Microscopes: Simple and compound microscopes for microscopic examination, Microbiology, Biochemistry (Light microscopes/Optical microscopes, Inverted microscopes, Compound microscopes)

1. Simple microscopes 

    Procedure:

a) Set up the microscope

• Simple microscopes come with 4x, 10x, 25x and 40x objective lenses, use the desired magnifying lenses to observe and examine the microorganisms.
• Avoid touching the lenses on the objectives and the eyepieces.
• Plug in the microscope to turn the unit on.
• With both eyes open, look into the eyepieces. Adjust the interpupillary distance by holding the eyepiece tubes and rotate the eyepiece tubes either towards or away from each other until only one circle of light is seen by both eyes.
• Place the specimen to be studied onto the stage of the microscope (slide, flask, vial, etc).
• Using the mechanical stage’s slide controls, center the specimen over the stage opening, lining it up with the light and the objective lens.
• To adjust the illumination, turn the dimmer knob on the left side of the body until the desired intensity of light is achieved.
• Adjust the iris on the light source itself by rotating the ring around the base lens or the iris on the condenser if needed.

b) Adjust the Focus

• First, use 4x lowest magnification objective to locate and focus of the specimen.
• Rotate the nosepiece to choose an objective.
• Adjust magnification and focus as needed.
• While looking into the eyepieces, turn the “fine/coarse focus knob” to bring the specimen into focus.
• Once the image is clear, use the fine focusing knob to tune it for best results.
• Inverted microscopes have long working distance objectives, it can take time to achieve focus on the correct plane of view (depth wise) when looking through a petri dish, slide, beaker, or other storage medium.

c) Adjust the Condenser and Diaphragm

• Move the 10X objective into the optical path.
• Center the condenser and lower the field diaphragm lever until the field diaphragm image appears in the field of view.
• Turn the “condenser focus knob” to bring the field diaphragm image into focus.
• Turn the two “condenser centering screws” to move the field diaphragm image to the center of the view field.
• Select the 40X objective. Adjust the position of the “field diaphragm lever” on the dia-illuminator until the size of the field diaphragm image is about the same as that of the view field.
• Turn the two “condenser centering screws” to move the field diaphragm image to the center of the view field.
• To adjust the iris diaphragm, move the slider to allow the desired amount of light to escape and reach the sample.

d) Trinocular Port (Microscopes with this feature)

• Use the eyepieces and the trinocular port to view the images through the microscope’s eyepieces, and those displayed on the computer screen or television.
• Conduct observation
• Select an objective of desired magnification.
• Move the “aperture diaphragm lever” and set the eyepiece position, turn the lens focus and perform focusing,
• Adjust the size of the image while viewing the exit pupil of the objective and aperture diaphragm image.
• Use 70% isopropyl alcohol and appropriate laboratory wipes to wipe the lens surface. Clean the specimen stage.

2. Compound microscopes

Procedure:

a) Set up the microscope

• Use the 4x, 10x and 20x magnifying objective lenses.
• Avoid touching the lenses on the objectives and the eyepieces.
• Switch on microscope, halogen lamp will be turned on.
• Adjust the brightness with the rotary knob.
• Use a specimen that contains areas of high and low contrast for initial adjustment.
• For incident-light fluorescence of transparent objects, adjust in transmitted light first.
• Place the specimen to be studied onto the stage of the microscope (slide, vial etc)
• Make sure all are adjusted to the correct positions.

b) Adjust the Focus

• Lower the nosepiece and set the desired objective.
• Use the knob/ grip to move the objective into the light path.
• Use the coarse and fine adjustment to focus on the object, this will vary the height of the objective nosepiece while the stage level will remain the same.

c) Adjust tubes and eyepieces

• Remove or fold back the glare protection on the eyepieces if wearing glasses, make sure to put it on if not wearing glasses.
• Set the interpupillary distance by pulling the eyepiece tubes apart or pulling them closer together until a single superimposed image is viewed.
• Note the interpupillary distance.
• Additional procedure for ergo tubes, set the viewing angle (0° – 35°) by tilting the binocular viewing port. To avoid fatigue symptoms, vary the viewing angle from time to time.
• Close any unused tube exits as stray light that may disturb viewing.
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• DM ILT trinocular tube:
• Switch the beam splitter to visual observation by moving the rod. The switching position is marked with symbols on the lateral face of the tube.
• Rod retracted = visual, Rod inserted = photo.
• Adjustment of the eyepieces is made in the same manner as for the binocular tube.
• Correct for defective vision by adjusting the eye lens of the eyepiece.

d) Operation of Objectives

    Immersion Objectives

• Use optical immersion oil, water, or glycerin for the immersion of objectives.
• Water immersion requires an objective with a ceramic front.
• Immersion objectives are also marked with a second, lower color ring:
• black: oil/water/glycerin. Black is the universal objective for oil, water, glycerin
• white: Water
• orange: Glycerin

  Locking of objectives:

Objectives with a knurled grip can be virtually shortened (locked).

Push in the front part by about 2 mm in the direction of the nosepiece.

Lock the objective by rotating it slightly. The locking mechanism must always be released before the immersion objective is used again.

  CORR Objectives

These are special objectives which can be adjusted to different cover glass thicknesses.

Adjust the correction mount coarsely to an average or estimated value by using the knurled grip.

Focus the preparation and readjust the correction mount until an optimum contrast is obtained, which may require refocusing with the fine adjustment.

e) Operation of Transmitted Light (Bright field illumination)

• Use this option for stained specimens.
• Press the catch lever and adjust the incident-light illumination carrier until the upper edge of the carrier and the corresponding condenser level marking match.
• Adjust the aperture diaphragm, for best resolution make the apertures of the objective and of the condenser to be the same size.
• Adjust the aperture diaphragm according to a subjective impression of the image.
• For visual comparison of the objective and the condenser, remove the eyepiece or use a focusing telescope and focus.
• Close or open the aperture diaphragm until its image is just visible in the objective pupil (= brighter circle).
• Reattach the eyepiece.
• For low-contrast objects, the aperture diaphragm can be closed further to highlight the faint specimen details.
• Use the rotary brightness adjustment knob or neutral density filter to set the image brightness in aperture diaphragm.
• For better results, do not use wrong cover glass thickness or wrong objective, do not place specimens on the stage with the cover glass upwards, do not open aperture diaphragm too wide or closed.

f) Operation of phase contrast

• Use phase contrast for unstained specimens.
• Adjust the condenser level, condensers have markings on the columns that indicate a liquid level (15mm).
• If two markings are present, the lower line indicates 15mm while the upper line indicates 50mm.
• The catch lever and illumination carrier should be level with the corresponding condenser level marking.
• Place the slide with the required light rings into the holder.
• Rotate the objective nosepiece to move the phase contrast objective with the lowest magnification into the light path.
• Open the aperture diaphragm marked “PH” .
• Focus the specimen using coarse and fine adjustment. If it proves difficult to find the object plane, temporarily narrow the aperture diaphragm or use a stained specimen. 
• Set the condenser disc to the BF position or pull out the light ring slide. Reopen the aperture diaphragm.
• Use the light ring that corresponds to the engraving on the objective.

    Centre the light ring:

• Remove one eyepiece from the tube. Insert the focusing telescope.
• Loosen the clamp ring on the focusing telescope and shift it until the light ring (bright) and the phase ring (dark) are in sharp focus.
• If the light and the phase rings are greatly different in dimension, they need to be matched by varying the condenser level.
• Do not use too thick or thin, brightly stained specimens.

 

g) Objective Phase Components

       Operation of Integrated Modulation (IMC)

• Use this option for both stained and unstained specimens.
• For high contrast and high resolution, to view the object through double-refracting plastic materials, such as Petri dishes.
• Remove the empty slide in the microscope stand if present and position the IMC modulator so that the lettering points forward. Lock the slide in position IMC (lettering IMC visible). 
• The IMC modulator will be flush on either side.
• Adjust the slit-diaphragm, fully open the aperture diaphragm.
• Select a medium brightness to avoid blurring of the image.
• Switch off the filters.
• Rotate the objective with the lowest magnification (10x) into the beam path.
• Move the slide of the IMC slit-diaphragm slide to the proper position for the objective to adjust the slit width.
• Remove one eyepiece and insert the focusing telescope, the line of light will appear as a bright line on the gray image of the modulator.
• Focus the line of light with the focusing telescope and adjust the position of the line of light with the adjusting screws to the right of the IMC slit-diaphragm slide with a fitting Allen key.
• Adjust the slit-diaphragm so that the boundary of the bright line of light is located near the darker edge.
• Move the other objectives with ascending magnification into the light path one after the other and check the position of the line of light. In case of a smaller deviation, find an intermediate position.
• Always make sure that the objective magnification and the slide position on the IMC slit-diaphragm slide match.
• Optimize the IMC by performing fine adjustments with adjusting screw (Allen key) on the IMC modulator slide if using the objective with the largest magnification.
• Once the IMC is perfectly adjusted, remove the focusing telescope and replace the eyepiece.
• If the image quality is poor, reverse the IMC modulator and adjust the slit to avoid glare and to achieve sufficient coverage.

Operation Of Incident-Light Fluorescence ( microscopes in facility with integrated incident-light fluorescence)

• Make an adjustment with transmitted-light first.
• Open the light stop by moving the lever.
• O = Light stop moved out of beam path.
• ⬤ = Light stop moved into beam path.
• Slide the filter block into the beam path.
• Position the specimen and focus. The field diaphragm is installed and pre-centered so that no adjustment is necessary.
• If a reddish background of the specimen becomes visible with UV excitation, eliminate this effect by moving the red attenuating filter into the beam path.
• O = Filter moved out of beam path
• ⬤ = Filter moved into beam path
• To switch on the 12 V/100 W halogen lamp at the power unit, open the light stop. Move the filter block into the beam path and remove the objective in the beam path.
• Place a white sheet of paper on the specimen stage.
• Rotate the collector adjustment until the lamp filament is clearly projected.
• Use a 3 mm fixed spanner to adjust the centering screws for level adjustment and for horizontal adjustment of the lamp until the lamp filament is in the center of the light spot.

Halogen, Xe and Hg lamps:

• Switch on the lamp at the power unit.
• Open the light stop and move the filter block into the beam path.
• Place a white sheet of paper on the specimen stage.
• Coarsely focus on the surface using a dry objective with low to medium magnification.
• Use a pen to draw a mark in the center of the bright surface.
• Remove the objective in the beam path.
• Rotate the collector adjustment until the lamp filament or the discharge arc is clearly projected.
• Move the reflection of the lamp filament or the discharge arc to the side by rotating the adjustment screws on the rear of the lamp housing.
• Focus the direct image of the lamp filament or the discharge arc and adjust.

Halogen lamp:

• Move the direct image to a position just below or above your center mark or, especially for higher objective magnifications such as with the Xe lamp, to the center, superimposed.
• Move the reflection into the brighter circular area.
• Align the reflection symmetrically with the direct image.

Mercury (Hg) and xenon lamps (Xe):

• Use the horizontal and vertical adjustment controls of the holder to move the direct image into the center of the brighter circular area.
• Move the reflection into the brighter circular area and focus on the reflection, adjust the mirror until the reflection is superimposed to the direct image.
• Remove the paper and position the specimen.
• Check with low objective magnification that the image is illuminated in a homogeneous manner.
• Adjust the collector if necessary.

Operating the camera

Start and Follow the instructions in the software for adjusting and acquiring an image.

For Camera Trigger Function, Start the microscope Hardware Configurator.

Clean the lens surface and specimen stage with 70% isopropyl alcohol and appropriate laboratory wipes.