Scanning Laser Confocal Fluorescence Microscope

E.S.Cole 2005

 

Warming Up

 

Turn on equipment from left to right:

            1) Turn ON the gas supply to the air table.   (Near sink, turn handle on tank).

            2) Turn ON mercury lamp for conventional imaging.  (Directly above scope).

            3) Turn on RT-SPOT camera power supply (for conventional fluorescence only).

                        Located above and to right on top of laser power supplies.

            4) Turn ON appropriate Laser power supply (there are three labeled for probes

                        being imaged).  Use toggle and then key to fire it up.

            5) Turn on compiler (large beige box above right of scope). Toggle.

            6) Turn on computer (note, there are two, one for confocal, above right, and one

                        for conventional fluorescence, black, under bench to right). 

                        On monitor:

                                    Input 1 = confocal/fluoview,  Input 2= Conventional SPOT-RT.

                        Obey computer prompts, for access codes, just hit return. 

            7) Turn on sub-stage illuminator (lower right of air-table).  Tiny box.

 

Finding specimen

 

Be sure sub-stage illuminator is ON.  This requires several steps.  First, hit toggle on power supply, (to the right of air table).  Second, if confocal/ Fluoview is ON, on the fluoview screen:  click “Optics” button at bottom left of screen, then click “Trans. Lamp”.

Third, make sure tiny switch on power supply is on manual, and rheostat is half up.  Sub-stage illuminator should now be lit. 

 

Focus on image, use bright-field or conventional fluorescence to find desired image. 

Note: two beam splitters:  top one is IN for Spot RT, OUT for confocal.  Lower beam splitter: IN for light to eyes, OUT for light to camera or laser access.

 

Switch to Confocal

 

Be sure monitor input is on 1. 

 

Pull out top beam splitter opening light-path to lasers. 

 

Pull out bottom beam splitter as well.  (light to camera/ lasers).

 

Dial in blank on filter changer dial.

 

(DIC analyzer is IN, polarizer is IN, turret dialed to correct mag). 

 

 

Configure mirrors and apertures for confocal:

           

For green fluorescent probes alone  (FITC-fluorescein/ GFP/ Sytox)

            Channel 1.

                        Large knob (beam splitter) is IN

                                    510 filter is IN.

                                    550 filter is OUT.

            Channel 2.        585 filter is OUT

                                    660 filter is OUT.

 

CA (channel aperture) is usually set at 2 (3 for 100X OIL).

 

For green AND  red fluorescent probes (TRITC-rhodamine, propidium iodide, Texas Red)

 

            Channel 1.       

                        Large knob (beam splitter) is Half-In/ Half-Out.

                                    510 filter is IN.

                                    550 filter is IN.

            Channel 2.        585 filter is IN.

                                    660 filter is OUT.

 

Set up Fluoview computer program for acquisition.

with image in focus:

(Work from bottom of left hand menu screen up).

 

1) Optics tab:  hit Trans Lamp (as mentioned above) to activate sub-stage illuminator. 

 

2) Dyes tab:  Clear/  Drag and Drop desired dye balloons into box/  Hit Apply.

 

3) Click on Z-Stage to access focusing motor.

 

(Jump to top of left side menu)

 

4) Select desired channels (FITC/ TRITC/ DIC).  Click on panel, click on tiny box in upper left             to activate/ inactive capture mode.

 

5) HIT XY Repeat under Acquisition tab.  (You are now imaging).

 

6) Zoom in/out to desired magnification.  Select proper lens setting in box. (eg. 60X oil).

 

7) Adjust brightness in channel boxes at top.

 

8) Go to Z-Series.  Focus down to mid section.  Re-adjust brightness.  Set bottom of stack, top of

            stack.   Set thickness of slices to achieve 20-30 sections max.  (Can go higher with stable

            fluors).  (Take single focus shot with DIC and save this for later super-imposing). 

 

9) Stop scan.

 

10) Go to Scan tab.  Select Kalman 4. 

 

11) Select XYZ feature.

 

12) Hit XYZ collect.

 

Process Image.

 

1) Choose Z-projection or flip through sections, select desired sections.  Can also select book-ends for Z-series.

 

2) Can show image with selected channels, or 2-3 separate tiles.

 

3) To save:  go to File I/O  (input/output).

            Save experiment only if necessary (takes huge space).

            Save Display:  choose where to save (folder/ desktop etc.) name and save.

 

4) Only output is a 250 Megabyte Zip disk.  (alas). 

 

5) After transferring to home computer via zip disk, erase files to create space.

 

SHUT DOWN.  Very Important.

 

 From right to left:

 

1) shut off sub-stage lamp.

 

2) shut off computer.

 

3) Shut off compiler.

 

4) Shut off Laser!   First turn key off (allow fan to run til it stops to cool laser, 10 minutes).

             Then, turn off toggle, ONLY after fan stops on its own.

 

5) Turn OFF mercury lamp.

 

6) Turn OFF SPOT-RT if it is ON.

 

7) Turn OFF gas supply to air table.

 

Leaving mercury lamps or lasers ON will destroy their useful lifetime.

 

Please LOG your hours specifying which lasers, or Hg lamp were used.