{"__v":11,"_id":"5716a534f5b57c20005390cc","category":{"__v":19,"_id":"54f7a7be0a3cbb0d00d666fb","pages":["54f7a95354182d2100c9d057","54f7ad293607243500de2496","54f7ae7d0a3cbb0d00d66705","54f7b0013607243500de249b","54f7b4360a3cbb0d00d6670c","54f7c71154182d2100c9d073","54fa5205961fea21009206a9","54fa55e1c6db4517005b0147","54fddf642804410d00ee8a2a","5509fd2bdfed731900b81863","552dfa862594f70d001b2c48","552dfab3a702770d00d96d5b","552dfaca2594f70d001b2c4a","55d68d2c250d7d0d00427478","55d68d3bae529e0d00d34edb","5611dd433ca69417008981af","5679bca976cd370d003c1183","56de30df9ca83e17000cbc59","56eadf62199f7317006eb4db"],"project":"5476bf0f817e8d080031f988","version":"5476bf10817e8d080031f98b","sync":{"url":"","isSync":false},"reference":false,"createdAt":"2015-03-05T00:47:58.582Z","from_sync":false,"order":4,"slug":"instructions","title":"Capabilities"},"parentDoc":null,"project":"5476bf0f817e8d080031f988","user":"56f57b8eab3f610e000a6596","version":{"__v":17,"_id":"5476bf10817e8d080031f98b","project":"5476bf0f817e8d080031f988","createdAt":"2014-11-27T06:05:04.263Z","releaseDate":"2014-11-27T06:05:04.263Z","categories":["5476bf10817e8d080031f98c","5477c46cf3736008009e9eb5","5477c474f3736008009e9eb6","5477c47ef3736008009e9eb7","5477c48ff3736008009e9eb8","5477c4948deb230800808bf0","54e68328154f8e0d0007b55c","54e90194c8e0c00d007ac061","54eed2275bf74a0d00ef4076","54f7a7be0a3cbb0d00d666fb","559b0ebf7ae7f80d0096d871","55d697f9ae529e0d00d34f03","562d4dcc8c6e5a0d00d6ed1d","562e591c4376430d006f17e0","568f0e73bdb9260d00149d8c","5719542aac1e2e0e001834c6","57a14a8ed778850e0047e230"],"is_deprecated":false,"is_hidden":false,"is_beta":false,"is_stable":true,"codename":"","version_clean":"1.0.0","version":"1.0"},"updates":[],"next":{"pages":[],"description":""},"createdAt":"2016-04-19T21:37:56.797Z","link_external":false,"link_url":"","githubsync":"","sync_unique":"","hidden":false,"api":{"results":{"codes":[]},"settings":"","auth":"required","params":[],"url":""},"isReference":false,"order":20,"body":"[block:api-header]\n{\n  \"type\": \"basic\",\n  \"title\": \"Instruction and Parameters\"\n}\n[/block]\n 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.\n\n##Absorbance\nAbsorbance readings can be taken for wavelengths between 300 nm and 1000 nm.\n[block:code]\n{\n  \"codes\": [\n    {\n      \"code\": \"{\\n  \\\"op\\\": \\\"absorbance\\\",\\n  \\\"object\\\": container,\\n  \\\"wells\\\": [wells],\\n  \\\"wavelength\\\": length,\\n  \\\"num_flashes\\\": integer, // default: 25\\n  \\\"dataref\\\": string,\\n  \\\"temperature\\\": temperature, // optional\\n  \\\"incubate_before\\\": { // optional\\n    \\\"duration\\\": time, // required if `incubate_before` specified\\n    \\\"shaking\\\": { // optional\\n      \\\"amplitude\\\": length, // required if `shaking` specified\\n      \\\"orbital\\\": bool // required if `shaking` specified\\n    }\\n  }\\n}\",\n      \"language\": \"json\"\n    }\n  ]\n}\n[/block]\n##Fluorescence\nFluorescence excitation wavelength can be between 230 nm and 600 nm, and emission can be between 330 nm and 850 nm.\n\nThe 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.\n\nThe `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.\n[block:code]\n{\n  \"codes\": [\n    {\n      \"code\": \"{\\n  \\\"op\\\": \\\"fluorescence\\\",\\n  \\\"object\\\": container,\\n  \\\"wells\\\": [wells],\\n  \\\"excitation\\\": length,\\n  \\\"emission\\\": length,\\n  \\\"num_flashes\\\": integer, // default: 25\\n  \\\"dataref\\\": string,\\n  \\\"temperature\\\": temperature, // optional\\n  \\\"incubate_before\\\": { // optional\\n    \\\"duration\\\": time, // required if `incubate_before` specified\\n    \\\"shaking\\\": { // optional\\n      \\\"amplitude\\\": length, // required if `shaking` specified\\n      \\\"orbital\\\": bool // required if `shaking` specified\\n    }\\n  },\\n  \\\"gain\\\": float // optional\\n}\",\n      \"language\": \"json\"\n    }\n  ]\n}\n[/block]\n##Luminescence\nThe luminescence mode has no wavelength parameter and measures all photon emissions in the detectable wavelength range of 380 nm - 600 nm.\n[block:code]\n{\n  \"codes\": [\n    {\n      \"code\": \"{\\n  \\\"op\\\": \\\"luminescence\\\",\\n  \\\"object\\\": container,\\n  \\\"wells\\\": [wells],\\n  \\\"dataref\\\": string,\\n  \\\"temperature\\\": temperature, // optional\\n  \\\"incubate_before\\\": { // optional\\n    \\\"duration\\\": time, // required if `incubate_before` specified\\n    \\\"shaking\\\": { // optional\\n      \\\"amplitude\\\": length, // required if `shaking` specified\\n      \\\"orbital\\\": bool // required if `shaking` specified\\n    }\\n  }\\n}\",\n      \"language\": \"json\",\n      \"name\": \"Luminescence\"\n    }\n  ]\n}\n[/block]\n## Optional Parameters\n\nAll 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.\n\nIf `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`. \n\nIncubation 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.\n[block:api-header]\n{\n  \"type\": \"basic\",\n  \"title\": \"Device\"\n}\n[/block]\nThe `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.\nMore information on this plate reader can be found [here](http://www.tecan.com/platform/apps/product/index.asp?MenuID=1813&ID=1918&Menu=1&Item=21.2.10.1.1).\n[block:parameters]\n{\n  \"data\": {\n    \"h-1\": \"Wavelength range\",\n    \"h-0\": \"Reading type\",\n    \"0-0\": \"Absorbance\",\n    \"1-0\": \"Fluorescence\",\n    \"0-1\": \"230 nm - 1000 nm\",\n    \"1-1\": \"Excitation: 230 nm - 600 nm\\nEmission: 330 nm - 600 nm\",\n    \"2-0\": \"Luminescence\",\n    \"2-1\": \"380 nm - 600 nm (data will be collected across this range)\",\n    \"h-2\": \"Accucracy\",\n    \"h-3\": \"Precision\",\n    \"0-2\": \"< ± 0,5 nm for λ> 315 nm\\n< ± 0.3 nm for λ≤ 315 nm\",\n    \"0-3\": \"< ± 0,5 nm for λ> 315 nm\\n< ± 0.3 nm for λ≤ 315 nm\",\n    \"1-2\": \"< ± 2 nm for λ> 315 nm\\n< ± 1 nm for λ≤ 315 nm\",\n    \"1-3\": \"< ± 1 nm for λ> 315 nm\\n< ± 0.5 nm for λ≤ 315 nm\"\n  },\n  \"cols\": 4,\n  \"rows\": 3\n}\n[/block]\n## Supported plates\nCurrently, only `96-flat` and `384-flat` plates can be used in a plate read command (see [Containers](doc: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.\n\n## Detectors\n[block:parameters]\n{\n  \"data\": {\n    \"h-0\": \"Reading type\",\n    \"0-0\": \"Absorbance\",\n    \"1-0\": \"Fluorescence\",\n    \"0-1\": \"UV silicon photodiode\",\n    \"1-1\": \"PMT, optionally UV and red-sensitive\",\n    \"2-1\": \"photon counting system with low dark current PMT\",\n    \"2-0\": \"Luminescence\",\n    \"h-1\": \"Detectors\",\n    \"h-2\": \"Detection mode\",\n    \"0-2\": \"bottom\",\n    \"1-2\": \"bottom\",\n    \"2-2\": \"top\"\n  },\n  \"cols\": 2,\n  \"rows\": 3\n}\n[/block]\nAt 260nm the absorbance detection shows less than 0.2% variance in precision and less than 0.5% variance in accuracy.\nFluorescence readings are sensitive down to 1.2 fmol / well.\nGlow luminescence readings are sensitive down to 225 amol ATP / well. Flash luminescence readings down to 12 amol ATP / well.","excerpt":"","slug":"spectrophotometry","type":"basic","title":"Spectrophotometry - Absorbance, Fluorescence, Luminescence"}

Spectrophotometry - Absorbance, Fluorescence, Luminescence


[block:api-header] { "type": "basic", "title": "Instruction and Parameters" } [/block] 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. [block:code] { "codes": [ { "code": "{\n \"op\": \"absorbance\",\n \"object\": container,\n \"wells\": [wells],\n \"wavelength\": length,\n \"num_flashes\": integer, // default: 25\n \"dataref\": string,\n \"temperature\": temperature, // optional\n \"incubate_before\": { // optional\n \"duration\": time, // required if `incubate_before` specified\n \"shaking\": { // optional\n \"amplitude\": length, // required if `shaking` specified\n \"orbital\": bool // required if `shaking` specified\n }\n }\n}", "language": "json" } ] } [/block] ##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. [block:code] { "codes": [ { "code": "{\n \"op\": \"fluorescence\",\n \"object\": container,\n \"wells\": [wells],\n \"excitation\": length,\n \"emission\": length,\n \"num_flashes\": integer, // default: 25\n \"dataref\": string,\n \"temperature\": temperature, // optional\n \"incubate_before\": { // optional\n \"duration\": time, // required if `incubate_before` specified\n \"shaking\": { // optional\n \"amplitude\": length, // required if `shaking` specified\n \"orbital\": bool // required if `shaking` specified\n }\n },\n \"gain\": float // optional\n}", "language": "json" } ] } [/block] ##Luminescence The luminescence mode has no wavelength parameter and measures all photon emissions in the detectable wavelength range of 380 nm - 600 nm. [block:code] { "codes": [ { "code": "{\n \"op\": \"luminescence\",\n \"object\": container,\n \"wells\": [wells],\n \"dataref\": string,\n \"temperature\": temperature, // optional\n \"incubate_before\": { // optional\n \"duration\": time, // required if `incubate_before` specified\n \"shaking\": { // optional\n \"amplitude\": length, // required if `shaking` specified\n \"orbital\": bool // required if `shaking` specified\n }\n }\n}", "language": "json", "name": "Luminescence" } ] } [/block] ## 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. [block:api-header] { "type": "basic", "title": "Device" } [/block] 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](http://www.tecan.com/platform/apps/product/index.asp?MenuID=1813&ID=1918&Menu=1&Item=21.2.10.1.1). [block:parameters] { "data": { "h-1": "Wavelength range", "h-0": "Reading type", "0-0": "Absorbance", "1-0": "Fluorescence", "0-1": "230 nm - 1000 nm", "1-1": "Excitation: 230 nm - 600 nm\nEmission: 330 nm - 600 nm", "2-0": "Luminescence", "2-1": "380 nm - 600 nm (data will be collected across this range)", "h-2": "Accucracy", "h-3": "Precision", "0-2": "< ± 0,5 nm for λ> 315 nm\n< ± 0.3 nm for λ≤ 315 nm", "0-3": "< ± 0,5 nm for λ> 315 nm\n< ± 0.3 nm for λ≤ 315 nm", "1-2": "< ± 2 nm for λ> 315 nm\n< ± 1 nm for λ≤ 315 nm", "1-3": "< ± 1 nm for λ> 315 nm\n< ± 0.5 nm for λ≤ 315 nm" }, "cols": 4, "rows": 3 } [/block] ## Supported plates Currently, only `96-flat` and `384-flat` plates can be used in a plate read command (see [Containers](doc: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 [block:parameters] { "data": { "h-0": "Reading type", "0-0": "Absorbance", "1-0": "Fluorescence", "0-1": "UV silicon photodiode", "1-1": "PMT, optionally UV and red-sensitive", "2-1": "photon counting system with low dark current PMT", "2-0": "Luminescence", "h-1": "Detectors", "h-2": "Detection mode", "0-2": "bottom", "1-2": "bottom", "2-2": "top" }, "cols": 2, "rows": 3 } [/block] 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.