Biology Reference
In-Depth Information
OH
HO
OH
COOH
OH
OH
pO
2
+ H
2
O
Methyl viologen
OH
HO
O
H
2
O
2
OH
O
OH
Glucose
Oxidase
−
H
2
O
Catalase
pO
2
OH
HO
O
1/
2
O
2
OH
Oxygen scavenger
1/
2
O
2
OH
OH
FIGURE 15.5
A motorized x-y stage increases throughput and facilitate experiments in high-density
microplates. The scheme shows in-scale the well geometry of the 384-well, small-volume,
glass-bottom microplate (SensoPlate, Greiner). The total internal reflection of the laser beam
(red) excites molecules in a 0.2 mm diameter spot. A 40
m square field of view is imaged
by the QuadView optics on the EM CCD camera. Trolox and methyl viologen form a reducing
and oxidizing system (ROXS) that in combination with an enzymatic oxygen scavenger
improves the photostability of fluorophores.
m
The combination of laser lines and optical filters in our system can be used to
image the popular fluorophores, Alexa 488, 555, 594, and 647 (
Fig. 15.6
). We have
used DIBO derivatives of these dyes to label the GPCR rhodopsin for the three-color,
single-molecule fluorescence images shown in
Fig. 15.7
.
15.8.1
Step 1: microscope optical setup
The following optical components are used in
Scheme 15.2
:
SEMROCK:
FF01-390/40-25 (BP7)
FF02-482/18-25 (BP8)
FF01-563/9-25 (BP9)
FF01-640/14-25 (BP10)
FF01-390/482/563/640-25 (BP1)
Di01-R405/488/561/635-25
36 (BS1)
FF484-FDi01-12.5D (BS3)
FF560-FDi01-12.5D (BS4)
FF640-FDi01-12.5D (BS5)
FF01-446/523/600/677-25 (BP2)
FF01-445/20-25 (BP4)
