Zeiss Axioskop Manual de usuario

ZEISS AXIOSKOP

Microscope’s User Manual

Managed by

For information about this instrument, please contact

Dr. Alloysius Budi Utama @ ext. 8232 or e-mail [email protected]

Use of the Axioskop (Zeiss)

The Axioskop is equipped for viewing samples with transmitted light (from a halogen lamp

source) and has optics for: Bright field, Phase Contrast and Differential Interference Contrast

(DIC) microscopy. Samples can also be viewed for fluorescence microscopy using the incident-

light system (front a mercury lamp source).

1) For transmitted light microscopy work, press the

green on/off light switch (1a) at the back-right side of the

microscope while light intensity / level knob is located

next to switch (1b). The halogen lamp is in the vented

box at the bottom rear of the scope (1c).

2) For fluorescence microscopy work, press green on/off light switch (2a) on the front of the

arc lamp power supply box behind the scope (right side- 2b). The mercury lamp is in the vented

housing on the rear of the scope and is above the halogen lamp box (2c).

*Note: the halogen lamp does not need to be on when only viewing fluorescence

Transmitted light microscopy (Brightfield/DIC)

Begin with glass slide/specimen securely in place on stage, light at medium

brightness/temperature (~6).

Ocular's (eyepieces) diopter rings set

at "white dot-to-0-position". In most

cases start with 10 x objective lens in

position. Obtain a "basic" focus using the coarse (large inner black knob), and then if desired,

the fine (smaller outer knob) coaxial focusing control knobs (on the back left and right side of

the microscope base).

*Note: one revolution of the course knob = ~2 mm of stage travel toward any Z-direction

Kohler Alignment

To achieve maximum even illumination of the sample, the condenser needs to be properly

focused and adjusted (Kohler Alignment)

1) Put the specimen in focus in the light path, then pull out the Analyzer [thin black plastic

slider with dark filter that is below and to right of the ocular and the large black filter slider].

Turn/rotate

counter-clockwise

the lower

Polarizer using its

black lever (at the

bottom of the

condenser and above the field diaphragm) so they are not in the

light path.

*Note: for Brightfield/DIC adjustment, the black horizontal metal filter slider should be in the

"no-filter/ open position" (last position on left or 5th slot to left; pulled to the extreme right).

**Note: After adjustment, for Brightfield use, you can leave analyzer & bottom polarizer in

OUT position. For DIC use, analyzer and polarizer can be returned (slide IN) to the light path.

2) Transmitted light can be controlled by adjusting the black circular field diaphragm lens

(3a) at the base of the microscope by turning its outer ring clockwise (opens) or

counterclockwise (closes). To adjust the condenser and focus the light, put the sample on the

stage look into the ocular and focus it.

Then close the field diaphragm,

center the "small" light spot by

turning the left and right silver

color centering screws (3b - Two

silver screws at 7 & 5 o'clock on

the front of the condenser).

Pull out

Analyzer

Raise /lower the condenser apparatus by turning the small black knob (3c - on the left back

side of the condenser below the stage) until the circumference of the light image is "sharp and

well defined as a hexagon shape."

* Note: During this process, the particular condenser ring optics can be selected to match the

type of imaging desired: turn the condenser (turret) control ring (3d) to: HDIC for brightfield

work; DIC for differential interference contrast work. For Phase Contrast work, use no “1” for

10X, no “2” for 20X and 40X and no “3” for 63X objective.

**Note: it might not be necessary to adjust/focus the condenser for different objective lenses

(magnifications). For example, if condenser is focused for 20x, that could be "acceptable" for

10x and 40x (air objectives); focus for 63x could be "acceptable" for 100x (oil objectives), but

NOT for the 10, 20, and 40x air. Minor re-centering with the condenser centering screws can be

performed: the light is best centered when the spot is opened up with the field diaphragm ring

to about 80- 90% full field illumination, and the light circumference is "concentric" with the

edge of the field boundary.

***Note: the silver ring on the condenser opens (clockwise) and closes (counter-clockwise) a

diaphragm for more or less light, respectively —lesser light might give best image (totally

counter-clockwise), however, adjust as necessary for the conditions.

Before

After

Sharp edge

DIC

Brightfield

Phase 1

Phase 3

The best DIC images are determined empirically, and important factors are: positions of the

prism (adjusted by turning the little silver screw above each

objective lens –red arrows in left image) and the condenser

aperture diaphragm. Turn the prism screw for the desired

image, and turn the diaphragm ring –3a [more open

diaphragm gives more contrast and depth of focus].

On the right side of the microscope base, there are 4 buttons for the filter magazine in the

illuminating beam path, and from front-to-back are: neutral density

filters 0.015, 0.06, 0.25, and a conversion filter which converts artificial

halogen light (color temperature of 3200 K) into natural outdoor light

(color temperature of 5500 K). When push buttons are IN or OUT, the

corresponding filters are IN or OUT as well.

Fluorescence microscopy

After turning ON the mercury arch lamp, record in the log hook the number on the time

counter and actual time of day.

* Note: once the arc lamp is ON, it must stay on for at least 30 minutes before being turned

off—when the lamp is turned OFF, it must remain off (to cool) for at least 30 minutes before

being turned ON again.

** Note: Pull out the analyzer (thin black plastic slider with dark

filter which is below and to the right of the ocular and the large

black metal filter slider) from the light path because it not only

blocks light, but continued exposure to UV light will damage the

filter.

Pull out

Analyzer

When the arc lamp is turned on, and the slide/specimen is placed in position on the stage,

illumination of the sample from the objective lens will be readily apparent. The slider, black and

marked L on left side and R on right side, can be used to block / interrupt the illumination beam

path (move to far right position) when not viewing the sample to prevent photobleaching. The

green filter/middle position slightly filters the light, but the open position (far right) is probably

best when examining the sample.

*Note: in fluorescence microscopy, the less exposure of the sample / fluorophore to excitation

light, the less photobleaching and subsequent loss of fluorescence and intensity of signal.

The fluorescence reflector or tiller slider [large horizontal black metal box between ocular and

objective lenses] has 4 positions for fluorescence that contain various excitation filter / dichroic

mirror / emission filter sets, from right to left:

// ~450-490 um ex. (green em.)

// ~510-560 nm ex. (red em.)

// ~450-490 nm ex. (green/yellow em)

// ~300-390 nm ex. (blue em.)

Decide which filter set is appropriate/best for exciting and

detecting the fluorophore(s) being used, and move the filter slider

to that position. [See fluorescence reflector slider sheet for filter set details.]

*Note: It should not be necessary to change/adjust the illumination beam, however, the

following controls affect the diameter and centering of the light. Levers for adjusting the

luminous field diaphragm (upper, left and right side) —move by pushing or pulling levers to

the right to reduce and to the left to increase the illuminated area [“normally," the area of light

should just fill the field of view, as for the halogen lamp beam with light microscopy]. Use the

two silver centering screws (lower, left and right side) to position the luminous field diaphragm

- to move and align the light beam, alternate twisting both screws.

Zeiss Axioskop Fluorescence Reflector Slider

The first (right-most) position: (for FITC/ AF488/ GFP - type fluorophores)

Excitation filter: 450 - 490 nm

Dichroic mirror: 495 nm

Emission filter: 500 - 550 nm

Second position: (for Rhodamine/ AF594/ Texas red - type fluorophores)

Excitation filter: 510- 560 nm

Dichroic mirror: 580 nm

Emission filter: 590 nm LP

Third position: (for YFP - type fluorophores)

Excitation filter: 450 - 490 nm

Dichroic mirror: 510 nm

Emission filter: 520 nm LP

Fourth position: (for DAPI / Hoeschst - type fluorophores)

Excitation filter: 300(365)390 nm

Dichroic mirror: 395 nm

Emission filter: 420 nm LP

Fifth position: (for DIC / Brightfield)

OPEN (no filters)

Objectives for Zeiss Axioskop

Plan - NEOFLUAR 10X, NA 03 Ph 1 (air; n=1)

Phase contrast: condenser position 1

Brightfield: condenser position HDIC