| Microscopea |
Olympus |
IX70/IX71/IX81 |
|
|
|
| Objectivesb |
|
|
| 100X (1.45 NA) |
Olympus |
PlanApo 100X 1.45 NA, ∞/0.17 |
| 60X (1.45 NA) |
Olympus |
PlanApo 60X 1.45 NA, ∞/0.17 TIRFM |
| 100X (1.65 NA) |
Olympus |
Apo 100X 1.65NA, ∞/0.15 |
| 100X (1.49 NA) |
Nikon |
Apo TIR 100X |
|
|
|
| CCD camerac |
Andor Technology |
iXonEM (DV887DCS-BV/DV887ECS-BV) |
|
Andor Technology |
iXonEM+ (DV-897E-CS0) |
|
Andor Technology |
iXonEM (DV860DCS-BV) |
|
|
|
| Lasers |
|
|
| Green laser (532 nm) |
CrystaLaser |
Diode-pumped solid-state laser (DPSS) |
| Red laser (633 nm) |
CrystaLaser / Melles Griot |
DPPS / Helium Neon (HeNe) (25-LHP-928-249) |
| Blue laser (488 nm) |
Melles Griot |
Argon-ion (35-LAS-450-120) |
|
|
|
| Mirrors, lenses, mounts, and posts |
|
|
| Broadband dielectric mirrors |
ThorLabs Inc. |
BB5-E02(1/2 in), BB1-E02(1 in), BB2-E02(2 in) |
| Kinematic mirror mounts |
ThorLabs Inc. |
KS05 (1/2 in), KS1(1 in), KS2(2 in) |
| Post holders |
ThorLabs Inc. |
PH6-ST/M - PH1-ST/M (ST = 6-1 in, M = 150-25 mm) |
| Posts |
ThorLabs Inc. |
TR6-TR075 (12.75 in) TR300/M - TR20/M (300-20 mm) |
| Manual linear translation stages |
Newport Corp. |
423/433/443 series (requires actuators) |
| TIR focusing lens |
Thorlabs Inc. |
Plano-Convex lens, AR-coated |
| Beam expander |
Thorlabs Inc. |
i.e., 5X/10X (BE05M, BE10M) |
|
|
|
| Optical tabled |
|
|
| Sealed-hole optical table |
Newport Corp. |
RS4000 |
| Pneumatic isolators |
Newport Corp. |
I-2000 LabLegs |
|
|
|
| Filters and dichroics: single color |
|
|
| Neutral density filters |
Thorlabs Inc. |
ND range 0.1-5 (NE01A - NE50A) |
| Excitation filters |
|
|
| 532 nm |
Chroma |
Z532/10X (Z532BP) |
| 633 nm |
Chroma |
Z633/10X (Z633BP) |
| 488 nm |
Chroma |
Z488/10X (Z488BP) |
| Dichroics |
|
|
| 532 nm; Cy3 |
Chroma |
Q565LP (41007) |
| 633 nm, Cy5 |
Chroma |
Q660LP (41008) |
| 488 nm, FITC/GFP |
Chroma |
Q505LP (41001) |
| Emission filters |
|
|
| 532 ex, 570 em (Cy3) |
Chroma |
HQ585/70M |
| 633 ex, 670 em (Cy5) |
Chroma |
HQ 690/90M |
| 488 ex, 520 em (GFP, FITC) |
Chroma |
HQ525/50M |
| aThe Olympus microscope stage has a variable amount of drift. We often test up to three stages and choose the best one.
|
| bOur standard objective is the 100X, NA = 1.45 objective, combined with a 1.5X (IX-71) or 1.6X (IX-70) magnifying lens in the
microscope. With 16-μm CCD pixels (Andor, 512 × 512 camera), this gives 100-nm effective pixel size. Pixel sizes >120 μm are
not recommended due to loss of resolution (Enderlein et al. 2006). We also use the 100X 1.65 NA, which requires special oil
(n = 1.78, Model #9999902/4113/7, SPI Supplies) and special coverslips. Rarely, we use a 1.40 NA superapochromat, which is a
very low chromatic aberration objective.
|
| cEMCCD detectors--that is, cooled, back-thinned CCDs with an electron multiplication factor--are ideal detectors. Back-thinning
allows for a quantum efficiency of ~90% (in the visible range). Thermoelectric cooling (to roughly -70ºC) reduces the noise
due to CCD dark-current to virtually zero. Electron multiplication facilitates detection of even a small number of photons,
which is enough to exceed the minimal background of the camera. The pixel size (16-24 μm) is generally reasonable, so that
160X magnification (100X of objective, plus 1.6X of internal microscope lens) provides an effective pixel size of 100-150
nm. This is reasonable, although the 24-μm pixel size generally requires an external lens in front of the camera.
|
| dOur RS4000 optical tables are either 4 ft × 6 ft × 8 in or 4 ft × 10 ft × 8 in.
|
