Table 1.

Suggested optimal settings and starting points for imaging on various platforms *

Platform To improve efficiency To improve sensitivity and S/N To minimize light exposure
All Hard-coated filters (see Box 1). General: Keep excitation light levels low.
Phenol-red-free medium.
Dye-specific filters. Avoid blue dyes.
Restorative deconvolution.
Avoid excess optical elements (lenses, DIC components). Minimum resolution (60×, 1.4 NA):
Camera-based systems: x,y ∼0.1-0.2 μm
100% light to detector port. Avoid color cameras. z ∼0.3-0.4 μm
t=2.3× timescale of events.
High NA objectives. Bin high-resolution cameras 2×2 (exposure times of 200-500 ms). Minimum number of probes.
Spectral un-mixing. Use oxygen-radical scavengers.
Slow camera read times.
Avoid phase objectives.
Use EM-CCD for high speed (exposure times of <100 ms).
WFM Remove DIC prism and analyzer when imaging fluorescence. Perform post-acquisition restorative deconvolution (Fig. 3C,D). Find cells with transmitted light.
Use ND filters (<10% lamp power).
Use UV- and IR-blocking filters.
Use halogen lamps if possible.
CLSM Use long-pass or wide band-pass filters and un-mix post acquisition. High PMT voltage (≥800 V). Low laser power.
Restorative deconvolution. AOTF laser blanking.
Open the pinhole ≥2 Airy units.
Avoid spectral-array detectors. No line or frame averaging.
Remove DIC prism.
Fast scan speed (≥8 μs/pixel).
Image regions of interest when possible.
Recommended laser powers:
405 nm Avoid
488 nm <2% (30 mW)
514 nm <2% (30 mW)
543 nm <50% (1 mW)
633 nm <5% (5 mW)
MP-CLSM Use long-pass filters and un-mix post acquisition. Use high PMT voltages. Refer to points for CLSM above.
NIR light is lower in energy. Inherently confocal excitation.
Use NDDs.
Restorative deconvolution. One excitation for multiple dyes.
For a 2 W multi-photon laser recommend ∼2 mW (<10 mW) at 860 nm for cellular work.
Higher powers are needed for intravital imaging deep inside tissue.
SD-CM Most current confocal heads (Yokogawa X1) or optimized versions of the Yokogawa CSU-10 (e.g. Quorum Technologies). Choose appropriate camera for resolution (scientific grade CCD) or speed (EM-CCD). Keep laser power low.
Excitation light spread over thousands of pixels.
Restorative deconvolution (Fig. 5C,D). Excitation light is confocal.
Use high-quality high-light-throughput camera-coupling lenses. Recommended laser powers:
405 nm Avoid
440 nm <70% (15 mW)
491 nm <30% (25 mW)
561 nm <20% (50 mW)
638 nm <20% (30 mW)
TIRFM Use broad band-pass or long-pass filters. Choose appropriate camera for resolution or speed. Only excite probes ∼100 nm from cover-slip surface.
TIRF filters to avoid laser reflections and interference patterns.
Little out-of-focus light. Recommended laser powers:
405 nm Avoid
488 nm <20% (30 mW)
514 nm <20% (30 mW)
543 nm ∼50-100% (1 mW)
633 nm <20% (5 mW)
  • * Note that these are only suggestions and the actual values will depend on the specific microscope, lenses, dyes, tissue and detectors. These settings are meant as a guideline, but all settings should be optimized further for each piece of equipment with specific samples under given experimental conditions