Differences between using Fluorescence and Brightfield

• When using fluorescence , you don't have to turn on the light source that is part of the microscope

  • aka , don't turn this on :

    

• We are doing "epifluoresence" , and the light source comes from the back ( mercury halide lamp ) :

  

• Light path travels into "filter turret" , where you can select different filters to use :

  

• Then light passes down into the objective , where it illuminates the sample :

  

• the fluorescent light is then collected by the objective , and reflected back up into the eye piece or camera

• Don't have to mess with preset button ( just leave it turned off ) :

  

• However , the mercury burner lamp still needs to be turned on when you first get to the microscope :

  • make sure "burner light" is solid green

  

• Sample we are using is in the back fridge

  • its a special sample they purchased of "cultured cells"

    • labeled with 3 different fluorescent markers

      1. Beta Tubulin
      2. Flamoydin ?
      3. DAPI = for nucleu

  

• MAKE SURE THE SAMPLE CONTAINER STAYS COLD

• WHEN YOU ARE DONE WITH THE SLIDE , PUT IT BACK IN THE TUBE , AND THEN BACK IN THE FRIDGE

  • otherwise it will fade

1. Start with the 10x , and focus and find your sample

   

2. Select appropriate channel / filter

   • use FITC ( blue light ) to see green fluorescence

   

   • use TRITC ( green light ) to see red fluorescence

   

   • DAPI ( blue light ) to see nuclei

     • chemical that labels double stranded DNA

   

3. To illuminate the sample , open the shutter

   

4. Once you have the sample in good focus , take pictures with 20x and 40x objectives

5. Pull light selector knob all the way out for fluorescence

   • the images will be much dimmer than they were for bright field

   

6. When you are in the fluorescence mode and using the CellSens software :

   • make sure you choose the appropriate acquisition setting :

     

7. Adjust exposure time so you can see the sample

   • if you sit down to the computer chair and you can't see anything on the computer screen

     • but you CAN see something via the eyepiece

       • then your exposure settings are messed up ( probably too low ) , so turn up

8. Ctrl+H keyboard shortcut = toggles "high/low" mode

   • applies lookup table to pixels

     • totally dark pixels are turned to blue color

     • saturated pixels are turned to red color

       • so minimize red color via exposure setting

     • toggle back and forth between "high/low" mode to get reasonable image

9. Save image file as usual

• might have to adjust focus as you change different filters

• CLOSE THE SHUTTER once you are done imaging

  • increases the lifespan of the slide

  • especially with DAPI ( uses short blue wavelengths )

    • can bleach the sample

• You can merge all three images ( if you want ) , all of the channels have to be open at the same time

• make sure you select "convert to RGB"

• click "ok"

  • creates a new merged image