RNA Spot Quality table
Requirement level: optional
Recommended: Yes
Namespace: 4dn_FOF-CT_rna_quality
Summary
This table is highly recommended and it is designed to provide quality metrics for RNA Spots localization, information about the optical Channel that was used to image a given RNA Spot, and various aberration corrections that might have been applied prior to localization (e.g., drift correction, chromatic correction, etc.).
Because the metrics used to quantify RNA Spot detection accuracy and precision are not trivial and lacking a widely shared consensus, the specific columns in this table remain largely at the user’s discretion and should be described with sufficient details to ensure interpretation and reproducibility.
However, in order to align with existing 4DN-BINA-OME Microscopy Metadata specifications, the use of specific column names and descriptions is conditionally required in case the described metric is reported. As an example, the column name X_Drift is conditionally required in case the user intends to report a comparison between the Observed vs. Expected (i.e., based on a fiducial reference) positions of a detected Spot.
The table is indexed by RNA_Spot_ID and each row corresponds to a RNA bright Spot. The order of all other columns (including those conditionally required) and of the rows are at the user’s discretion.
Warning
All MANDATORY header fields and column names are indicated in bold. All conditionally required header fields and column names are indicated in italics.
File Header
For full instructions see File Header
The first line in the header is always
##FOF-CT_Version=vX.X.The second line in the header is always
##Table_Namespace=4dn_FOF-CT_rna_quality.
Tip
The header MUST contain a mandatory set of fields that describe any Software tool that was used to produce/process data in this table. If more than one software tool was used, please repeat a set of Software-fields for describing each of them.
The header MUST include a detailed description of each optional columns used.
Tip
The table MUST contain at least 1 Optional or Conditionally Required Column.
Name |
Description |
Example |
Conditional requirement conditions |
|---|---|---|---|
##FOF-CT_Version= |
Version of the FOF format used in this case. |
v1.0 |
|
##Table_Namespace= |
Identifier for this type of table. Value must be as in the example. |
4dn_FOF-CT_rna_quality |
|
##XYZ_Unit= |
If relevant, the unit used to represent XYZ locations or distances in this table. Note: use micron to avoid problem with special, Greek symbols. Other allowed values should be drawn from SI units of Length. Examples: ‘nm’, ‘micron’ ‘mm’ etc. |
micron |
Conditional requirement: this MUST be reported if any locations metrics are reported. |
##Time_Unit= |
If relevant, the unit used to represent a time interval in this table. Allowed values should be drawn from SI units of Time or be one of the following: ‘min’, and ‘hr’. |
sec |
Conditional requirement: this MUST be reported if any time metrics are reported. |
##Intensity_Unit= |
If relevant, the unit used to represent intensity measurements in this table. |
a.u. |
Conditional requirement: this MUST be reported if any intensity metrics are reported. |
#Lab_Name: |
name of the lab where the experiment was performed. |
Nobel |
|
#Experimenter_Name: |
name of the person performing the experiment. |
John Doe |
|
#Experimenter_Contact: |
email address of the person performing the experiment. |
||
#Description: |
A free-text, description of the experiment and of the data recorded in this table. This description should provide a clear understanding of the process utilized to produce the data and contain sufficient details to ensure interpretation and reproducibility. |
||
#Software_Title: |
The name of the Software tool that was used to produce the results reported in this table. If more than one software tool was used, please repeat a set of Software-fields for describing each of them. |
AlgorithmXYZ |
|
#Software_Type: |
The type of this Software used to produce results recorded in this table. Allowed values: SpotLoc, Tracing, SpotLoc+Tracing, Segmentation, QC, Other |
Segmentation |
|
#Software_Authors: |
The Name(s) of the individual Author(s) of this Software. In case there are more than one Authors, individual names should be listed as follows, Doe, John; Smith, Jane; etc,. |
John Doe |
|
#Software_Description: |
A free-text, description of this Software. This description should provide a detailed understanding of the algortithm and of the analysis parameters that were used, in order to guarantee interpretation and reproducibility. |
A pretty clear description |
|
#Software_Repository: |
The URL of any repository or archive where the Software executable release can be obtained. |
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#Software_PreferredCitationID: |
The Unique Identifier for the preferred/primary publication describing this Software. Examples include, Digital Object Identifier (DOI), PubMed Central Identifier (PMCID), ArXiv.org ID etc,. |
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#Intensity_Measurement_Method: |
If relevant, the method that was used to perform intensity measurements. In particular, sufficient information should be provided to document how digital intensity signals were converted to Photon counts. |
Spot centroid intensity. |
Conditional requirement: this MUST be reported if any intensity metrics are reported. |
#^Centroid_Intensity: |
The signal intensity of the pixel occupying the center-of-mass within a bright Spot (i.e. centroid). |
Conditional requirement: when this metric is reported, the use of this column name MUST be used. |
|
#^Peak_Intensity: |
The signal intensity of the brightest pixel within a bright Spot (i.e. local maximum). |
Conditional requirement: when this metric is reported, the use of this column name MUST be used. |
|
#^Raw_X: |
The Raw sub-pixel X coordinate of this bright Spot relative to the optical system (i.e., Objective and Detector), as determined before any performed post-processing correction procedures (i.e. drift correction, chromatic correction etc). This is the appropriate coordinate system for correcting optical aberrations. |
Conditional requirement: when this metric is reported, the use of this column name MUST be used. |
|
#^Raw_Y: |
The Raw sub-pixel Y coordinate of this bright Spot relative to the optical system (i.e., Objective and Detector), as determined before any performed post-processing correction procedures (i.e. drift correction, chromatic correction etc). This is the appropriate coordinate system for correcting optical aberrations. |
Conditional requirement: when this metric is reported, the use of this column name MUST be used. |
|
#^Raw_Z: |
The Raw sub-pixel Z coordinate of this bright Spot relative to the optical system (i.e., Objective and Detector), as determined before any performed post-processing correction procedures (i.e. drift correction, chromatic correction etc). This is the appropriate coordinate system for correcting optical aberrations. |
Conditional requirement: when this metric is reported, the use of this column name MUST be used. |
|
#^X_Drift: |
This field captures the offset in the observed X-coordinate of the Intensity maxima or the Intensity centre of gravity of the bright Spot when comparing the Observed vs. Expected (i.e., based on a fiducial reference) positions. This shall be calculates as: √(Xe - Xo)^2, and reported in physical distance using the unit indicated in the header. |
Conditional requirement: when this metric is reported, the use of this column name MUST be used. |
|
#^Y_Drift: |
This field captures the offset in the observed Y-coordinate of the Intensity maxima or the Intensity centre of gravity of the bright Spot when comparing the Observed vs. Expected (i.e., based on a fiducial reference) positions. This shall be calculates as: √(Ye - Yo)^2, and reported in physical distance using the unit indicated in the header. |
Conditional requirement: when this metric is reported, the use of this column name MUST be used. |
|
#^Z_Drift: |
This field captures the offset in the observed Z-coordinate of the Intensity maxima or the Intensity centre of gravity of the bright Spot when comparing the Observed vs. Expected (i.e., based on a fiducial reference) positions. This shall be calculates as: √(Ze - Zo)^2, and reported in physical distance using the unit indicated in the header. |
Conditional requirement: when this metric is reported, the use of this column name MUST be used. |
|
#^X_Chromatic_Shift: |
This field captures the offset in the observed X-coordinate of the Intensity maxima or the Intensity centre of gravity of the bright Spot when comparing the Reference (λR) vs. the Test (λT) wavelengths. This shall be calculated as: √(XλT - XλR)^2. This offset could be reported either in number of Pixels or in physical Distance, when a sub-Pixel offset needs to be calculated. |
Conditional requirement: when this metric is reported, the use of this column name MUST be used. |
|
#^Y_Chromatic_Shift: |
This field captures the offset in the observed Y-coordinate of the Intensity maxima or the Intensity centre of gravity of the bright Spot when comparing the Reference (λR) vs. the Test (λT) wavelengths. This shall be calculated as: √(YλT - YλR)^2. This offset could be reported either in number of Pixels or in physical Distance, when a sub-Pixel offset needs to be calculated. |
Conditional requirement: when this metric is reported, the use of this column name MUST be used. |
|
#^Z_Chromatic_Shift: |
This field captures the offset in the observed Z-coordinate of the Intensity maxima or the Intensity centre of gravity of the bright Spot when comparing the Reference (λR) vs. the Test (λT) wavelengths. This shall be calculated as: √(ZλT - ZλR)^2. This offset could be reported either in number of Pixels or in physical Distance, when a sub-Pixel offset needs to be calculated. |
Conditional requirement: when this metric is reported, the use of this column name MUST be used. |
|
#^X_Loc_Error: |
Metric used to quantify the Error associated with the estimation of the X-axis localization of this bright Spot. Whatever method is used, a description of how this metric was computed and of the Software that was employed must be provided in the header of the table. Such description must contain enough details to allow interpretation and reproducibility. |
Conditional requirement: when this metric is reported, the use of this column name MUST be used. |
|
#^Y_Loc_Error: |
Metric used to quantify the Error associated with the estimation of the Y-axis localization of this bright Spot. Whatever method is used, a description of how this metric was computed and of the Software that was employed must be provided in the header of the table. Such description must contain enough details to allow interpretation and reproducibility. |
Conditional requirement: when this metric is reported, the use of this column name MUST be used. |
|
#^Z_Loc_Error: |
Metric used to quantify the Error associated with the estimation of the Z-axis localization of this bright Spot. Whatever method is used, a description of how this metric was computed and of the Software that was employed must be provided in the header of the table. Such description must contain enough details to allow interpretation and reproducibility. |
Conditional requirement: when this metric is reported, the use of this column name MUST be used. |
|
#^X_Loc_Precision: |
Metric used to quantify the Precision associated with the estimation of the X-axis localization of this bright Spot. Different methods might be used. The Cramer-Rao Lower and Upper Bounds methods is widely accepted, but it tends to overestimate the Precision value. Alternatively, the Thompson method, by which Precision is estimated to be proportional to Photon Count, can also be used even though this method highly overestimates the Precision. Whatever method is used, description of how this metric was computed and of the Software that was employed must be provided in the header of the table. Such description must contain enough details to allow interpretation and reproducibility. |
Conditional requirement: when this metric is reported, the use of this column name MUST be used. |
|
#^Y_Loc_Precision: |
Metric used to quantify the Precision associated with the estimation of the Y-axis localization of this bright Spot. Different methods might be used. The Cramer-Rao Lower and Upper Bounds methods is widely accepted, but it tends to overestimate the Precision value. Alternatively, the Thompson method, by which Precision is estimated to be proportional to Photon Count, can also be used even though this method highly overestimates the Precision. Whatever method is used, description of how this metric was computed and of the Software that was employed must be provided in the header of the table. Such description must contain enough details to allow interpretation and reproducibility. |
Conditional requirement: when this metric is reported, the use of this column name MUST be used. |
|
#^Z_Loc_Precision: |
Metric used to quantify the Precision associated with the estimation of the Z-axis localization of this bright Spot. Different methods might be used. The Cramer-Rao Lower and Upper Bounds methods is widely accepted, but it tends to overestimate the Precision value. Alternatively, the Thompson method, by which Precision is estimated to be proportional to Photon Count, can also be used even though this method highly overestimates the Precision. Whatever method is used, description of how this metric was computed and of the Software that was employed must be provided in the header of the table. Such description must contain enough details to allow interpretation and reproducibility. |
Conditional requirement: when this metric is reported, the use of this column name MUST be used. |
|
#^Optional_Column_1: |
Optional column 1 description. This description should provide a clear understanding of the process utilized to produce the data and contain sufficient details to ensure interpretation and reproducibility. |
||
#^Optional_Column_2: |
Optional column 2 description. This description should provide a clear understanding of the process utilized to produce the data and contain sufficient details to ensure interpretation and reproducibility. |
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#^Optional_Column_3: |
Optional column 3 description. This description should provide a clear understanding of the process utilized to produce the data and contain sufficient details to ensure interpretation and reproducibility. |
||
#Additional_Tables: |
list of the additional tables being submitted. Note: use a comma to separate each table name from the next. |
4dn_FOF-CT_core, 4dn_FOF-CT_rna, 4dn_FOF-CT_bio, 4dn_FOF-CT_trace, 4dn_FOF-CT_cell |
|
##Columns= |
list of the data column headers used in the table. Note: enclose the column headers and use a comma to separate each header name from the next. |
(RNA_Spot_ID, Channel_Name, Fluorophore_Name, Optional_Column_1, Optional_Column_2, Optional_Colummn_3) |
Data Columns
For full instructions see Data Columns
As with all other RNA Spot Data tables in this format, each row corresponds to data associated with an individual RNA_Spot.
The first columns of this table are always Spot_ID, Channel_Name, Fluorophore_Name.
This table MUST contain at least 1 Optional or Conditionally Required Column.
The content and order of all other columns is largely at user’s discretion. However, in order to align with existing 4DN-BINA-OME Microscopy Metadata specifications, the use of specific column names and descriptions is conditionally required as indicated below. The order of the rows are at the user’s discretion.
Name |
Description |
Example |
Conditional requirement conditions |
|---|---|---|---|
RNA_Spot_ID |
A unique identifier for this bright Spot. |
1 |
|
Channel_Name |
The name of the Channel that was used to image this Spot. |
510/25 |
|
Fluorophore_Name |
The name of the Fluorophore whose Emission is utilized to detect this Spot. |
AlextFluor_488 |
|
Conditionally_Required_Column_1 |
one of the conditionally required columns desribed in the header |
||
Conditionally_Required_Column_2 |
one of the conditionally required columns desribed in the header |
||
Conditionally_Required_Column_3 |
one of the conditionally required columns desribed in the header |
||
Optional_Column_1 |
|||
Optional_Column_2 |
|||
Optional_Column_3 |
Example
The only mandatory column in this table are RNA_Spot_ID, Channel_Name, and Fluorophore_Name.
All other columns are either Conditionally required or Optional. Conditionally required columns should be drawn from the list provided in the file header or MUST be defined by the user using a Header line starting with #^.
Tip
The optional columns in this example table are included for illustrative purposes only and describe a case in which the user is reporting the RNA Spot fit quality using metrics that align with existing 4DN-BINA-OME Microscopy Metadata specifications.
##FOF-CT_Version=v1.0
##Table_Namespace=4dn_FOF-CT_rna_quality
##XYZ_Unit=micron
##Intensity_Unit=photonCount
#Lab_Name: Nobel
#Experimenter_Name: John Doe
#Experimenter_Contact: john.doe@email.com
#Description: Lorem ipsum dolor sit amet, consectetur adipiscing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua.
#Software_Title: SpotQualityCheck
#Software_Type: QualityControl
#Software_Authors: John Doe et al.
#Software_Description: A pretty clear description.
#Software_Repository: https://github.com/repo_name_goes_here
#Software_PreferredCitationID: https://doi.org/doi_goes_here
#Intensity_Measurement_Method: Spot peak intensity
#^Peak_Intensity: The signal intensity of the brightest pixel within a bright Spot (i.e. local maximum).
#^Raw_X: the original fit x-position relative to the camera and objective, (prior to drift correction, chromatic correction, or conversion to stage coordinates). This is the appropriate coordinate system for correcting optical aberrations.
#^Raw_Y: the original fit y-position relative to the camera and objective, (prior to drift correction, chromatic correction, or conversion to stage coordinates). This is the appropriate coordinate system for correcting optical aberrations.
#^Raw_Z: the original fit z-position relative to the camera and objective, (prior to drift correction, chromatic correction, or conversion to stage coordinates). This is the appropriate coordinate system for correcting optical aberrations.
#^X_Drift: the distance in nm the spot was moved in x based on fiducial tracking
#^Y_Drift: the distance in nm the spot was moved in y based on fiducial tracking
#^Z_Drift: the distance in nm the spot was moved in z based on fiducial tracking
#^X_Chromatic_Shift: the distance in nm the spot was moved in x based on chromatic correction map
#^Y_Chromatic_Shift: the distance in nm the spot was moved in y based on chromatic correction map
#^Z_Chromatic_Shift: the distance in nm the spot was moved in z based on chromatic correction map
#^X_Loc_Precision: lower and upper bound of 95% confidence interval on X-position after fit
#^Y_Loc_Precision: lower and upper bound of 95% confidence interval on Y-position after fit
#^Z_Loc_Precision: lower and upper bound of 95% confidence interval on Z-position after fit
#Additional_Tables: 4dn_FOF-CT_core, 4dn_FOF-CT_rna, 4dn_FOF-CT_trace, 4dn_FOF-CT_cell
##Columns=(RNA_Spot_ID, Channel_Name, Fluorophore_Name, Peak_Intensity, Raw_X, Raw_Y, Raw_Z, X_Drift, Y_Drift, Z_Drift, X_Chromatic_Shift, Y_Chromatic_Shift, Z_Chromatic_Shift, X_Loc_Precision, Y_Loc_Precision, Z_Loc_Precision)
001, 510/25, AlexaFluor_488, 1.1, 1.05, 1.2, 0.1, 0.05, 0.2, 0.2, 0.2, 0.2, 0.01, 0.01, 0.01
002, 510/25, AlexaFluor_488, 1.11, 1.055, 1.22, 0.11, 0.055, 0.22, 0.22, 0.22, 0.22, 0.012, 0.012, 0.012
003, 510/25, AlexaFluor_488, 1.12, 1.054, 1.21, 0.12, 0.054, 0.21, 0.22, 0.22, 0.22, 0.012, 0.012, 0.012
004, 510/25, AlexaFluor_488, 1.13, 1.15, 1.202, 0.13, 0.15, 0.202, 0.23, 0.23, 0.23, 0.013, 0.013, 0.013