ELYRA 7
Left Tool Area and Hardware Control Tools
ZEISS
03/2019 V_02
000000-2262-999
79
You can exced the boundaries of the slider only when going to one of its end and pressing
Save
TIRF angle
, as than the current poisition will be the center position. Continue until you have
reached the desired angle.
The TIRF angle can be adjusted using the
Specimen kit TIRF
(order number: 433800-0000-000).
New calibration might be necessary, if the TIRF angle was completely de-adjusted, if the TIRF objective is
changed or after new installation of the software.
Proceed according to the protocol below:
1.)
Put a drop of TIRF beads from the bottle
onto the glass section of a glass bottom
dish.
2.)
Dilute the TIRF beads with ca. 500 µl tap
water; do not use distilled water, as the
beads would settle only very slowly. Allow
beads for a few seconds to settle. If beads
settle too fast and only few remain in
solution, you might dilute tap water with
distilled water until you have a good ratio
between settled and floating beads.
3.)
Mount the glass bottom dish onto the
microscope using an appropriate
TIRF objective (e.g. objective alpha Plan-
Apochromat 100x/1,46 Oil DIC M27; order
number 420792-9800-720) and
appropriate oil immersion.
4.)
Select
EPI
mode (
TIRF angle
slider to the
very left) in the
Channels
menu
A
) and focus on the settled beads
using the 488 nm laser line (Fig. 106/
A
).
Besides the settled in-focus beads you also
should see blurry floating out-of-focus
beads.
5.)
Start moving the
TIRF angle
slider to the right. The image appearance will change as follows
B
-
E
):
a.
EPI
mode: on coverslip settled beads in focus plus floating blurry beads in solution with
prominent haze from out-of-focus regions (Fig. 106/
A
)
b. Settled beads become brighter as HILO mode is approached and out-of-focus haze decreases.
Fig. 105
"TIRF angle (°)" adjustment slider
with "EPI" (A-E) and "TIRF" (F) mode
activated.
Fig. 106
TIRF beads imaged in "EPI" (A), "HILO" (B), "TIRF" (C), "hyperTIRF" (D) and "out of TIRF" (F)
positions of the "TIRF angle (°)" adjustment slider