Since a user who has just taken control of the microscope does not know in what configuration it was left, the following steps are offered as suggestions for a smooth start.
And most importantly, each user is a front-line soldier in the battle to keep the lenses in fine working order.
N.B. Numbers in parenthesis refer to the figure at http://confocal.med.unc.edu/wwwLeica/Operation/OP_dmirbe.html
First, follow the Brief Check List to turn on the electronics and TCS program (see Note 1, "Loss of focus", below).
Then, at first approach to the microscope, follow these three lists:
For protection of the microscope, verify that
If the conventional microscope is on, turn it off before you ignite the mercury arc lamp. The electro-magnetic surge could damage the microscope's controls, Leica informs us. The lamp's power supply is far enough away from the computer that you may turn it on even if the computer is on. Since the lamp has significant warm-up time, turn it on first thing.
Using the condenser height adjustment knob (5), the condenser can be lowered below the stage. If it is so lowered when you swing the transmitted illumination arm into working position, you could destroy both the condenser and your sample.
Most of the lenses are spring-loaded and retractable. To lock them in the retracted position, push in the end and turn clockwise. Unfortunately, the lenses are functional, but poorly so, even in the retracted position. If you should set up with a lens in this position and then change to a lens that is not retracted using the motor, the second lens may smash the stage. You may wish to examine the tip of the 40x lens to see the least worrisome damage that can be caused by such a maneuver. In general, the lenses should never be retracted.
Warning: The focus positions of the 20X, 40x and 100x lenses are higher than the focus position of the 10x lens by approx. 120 m m (+120 m m on the LCD) or less; if the LCD says the lens is higher than this (more positive than 120 m m) and you are still groping for focus, stop immediately, drop the stage and un-retract the lens or lenses. Start over.
For an irritation-free startup, verify that the
- mirror to the transmitted light detector is out of the way (8)
Located on the right hand side, about 6 inches above the condenser. If the white line is toward you, the light path is blocked. Turn the knob 90o clockwise.
- transmitted light field diaphragm iris (9) is wide open and and the condenser iris (12) is set to minimum or set the way you want them
Closing these diminishes the transmitted light intensity and you may see the field diaphragm if the lens in place is near focus. Improper settings here can lead you to say "The lamp isn't working."
- condenser ring is on a click stop position (11).
Use HF position normally. 100 and 40 are for DIC. A sometimes useful dark field view can be obtained on the cheap by moving the condenser ring between click stop positions, and a previous user might have left it in that position.
- upper beam splitter switch rod is pushed in (scan head shutter, 17)
Located on the right hand side, its action is to deflect the laser beams from the oculars and block off the oculars during scanning, all for your protection The lower rod has no function for the moment
- wheel above the right hand sliding rods is in the SCAN position (or the Bertrand lens position if you will need that function; see Note 2, "Kohler illumination", below)
- Walliston prisms (13) are out of the path or are appropriate to your need
If you are not using DIC, you want position "H" (for brightfield). Two other positions are identified as being for the 40x and 100x lenses, and the 4th is blank for now (equivalent to H).
- polarizer and neutral density filter (10) (just above the condenser) are out of the path
- quarter waveplate slider (16) (left side, just below the fluorescent filters) is out of the path
- lens collars are set to fully clockwise position (best done when checking lens retraction)
The collars on the 100x, 40x and the 20x objectives are intended to give greater depth of focus by closing down a diaphragm. This reduces the N.A., however, and creams resolution. Use them with the diaphragm wide open (fully clockwise). The obvious effect of having the collar at any other position is decreased light. Warning: These collars, which can not be locked, are going to be a nuisance for high resolution users.
And do the following:
- Clear both travel limits on the lenses and drop lens turret to a low level (see LCD display & controls)
- Insert your slide only if you know it does not have any non-Leica immersion oil on it.
If you have examined the slide previously with an oil immersion lens, clean it off with alcohol (found in the confocal room). The presence of two different oils having different viscosities and possibly other properties, even though they have the same refractive index, leads to a very disconcerting overall background which renders the image essentially useless.
- Move the 10x lens to the viewing position and find focus
- Set upper limit; "0 m m" will be displayed
- Close down the field diaphragm (9) and raise or lower the condenser to bring the diaphragm into focus (while keeping the objective lens focused on your sample)
- Move to the lens you will be using and apply oil properly; see Lens handling and Immersion oil.
- Focus with this lens until the field diaphragm is again in focus (the lenses are approximately parfocal to one another) or focus on the sample directly.
If you will be using transmitted light for anything more than just finding fluorescent samples, complete the Kohler illumination procedure (see Note 2 below).
- Reset the upper limit
Note that the electronic focus (3) will not move the lens higher than the set upper limit. This is a major lens protection feature and proper setting of the upper limit is crucial. The manual focus wheel (18) will go anywhere.
- Ensure lower limit is not set.
Distance is displayed on the LCD; lens retraction is indicated by negative numbers.
To clear the travel limits
To set travel limits
With the lens turret actually at the desired upper or lower limit position, push the corresponding button until SET? is displayed. Release the button and push again until the upward or downward pointing arrow reappears
Set the oculars to be parfocal with scanned images.
With each new element you want to image, your movement from the conventional microscope to scanning will be easiest if focus in the former puts you real close to focus in the latter. To do this, get an initial scan of your first sample, then return to the conventional microscope and adjust the oculars to give a sharply focused image.
For a clean finish (leave nothing but footprints, take nothing but memories)
- Check the calendar to see when the next user is on board. If more than 1 hour away, kill the mercury arc lamp (you do not need to kill the microscope before turning off the lamp)
- Turn lasers down to minimum power. If no user will be on for 3 hours, turn off lasers, leaving the fans on for now
- If you are the last user for the day, start shut down by killing the mercury lamp and turning the power control on the two large lasers to minimum (leaving the fans on for now). Lasers should be run at minimum power for 10 minutes before keying off the ignition switch but leaving the fan(s) running. If you have been scanning at minimum power the 10 minute period has been met! By the time you finish everything else, the mercury lamp will have cooled enough that it won't melt the dust cover
- Before removing your last sample from the stage, put the 10x lens in the viewing position, drop the lenses to their lowest position, and clear the travel limits from the LCD
- Remove your sample; be sure you don't set it on the table oil-side down
- Remove oil from all lenses (lens paper only, no Kimwipes; they contain grit). Do not put lenses in retracted position
- Turn off lens controls and LCD display
- Key off laser ignition if they have been run for at least 10 minuntes at minimum power.
- Put on the dust cover if all is done for the day
- Kill the laser fans when exhaust pipes at back of table are not warm (less than body temperature)
- Close the door on the way out
Note 1: Loss of focus in going from the microscope to scanned images?
Think first about movements of the galvo stage rather than random focus drift in the microscope.
When the scan electronics are off or the TCS program is closed, the galvo stage is parked. The parked position is about 80 m m below the position during XY scans, which is also the "holding" position between scans. If you set up the microscope when the stage is parked, the stage will rise to the XY scan position when you start the scan and your first scanned image will be miles out of focus (you probably won't see any image at all). Solution: Turn on the scan electronics and activate the TCS program before setting up. If for some reason you have to turn on the scan electronics with the TCS program already loaded, move the stage to the "holding" position by doing one scan before anything else (no sample needed, the lasers don't even have to be on).
Note 2: Kohler illumination
All but the crudest uses of transmitted light require that the microscope be in the Kohler illumination configuration. For an optical explanation, see XXXXXX (in preparation). To set up the Leica properly, do the following:
- Focus on the specimen
- Close down the field diaphragm (9) and raise or lower the condenser (5) to bring the diaphragm into focus (while keeping the objective lens focused on your sample)
- Open the field diaphragm (9) until it can just be seen at the edge of the field
- Center the field diaphragm using the black centration screws on the condenser
- Using the dial on the right hand side of the microscope (above the beamsplitter rods), bring in the Bertrand lens, which images the diaphragm in the condenser
- Focus the image of the diaphragm using the black wheel on the big dial
- Open the condenser diaphragm until its image can be seen at the edge of the field of view
- Return the dial to the SCAN position
- You will probably have to repeat the adjustment each time you switch lenses
7/28/97 final version (modified by m.c. 98-04-29, 99-06-01, 99-06-23, 00-08-22)