Read Step
Protocol> Procedure> Read
Define the reading parameters based on the capability of the current reader:
1. (Optional) Enter a Step Label or unique name for this step. Data sets based on the reading results will use the label in online views, reports, and export files.
2. Keep the Full Plate or set a portion of the plate to process.
(The Plate Type is set for all steps in the Procedure.)
3. Select the Detection Method. Options are controlled by the current reader.
4. Select the Read Type. Options are controlled by the current reader and the detection method selected above.
5. Select the Read Speed from the list offered for the current reader 6. Set the Wavelengths or Filter Sets:
1 Use the numbered buttons to set the number of wavelengths/filter sets to obtain measurements with. Kinetic, Spectrum, Area and Linear Scans limit this option.
2 Click the down arrow or type in the text field to set the wavelengths 7. If applicable, define:0.
• Pathlength Correction
• Optics Position
• Sensitivity and Filter Set Options
• Top Probe Vertical Offset
Read Types
Depending on the reader, detection method, Gen5 product level, and the type of analysis you're conducting, one of several read types can be selected:
• Endpoint
The most commonly used Read Type, Endpoint, performs one read in the center of the well for each wavelength. It is the only read type that
supports Pathlength Correction.
Check your assay kit instructions to determine if this type of reading is required. Endpoint reads are generally conducted after a Stopping Solution is applied to the samples or when the effects of the chemistry occur at an expected time point.
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• Area Scan
When performing an Area Scan, the reader takes multiple measurements down and across each well, in a “matrix” format. This method is more effective for cellular assays than reading once in the center of the well.
• Learn more in the Scanning Analysis Options and Features chapter Readers that support Area Scanning include the ELx800, µQuant, FLx800, SynergyHT, and Synergy 2.
Note: the Synergy 2's probe size limits its ability to perform Fluorescence area scan in plates with a small well diameter.
Generally, this means you must use a plate with fewer than 96 wells.
Read Matrix Size represents the number of measurements taken across and down each well. If, for example, the Read Matrix Size is 5 x 5 a total of 25 measurements are taken. The potential Read Matrix Size is a function of the well size of the current plate.
• Linear Scan
When performing a Linear Scan, the reader takes multiple measurements in a line across the center of each well. Linear scanning allows you to observe a pattern that may be present in the well bottom, such as an agglutination pattern.
• Learn more in the Scanning Analysis Options and Features chapter Readers that support linear scanning include the ELx808 and all
PowerWave models. Note for PowerWave X Select: Linear scanning is supported for the 96-well plate type only.
Horizontal Reading Points setting represents the total number of points to be read across the center of each well. Valid entries are odd integers from 1 to 39.
Note: If the Scanning options are inaccessible, well scanning cannot be performed with the currently defined plate type. This may be due to a hardware limitation or an unacceptable
combination of optic probe size and well diameter.
• Spectrum
During a Spectrum Read, multiple readings are taken across a wavelength range. The objective is to plot a graph with absorbance versus wavelength.
• Learn more in the Scanning Analysis Options and Features chapter
• The Stop wavelength must be greater than or equal to the Start wavelength + the Step
Readers that support spectrum reads are µQuant, and all models of the PowerWave, Synergy HT and Synergy 2.
Read Step | 125
Read Step for Fluorescence for FLx800 and Synergy HT
Synergy 2 users find instructions on page 127 Protocol> Procedure> Read
When defining reading parameters for
Fluorescence analysis, setting the PMT Sensitivity (for the Filter Sets) is important for obtaining useful measurements. The valid range is 25 to 255, but too low a setting, like 25, can result in insufficient readings, and too high a setting, >120, can damage the PMT. BioTek recommends a setting between 40 -120 for Fluorescence assays and between 150 - 255 for Time Resolved Fluorescence.
1. (Optional) Enter a Step Label or unique name for this step. Data sets based on the reading results will use the label in online views, reports, and export files.
2. Keep the Full Plate or set a portion of the plate to process.
(The Plate Type is set for all steps in the Procedure.)
3. Click the down arrow at Detection Method to select Fluorescence
4. Select Time Resolved to perform this type of fluorescence analysis (learn more in the Florescence and Luminescence chapter)
5. In Synchronized mode, you can select Close Light Shutter to turn off the light between reads. Optionally, to protect the fluorescent nature of your samples, use this feature to block the light between measurements to prevent photo-bleaching effects. Gen5 blocks the light with a Plug in the filter wheel.
Important:
A plug or blocking filter in the excitation filter wheel must be adjacent to the filter used in the reading. Two plugs must be placed next to each other (which ensures they are adjacent to the two filters used) in a dual-filter-set read step. Define the Reader Settings
5. For the Read Type select:
• Endpoint
The most common read type, Endpoint, performs one reading per well for each filter set defined.
• Area Scan (not available in Synchronized mode)
When performing an Area Scan, the reader takes multiple measurements down and across each well, in a “matrix” format.
This method is more effective for cellular assays than reading once in the center of the well.
6. Set the Filter Sets:0.
1 Use the numbered buttons to set the number of wavelengths.
126 | Chapter 7: Defining the Procedure
2 Click the down arrow to select the filter 3 If applicable, define:0
• Optics Position
• Sensitivity or click Options to let Gen5 determine the optimal setting
• Filter Switching: reading each well with both filters before moving to the next well, is offered when only two filters are selected.
In Synchronized mode, the read step settings for the first read in a Plate or Well mode block are applied to any subsequent read steps in the block.
Learn more in the Florescence and Luminescence chapter
Read Step | 127
Read Step: Synergy 2 Fluorescence
Protocol> Procedure> Read
When defining reading parameters for Fluorescence analysis, setting the PMT Sensitivity (for the Filter Sets) is important for obtaining useful measurements. The valid range is 25 to 255, but too low a setting, like 25, can result in insufficient readings, and too high a setting, >120, can damage the PMT. BioTek
recommends a setting between 35 -120 for Fluorescence assays and between 100 - 160 for Time Resolved Fluorescence (TRF).
1. (Optional) Enter a Step Label or unique name for this step. Data sets based on the reading results will use the label in online views, reports, and export files.
2. Keep the Full Plate or set a portion of the plate to process.
(The Plate Type is set for all steps in the Procedure.)
3. Click the down arrow at Detection Method to select Fluorescence 4. Optionally, select Time Resolved or Polarization to perform this type of
fluorescence analysis. Your choice enables or disables related options as appropriate for the process.
5. Read Speed: Use the drop-down list to make a selection and/or click the 3-dot button to change the default settings for Measurement Options
6. For the Read Type select:
• Endpoint: The most common read type, Endpoint, performs one reading per well for each filter set defined.
• Area Scan: (not available in Kinetic or Synchronized mode): When performing an Area Scan, the reader takes multiple measurements down and across each well, in a “matrix” format. This method is more effective for cellular assays than reading once in the center of the well. Also, The Synergy 2's probe size limits its ability to perform Fluorescence area scan in plates with a small well diameter. Generally, this means you must use a plate with fewer than 96 wells.
7. Light Source: except for TRF, you can select the lamp to use for this read step:
Xenon Flash (Xe) or Tungsten (Tg) Using the Xenon Flash (Xe)
Advantages Disadvantages
Enables Sweep mode as a Read Speed Prohibits use of the Extended Range Very high energy, slightly more
sensitive than Tg bulb
It is expensive compared to the Tg
128 | Chapter 7: Defining the Procedure
High light output below 300 nm (UV Fluorescence)
Noise
Performs direct protein and amino acid quantification assays
Using the Tungsten Lamp (Tg)
Advantages Disadvantages Inexpensive, with high sensitivity for
Fluorescence Intensity (FI) and Fluorescence Polarization (FP)
Sweep read speed is prohibited
Enables Extended Dynamic Range Cannot perform TRF Strong and stable light output in
visible range
Slightly less sensitivity than Xe Flash
No light output below 300 nm
8. In Synchronized (non-kinetic) mode, you can select Close Light Shutter to turn off the light between reads. To protect the fluorescent nature of your samples, use this feature to block the light between measurements to prevent photo-bleaching effects. Gen5 blocks the light with a Plug in the filter wheel.
9. Set the Filter Sets:
10. Use the numbered buttons to set the number of wavelengths 11. Click the down arrow to select the filter
12. If applicable, define:
• Optics Position: for top reading select the mirror
• Sensitivity or click to let Gen5 determine the optimal setting
• Filter Switching: reading each well with both filters before moving to the next well, is offered when only two filters are selected.
• Top Probe Vertical Offset
In Synchronized mode, the read step settings for the first read in a Plate or Well mode block are applied to any subsequent read steps in the block.
The Filters offered for selection are defined by the Filter Wheel Library or Reader Configuration
Read Step | 129
Read Step for Luminescence
Protocol> Procedure> Read
When defining reading parameters for
Luminescence analysis, setting the PMT Sensitivity is important for obtaining useful measurements. The valid range is 25 to 255, but BioTek recommends a setting between 100 - 160 for Luminescence assays.
1. (Optional) Enter a Step Label or unique name for this step. Data sets based on the reading results will use the label in online views, reports, and export files.
2. Keep the Full Plate or set a portion of the plate to process.
(The Plate Type is set for all steps in the Procedure.)
3. Click the down arrow at Detection Method to select Luminescence The Read Type must be set to Endpoint
4. Enter the Integration Time: to set the read duration for each well in seconds or milliseconds. Click in the field and enter the Sec.Msec or use the spin buttons to set the duration.
Valid values:
• Synergy HT: 0.1 - 19.9 seconds;
• Synergy 2: 0.1 - 99.9 seconds, in 20 ms intervals
• FLx800: 0.1 - 6.0 seconds
5. Synergy 2: Click the 3-dot button to change the default settings for Measurement Options
6. Set the Filter Sets:0.
1 Use the numbered buttons to set the number of wavelengths.
2 Click the down arrow to select the filter or Hole (to not filter the light) 3 If applicable, define: (Learn more in the Florescence and Luminescence
chapter)0
• Optics Position
• Sensitivity or click to let Gen5 determine the optimal setting
In Synchronized Mode, the read step settings for the first read in a Plate or Well mode block are applied to any subsequent read steps in the block.
130 | Chapter 7: Defining the Procedure
Read Step for Spectrum Analysis
Protocol> Procedure> Read
Define the reading parameters based on the capability of the current reader:
1. (Optional) Enter a Step Label or unique name for this step.
2. Keep the Full Plate or set a portion of the plate to process.
3. Set the Detection Method to Absorbance
4. Click the down arrow to set the Read Type to Spectrum. Options are controlled by the current reader — if Spectrum is not available, the current reader does not have this capability.
5. Set the range of Wavelengths:
Acceptable Values:
• The acceptable range for the Start wavelength is from the lowest wavelength the reader supports to one less than the Stop wavelength selected.
• The acceptable range for the Stop wavelength is any wavelength greater than the Start wavelength to the highest wavelength allowed by the reader.
• The acceptable range for the Step value is any number equal to or less than the difference between the Start and Stop values.
• The only read speeds available for Spectrum reads are Normal and Sweep.
6. Enter the Start and Stop wavelengths to define the spectral range
7. Enter the number of Steps to define the number of measurements to take 8. Set:0.
• Read Speed from the list offered for the current reader
• Calibrate Before Read: When selected, the reader will always perform calibration at the wavelengths specified in the protocol, just prior to plate reading. If Calibrate is not selected, the reader will calibrate at only those wavelengths specified in the protocol that have not yet been calibrated since the reader was turned on.