… (the t being transmission).
Last menu is Scan control. (has its own page in binder). Mode or Channels. Channels eg. Red and Transmitted light. In Mode – other parameters eg. Image definition (eg 512x512 pixels) For a publication quality image, click on Optimal to get the best pixel definition. By default 8 bits but if quantify image intensity can use 12 bits. This is where the rotation of the plateau is as well. Scan control, often default speed is at 7. 4 is very slow.
Pinhole is principle of the confocal (this in microns is indicated when set to 1 unit) – only one focal plane. Laser is focussed through a lens first to excite the specimen then the epifluorescence bounces back and only get through the confocal plane (the pinhole) only the things that are in one focal plane. It’s actually a diaphragm. More pinhole, more light. But more focal planes, too. Click on 1 which is one Airy unit. The best resolution of the system. Depends on both objective and on the color – so click on 1 each time. Indicates the thickness of the optical slice. Fairly flat cells are about 5 microns thick. A platelet is more like 1 micron.
Choose a focal plane with Cont but take a single image, swept with laser from L to R and top to bottom, just did it once.
Fast XY is quicker sweep (vitesse 9 or 10) if want to position and screen a given field.
Back in Channel, can also work with Gain and Offset. This must be in Cont. Gain is sensitivity to electrons. Also leads to saturation of captor. Use Palette tool on the R of the image. Click “Range Indicator” red/blue. Spread out signal so goes just between red and blue by diminishing the gain until most red is gone – just a few red pixels. The background is adjusted by amplifier offset but not too much blue otherwise will lose some low intensity pixels. Take away the palette.
All colors are false colors – the eye sees cyan very well too, rather than blue, if you want the false color.
No pinhole in the detector for transmitted light, of course. However, can get better contrast. Nomarski filter with twist knob under each objective. And can regulate the gain/offset as well.
For the 63x objective, need DIC in white. For the 10x filter sometimes can take away the polarizer above and H for the wheel. No phase contrast in the objectives, though the Ph1, Ph2 etc options are in the wheel.
Last thing, in Scan Control = Mode – make a mean under Pixel Depth, Scan direction etc. using 4-8 images. Make a single scan. Now save the image!
Save as: Open MDB or New MDB. Make a new individual folder. Can open an old file and click on the right to “reuse” which will have kept all gain/offset settings, laser power, all the rest as well. Better than using the “Config” diskette. The Reuse will save 5-10 minutes of configuration!
Use Zoom: first separate the channels by doing “split” – the transmitted light is actually a reuse of the laser reflected light. So doesn’t additionally bleach. Don’t zoom over 3x by using the crop function. However, at 1024 not more than 2x. Press crop and then position. Can remove these by clicking on Reset.
Save all the channels, can remove the DIC image later if so wish. Or remove it from the outset by clicking on Channel column to the right of the image and unchecking (don’t do it at the level of the light).
Go to Multitrack and call up a config. Green red blue = green red far-red. Make sure to put each channel on 1. Must readjust each channel (after split).