Field diaphragm

STEP 3 Adjust the condeser hight by using the condenser focus knob to bring the edges of the field diaphragm into the best focus possible. Both the specimen and the field diaphragm should be in focus. 

STEP 4 Use the two condenser-centering knobs to center the image of the closed field diaphragm in the field of view. 

STEP 5 Open the field diaphragm just enough so that its edges are just beyond the field of view. 

Telescope objective variable image-field diaphragm, ens Light Stop-... 

Figure 1.13 Image of the field diaphragm with an image of the specimen. Magnification 100 x.

Adjust the field diaphragm for maximum contrast and the aperture diaphragm for maximum resolution and contrast and... 

Fig. 1. TIRF microscope. (A) A view of the TIRF system. (B)View looking down on the back of the microscope (environmental chamber at bottom, back of the system at the top). Arrow b indicates the location of the Selection Prism containing the 80%/ 20% beamsplitter. Pulling the knob at the top to its full upwards position sets the beamsplitter to 100%/ 0% (all Epi-illumination input to the microscope). Pushing it downward sets the beamsplitter to 80% laser and 20% epifluo-rescence illumination. (B ) Location of the Field Diaphragm and the Field Stop on the epifluorescence arm of the split box. Two arrows AS and FS indicate Aperture Stop and Field Stop, respectively. Adjustment of these is vital for good IRM imaging. (B ) The laser input from which the screw white arroW) for TIRF angle adjustment projects. This pair of screws laterally translocates the laser path off center in the objective so that the angle of reflection is altered.

Adjust the Field Diaphragm or Field Stop (FS, a second light intensity control after AS before 20/80 prism in the light path) to obtain the best contrast of adhering membranes or structures and background (Fig. lb and B , see FS). 

Turn on the illuminator, place an object on the stage, select the 10 x objective by rotating the objective nosepiece, fuUy open the field diaphragm and check that the sample is illuminated by placing a piece of paper over the sample to see if the hght is reaching it. 

Start to close the field diaphragm until visible in the field of view. Focus the condensing system until the diaphragm is in sharp focus with no color fringes. 

Center the image of the field diaphragm using the two knobs on the condenser. Closing down the field diaphragm will assist this procedure. 

Open up the field diaphragm until its edge is just outside the field of view. 

Bringing the condenser into focus is simply a matter of bringing the edges of the field diaphragm into sharp focus (Fig. 3A). 

Fig. 3. The steps involved in focusing the condenser, optimizing the field diaphragm (A) and the condenser aperture diaphragm (B) (see Subheading 3.4.1. and 3.4.2.). Schematic views through the eyepiece for each step are shown below.

Close the field diaphragm so that its edges are in view, and center the circle of illumination with adjnster screws on the snbstage condenser. 

Open the field diaphragm to illuminate the field of interest but minimize the amount of stray light The edges of the field diaphragm should remain just inside the field of view. 

Remove the eyepiece and ensure that the field diaphragm is sufficiently open to fully illuminate the field of view. 

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