FPBASE
Optics
Objectives
Plan ApoN 60x/1.42
TIRF Apo 60x/1.49
Cameras and Emission Filters
Camera 1: DAPI: 435/31 Cy5: 683/40 DIC
Camera 2: A488: 528/48
Camera 3: A568: 609/37
Lasers
405 nm
488 nm
568 nm
640 nm
Sample holder compatibility
Standard microscope slides
35 mm glass bottom dishes
Multiwell chambered coverslips
Interface Requirements
PC
Linux Centos 6.8
Kernel Linux 2.6.32-642.13.1.e16x86_64
GNOME 2.28.2
- Intel Core i7 2600 CPU
- NVidia NVS300
Drives 1 TB SATA boot drive
RAID 3 × 1 TB SATA in RAID5 configuration
Notes on usage:
SIM imaging is extremely sensitive to spherical aberration. To get the highest quality images you need to minimize spherical aberration. This means:
You should use high precision 0.17 mm coverslips
You should mount only a single coverslip in the center of the slide.
You should mount in an appropriate mounting medium. Fixed samples should be mounted in high refractive index mounting media with antifade. Be aware that hard-set mounting medium can cause shrinkage when it cures. We also recommend that you do not use mounting medium which contains DAPI or other fluorophores.
You should have your sample as close to the coverslip as possible (e.g. cells grown on coverslip not on slide).
You should use bright and stable fluorophores.
You should select the correct immersion oil for you sample. You can use the GE immersion oil calculator to determine the best oil to start with. The also have the calculator as a phone app.
As a general rule, if the sample doesn't look good in widefield or confocal, SIM imaging will not improve it. You should make every attempt to optimize your staining and reduce background from out-of-focus fluorescence for the best results.
