Spectrophotometry - Absorbance, Fluorescence, Luminescence

Instruction and Parameters

The bandwidth for wavelength selection in all following instructions is < 5 nm for λ ≤ 315 nm and < 9 nm for λ > 315 nm. Currently, all measurements are made in white, clear-bottom plates, and all absorbance and fluorescent readings are made from below. Luminescent readings are aquired from the top.

Absorbance

Absorbance readings can be taken for wavelengths between 300 nm and 1000 nm.

{
  "op": "absorbance",
  "object": container,
  "wells": [wells],
  "wavelength": length,
  "num_flashes": integer, // default: 25
  "dataref": string,
  "temperature": temperature, // optional
  "incubate_before": { // optional
    "duration": time, // required if `incubate_before` specified
    "shaking": { // optional
      "amplitude": length, // required if `shaking` specified
      "orbital": bool // required if `shaking` specified
    }
  }
}

Fluorescence

Fluorescence excitation wavelength can be between 230 nm and 600 nm, and emission can be between 330 nm and 850 nm.

The optional gain parameter represents the fraction of maximal signal amplification that the plate reading device can provide. This can be specified to control amplification of the signal from fluorescence emission. If gain is not specified, signal amplification will employ the optimal gain finding feature on the plate reading device. Gain is limited to 0.002-1.000.

The gain parameter can be set to ensure that amplification remains consistent over time on the same device. Please check the instruction to ensure that the same device was used for the reads you employed. We recommend in line controls for all experiments and strongly encourage their use when normalization is necessary. Kinetic reads incubated solely on the plate reader are more likely to use the same device when compared to kinetic reads incubated outside of the plate readers. Kinetic reads across multiple experiments can not be guaranteed to run on the same device.

{
  "op": "fluorescence",
  "object": container,
  "wells": [wells],
  "excitation": length,
  "emission": length,
  "num_flashes": integer, // default: 25
  "dataref": string,
  "temperature": temperature, // optional
  "incubate_before": { // optional
    "duration": time, // required if `incubate_before` specified
    "shaking": { // optional
      "amplitude": length, // required if `shaking` specified
      "orbital": bool // required if `shaking` specified
    }
  },
  "gain": float // optional
}

Luminescence

The luminescence mode has no wavelength parameter and measures all photon emissions in the detectable wavelength range of 380 nm - 600 nm.

{
  "op": "luminescence",
  "object": container,
  "wells": [wells],
  "dataref": string,
  "temperature": temperature, // optional
  "incubate_before": { // optional
    "duration": time, // required if `incubate_before` specified
    "shaking": { // optional
      "amplitude": length, // required if `shaking` specified
      "orbital": bool // required if `shaking` specified
    }
  }
}

Optional Parameters

All spectrophotometry instructions have the optional parameters of temperature and incubate_before. Fluorescence has the additional optional parameter of gain. For more on gain, please see the "Fluorescence" section above.

If temperature is specified, the plate environment will be heated to this temperature during reading and any incubation specified by incubate_before. Temperature is limited from 30:celsius to 42:celsius.

Incubation prior to a spectrophotometry reading requires the inclusion of the incubate_before parameter, with a corresponding duration. Shaking can be included in incubation, and will occur at the specified amplitude and orbital. Amplitude is limited to 1-6:mm. Specifying orbital as true will result in orbital shaking, while false will result in linear shaking.

Device

The absorbance, fluorescence, and luminescence instructions are all handled by Tecan Infinite 200M Pro instruments. This plate reader uses a Quad-4 Monochromator system with 2 excitation and 2 emission monochromators. Traditional plate readers use filter pairs and the number of filters that fit in a given system will limit the wavelength range attainable by that instrument. A monochromator transmits a mechanically selectable narrow band of wavelengths of light chosen from a wider range of wavelengths available at the input. Thus monochromator based systems allow for much greater flexibility in selecting analysis parameters within a single device.
More information on this plate reader can be found here.

Reading type	Wavelength range	Accucracy	Precision
Absorbance	230 nm - 1000 nm	< ± 0,5 nm for λ> 315 nm < ± 0.3 nm for λ≤ 315 nm	< ± 0,5 nm for λ> 315 nm < ± 0.3 nm for λ≤ 315 nm
Fluorescence	Excitation: 230 nm - 600 nm Emission: 330 nm - 600 nm	< ± 2 nm for λ> 315 nm < ± 1 nm for λ≤ 315 nm	< ± 1 nm for λ> 315 nm < ± 0.5 nm for λ≤ 315 nm
Luminescence	380 nm - 600 nm (data will be collected across this range)

Supported plates

Currently, only 96-flat and 384-flat plates can be used in a plate read command (see Containers). The fastest read-times for a 96-well plate (single flash) is 20 seconds and 30 seconds for a 384 well plate. An excitation/emission scan from 450nm to 550nm in 5nm increments on a 96 well plate will take approximately 150 seconds.

Detectors

Reading type	Detectors
Absorbance	UV silicon photodiode
Fluorescence	PMT, optionally UV and red-sensitive
Luminescence	photon counting system with low dark current PMT

At 260nm the absorbance detection shows less than 0.2% variance in precision and less than 0.5% variance in accuracy.
Fluorescence readings are sensitive down to 1.2 fmol / well.
Glow luminescence readings are sensitive down to 225 amol ATP / well. Flash luminescence readings down to 12 amol ATP / well.