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ZEISS
Left Tool Area and Hardware Control Tools
ELYRA 7
80
000000-2262-999
03/2019 V_02
c.
HILO
mode: settled beads and out-of-focus beads become brightest (Fig. 106/
B
). Haze is
reduced further.
d. Brightness of settled and floating beads stay approximately the same, but out-of-focus haze
becomes fainter as one approaches TIRF.
e.
TIRF
mode: Signal-to-noise ratio improves tremendously as out-of-focus haze disappears.
Settled beads are clearly visible, whereas floating beads are not detected any longer
(Fig. 106/
C
).
f.
HyperTIRF
mode. Signal of settled beads is fainting (Fig. 106/
D
).
g.
Out of TIRF
mode: Settled beads are no longer visible (Fig. 106/
E
).
6.)
Go back to slider position, where beads are the brightest without noticeable out-of-focus haze.
Recall TIRF angle position for the next step.
7.)
Go to
TIRF
mode (Fig. 105/
F
) and enter the TIRF position determined in step 6 in the input box.
Press
Enter
to move slider to the position.
8.)
By using the arrows of the
TRIF angle (
o
)
input box fine-tune the optimal TIRF angle position by
fine stepping back and forth.
9.)
Press the
Save TIRF angle
button to store the setting. Whenever the same laser line is chosen,
the TIRF angle will be set to the stored position.
10.)
You might repeat with other laser lines, albeit the beads of the kit have an excitation maximum
around 488 nm. Increase laser powers accordingly to visualize beads.
Please note, that the optimal TIRF angle is sample dependent. Therefore, it is recommended to fine-tune
the TIRF angle for each sample. If the cover glass is tilted or uneven, TIRF will also depend on the actual
position of the sample.
In order to fine-tune the TIRF angle, proceed as
follows:
1.)
Mount sample on microscope and focus in
EPI
mode.
2.)
Switch to TIRF mode and move
TIRF angle
(
o
)
slider back and forth to find the best
TIRF setting. In
EPI
or
HILO
conditions,
there is out-of-focus light visible and by
defocusing up other structures, if stained,
will appear (Fig. 107/
A
). In
TIRF
condition
out-of-focus haze is not visible and signal-
to-noise is maximal. By defocusing the
signal is lost (Fig. 107/
B
). In
hyperTIRF
condition, the signal will be fainter and
signal-to-noise will decay (Fig. 107/
C
).
3.)
Set the TIRF angle to the optimal position
and press the
Save TIRF angle
button if
you want to recall the position later.
Fig. 107
3T3 cells stained for tubulin imaged in
"HILO" (A), "TIRF" (B) and "hyperTIRF"
(C) mode.