The multicolor 4Pi-SMS system was built based on the previously
described instrument8 (link) with
minor modifications (Supplementary Fig. 1 ). The oil immersion objectives were replaced
with high numerical aperture (NA) silicone immersion objectives
(100×/1.35 NA, Olympus) for better refractive index matching. The system
was equipped with two excitation lasers at 560 nm (MPB Communications,
2RU-VFL-P-2000–560-B1R), 642 nm (MPB Communications,
2RU-VFL-2000–642-B1R) and an activation laser at 405 nm (Coherent OBIS
405 LX, 50 mW). Details about the dichroic beamsplitter and emission filters
used in the system are shown inSupplementary Fig. 1b . The
conventional fluorescence follows the same emission path as the previous design
and is collected by a sCMOS camera (ORCA-Flash 4.0v2, Hamamatsu) (Supplementary Fig. 1a ,
Camera 1). The salvaged fluorescence is reflected by the dichroic beamsplitter
to the back side of the system and collected by an EMCCD camera (128 ×
128 pixels, iXon DU860, Andor) (Supplementary Fig. 1c , Camera 2).
Both cameras were controlled by custom-written LabVIEW (National Instruments)
programs.
described instrument8 (link) with
minor modifications (
with high numerical aperture (NA) silicone immersion objectives
(100×/1.35 NA, Olympus) for better refractive index matching. The system
was equipped with two excitation lasers at 560 nm (MPB Communications,
2RU-VFL-P-2000–560-B1R), 642 nm (MPB Communications,
2RU-VFL-2000–642-B1R) and an activation laser at 405 nm (Coherent OBIS
405 LX, 50 mW). Details about the dichroic beamsplitter and emission filters
used in the system are shown in
conventional fluorescence follows the same emission path as the previous design
and is collected by a sCMOS camera (ORCA-Flash 4.0v2, Hamamatsu) (
Camera 1). The salvaged fluorescence is reflected by the dichroic beamsplitter
to the back side of the system and collected by an EMCCD camera (128 ×
128 pixels, iXon DU860, Andor) (
Both cameras were controlled by custom-written LabVIEW (National Instruments)
programs.
