Fluorescence Microscopy for the Axioplan 2
Reflected light illuminator.
- Identify the
object with bright field, then block transmitted light.
- Center and
focus epi-illuminating arc
- Select fluorescence
filter cube and filter wheel setting.
- Set up Koehler
- Center epi-path
field iris and open epi-path aperture iris
Step 1. Identify the object for fluorescence
with bright field or DIC objectives, then block transmitted light.
Step 2. Center and
focus epi-illuminating HBO arc source using adjustment aid.
Step 3. Select fluorescence filter
cube and filter wheel setting appropriate to the fluorophore in the specimen.
- The filter wheel and filter cubes have been selected
to allow optimal use of a variety of fluorophores. If you wish to install
other filter combinations, please see Dr. Griffing.
- Filter wheel settings:
- Home position: Triple Band Pass excitation. Maxima at 330-385 BP, 475-495 BP, 540-565 BP (CFP, GFP, mRFP or DAPI, FITC, Rhodamine)
- Deep Blue 442 10nm BP for CFP (or pH insensitive Fluorescein)
- UV excitation 360-370 BP for DAPI
- Blue 485-505nm excitation for GFP or pH sensitive Fluorescein
- Green/Yellow 565-585nm BP for mCherry or Texas Red
- Double Band Pass excitation at 475-495 BP and 545-565 BP for GFP and mCherry or mRFP - or Fluorescein and Rhodamine
- Filter cube settings:
- There are two emission-side filter cubes labeled GFP and Triple Pass. The GFP transmits light between 505 and 535nm. The Triple Pass filter transmits light between 445-470 BP (just barely gets CFP -does well with DAPI) 505-535 BP (GFP and Fluorescein), and 580-620 BP (mCherry, Texas Red, and Rhodamine)
- Spectral Characteristics
of all of the filters.
- Spectral Characteristics of a few common fluorophores.
Step 4. Set up Koehler epi-illumination
by focusing sample, removing diffusing screen and closing down the epi-path
- Focus the specimen with the focus knob.
- Remove diffusion screen by turning the screw on the epi-illuminator
to the black dot.
- Close down the epi-path field iris by pulling out the
rod near the screw.
Step 5. Center epi-path field iris
and open it so that it is just outside the object or field of interest.
Open epi-path aperture iris to level of desired excitation light (for faint
specimens, open it, or push in, all the way).
- Center epi-path field iris with the aid of the centering
screws near the push-rod.
- Open the field iris to just outside the field of view.
This is important to reduce glare from adjacent and out-of-focus cells.
- Adjust the intensity of light reaching the sample either
by opening the epi-path aperture iris by pushing in the push rod (less
light exciting the sample), or by closing the field iris by pushing in