Fluorescence Microscope Nikon Ti-U

While the ultraviolet light is passing through the objective, do not view the light directly unless using the ultraviolet light shield.

Be aware and adhere to all other warnings and cautions as stated in the product manual.

Images used in this tutorial have been taken from the Nikon Ti-U and Nikon TI-FL Epi-fl product manuals.

This microscope is capable of various imaging configurations including bright-field and phase contrast which will not be used here.

Stage - Platform where prepared microscope slides are placed and controlled.

Objective - Used to magnify image.

Focus - Adjusting distance between the lenses and the stage which holds the sample in order to make image look clear.

Shutter - A mechanical cover which blocks light from passing through.

Filter Block - Directs and filters light allowing only a particular wavelength section to be seen.

Make sure that the shutter is closed by positioning the lever on the Epi-fl turret to the "C" position as shown in the picture.

"C" is for closed while "O" is for Open

To avoid stray light entering the image, shut the curtain and turn off the light.

Turn on the mercury lamp using switch as shown in the picture.

This lamp must stay on for at least an hour before turning off. Failure to do this may cause the bulb to prematurely fail.

There are three commonly used wavelengths in florescent microscopy which this microscope is capable of producing including green(FITC), blue(DAPI) and red.

Producing these wavelengths is done using filter cubes which are located within the Epi-fl Turret.

Only 3 of the available 6 slots are filled with a filter cube.

Position 3 contains blue (DAPI)

Position 4 contains green (FITC)

Position 5 contains red

Place filter cube of choice within the optical path by rotating turret into position as shown in the picture.

Place sample onto stage within optical path.

Insert aperture diaphragm by pulling slider as shown in the picture.

Make sure light filters are not in the optical path.

The filters are located next to the aperture diaphragm and should be in the "pulled out" position.

These filters are used to create repeatable brightness.

Rotate nosepiece until the 10x objective is in the optical path. Nosepiece location is shown in picture.

It is best to start with a low magnification objective in order to more easily resolve the image and verify the microscope is working as intended.

Be mindful that the objective should not touch the sample while changing objectives or while focusing.

Open the shutter by positioning the lever to the "O" position.

If all of the steps were correctly followed prior to this point, looking through the eyepiece should result in some sort of image.

Adjust the focus using knobs as shown in the picture.

The larger knob is coarse while the inner knob is fine.

Avoid colliding objective with the sample while focusing.

In conjunction with the previous step, adjust sample position by using the stage positioning knobs.

Stray light can be further mitigated by centering and circumscribing the field diaphragm.

The field diaphragm lever is located on the left side of the microscope. Picture shows location.

Too much light exposure can result in a bleached sample. Try and minimize the light exposure by utilizing the shutter lever.

Change objectives and refocus for greater magnification.

Again, avoid touching the sample with objective.