For measuring BPE, the NightShade LB 985 in vivo Plant Imaging System (Berthold Technologies GmbH & Co.KG, 75323 Bad Wildbad, Germany), equipped with a sensitive, thermoelectrically cooled slow-scan NighOwlcam CCD device, was employed. The instrument was controlled by the IndiGo™ 2.0.5.0. software. Intensities of light were converted into counts per second (cps) by using the controlling software. The exposure time was kept at 60 s using a pixel binning of 4 × 4. In the duration of taking the images, both the “background correction” and the “cosmic suppression” options were enabled to ensure the elimination of high intensity pixels potentially caused by cosmic radiation. One pot for each treatment of the seedlings to be imaged was placed in the dark imaging box for 30 min in order to have dark adaptation, after which luminescence data were acquired for 10 min.
Nightshade lb 985 in vivo plant imaging system
Manufactured by Berthold Technologies
Sourced in Germany, United States
The NightShade LB 985 In Vivo Plant Imaging System is a laboratory equipment designed for the non-invasive imaging of plants. It is capable of capturing high-resolution images and data related to plant growth, development, and biological processes.
Automatically generated - may contain errors
Lab products found in correlation
Nighowlcam ccd device, by Berthold Technologies (3 mentions)
D luciferin potassium salt, by Thermo Fisher Scientific (1 mentions)
Clonexpress 2 one step cloning kit, by Vazyme (1 mentions)
D luciferin, by Duchefa Biochemie (1 mentions)
Chlorophyll assay kit, by Solarbio (1 mentions)
Du800, by Beckman Coulter (1 mentions)
Ccd camera, by Teledyne (1 mentions)
Indigo software, by Berthold Technologies (1 mentions)
Luciferin, by Promega (1 mentions)
10 protocols using nightshade lb 985 in vivo plant imaging system
1
In vivo Bioluminescence Imaging of Plants
2
CmNAC34 Transcription Factor Regulation
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The CDS of CmNAC34 was amplified and ligated into the PRI101 vector to yield an effector construct, 35S::CmNAC34. The promoter region of CmLCYB was cloned into the pRI-mini35S-LUC vector to produce a reporter construct, and the empty pRI-mini35S-LUC was used as a control. All resulting constructs were transformed into Agrobacterium tumefaciens stain EHA105. Tobacco leaves were used for co-infiltration and treated with 1 mM D-Luciferin Potassium Salt (Thermo Fisher Scientific, Waltham, MA, USA) after three days. The luciferase fluorescence signal was detected using the NightShade LB 985 In Vivo Plant Imaging System (Berthold, Bad Wildbad, German), and the fluorescence image was taken using the IngiGo program.
3
Cloning and Co-Infiltration of TF-LUC Vectors
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The coding region of 12 TFs was amplified from P. alba × P. glandulosa xylem cDNA, and, by using the ClonExpressII One Step Cloning Kit (Vazyme Biotech Co., Ltd., Nanjing, Jiangsu, China), they were cloned into pCAMBIA1300-cLUC and pCAMBIA1300-nLUC, which were pre-linearized by KpnI/SalI (Song et al., 2011 (link)), generating TF-nLUC and TF-cLUC. Primers used for the vector construction are shown in Supplementary Table 1 . After confirmation by sequencing, the vectors were transformed into Agrobacterium GV3101. Equal concentrations and volumes of Agrobacterium cultures were mixed and co-infiltrated into the N. benthamiana leaves. After incubation for 36 h, the LUC fluorescence was detected under a Berthold NightSHADE LB985 in vivo Plant Imaging System (Berthold Technologies GmbH, Bad Wildbad, Germany).
4
Quantifying Prebranching Sites in Roots
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To quantify the prebranching sites, we used 2DAG pDR5:LUC seedlings that were transferred to T6P 1 mM in 0.5XMS solid medium for 6 d. The seedlings were sprayed with 1 mM D-Luciferin (Duchefa Biochemie) solution [D-Luciferin dissolved in 0.01% (v/v) Tween80 and 0.1% (v/v) DMSO]. Then, they were kept in darkness for 10 min to allow the appropriate absorption of D-Luciferin by the roots (47 (link)). Emitted luminescence was captured using ANDOR iKon-M 934 charge-coupled device (CCD) camera (Oxford Instruments) paired with a fixed lens (Spacecom 43F2409M-MP C 4/3” 24 mm F0.9) over a 20-min exposure time. Bright-field images of the roots were taken in each condition to be able to measure the primary roots and quantify the density of DR5:LUC sites. DR5:LUC oscillation frequencies and amplitudes were determined via 22-h time-lapse assays. 3DAG seedlings were transferred to plates with or without 1mM T6P, sprayed with 1 mM D-Luciferin, and incubated in the dark for 1 h. Next, emitted luminescence was captured every 15 min with a 10-min exposure time using a NightSHADE LB985 in vivo plant imaging system (Berthold technologies) equipped with a deep-cooled slow scan CCD camera and the accompanying lens (Andor Instruments). Based on these time-lapses, the amplitude of and period between oscillation peaks in the oscillation zone were determined using ImageJ
5
Delayed Fluorescence for Assessing Wheat Stress
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Delayed fluorescence is associated with extremely weak light emitted by chlorophyll molecules in plants and can reflect the chlorophyll content, providing a powerful tool for studying stress reactions in plants. The chlorophyll content in wheat leaves were each infested by 20 aphids as described above was first detected using the NightShade LB 985 In vivo Plant Imaging System (Berthold Technologies, Bad Wildbad, Germany). After 48 h of aphid feeding, the leaves were cut and immediately illuminated for 30 s with an LED panel. After the light was switched off, the delayed fluorescence was measured immediately using the NightShade system. The exposure time was set to 30 s using 4-by-4 pixel binning. The total chlorophyll content in wheat leaves after aphid infestation was also examined using a Chlorophyll Assay Kit (Solarbio, Beijing, China) according to the manufacturer’s instructions. In brief, 0.1 g of fresh leaf tissues was ground to a fine powder and extracted with 2 mL of 80% acetone (v/v) at 4 °C for overnight. The homogenate was centrifuged at 4000 g for 10 min at 4 °C, and the supernatant was used for the chlorophyll assay. The amounts of chlorophyll were detected spectrophotometrically, by reading the absorbance at 645 and 663 nm (DU800, Beckman, USA), and then calculated as described previously [58 (link)].
6
Measuring Cadmium Stress-induced BPE
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For measuring BPE the NightShade LB 985 in vivo Plant Imaging System (Berthold Technologies GmbH & Co.KG, 75323 Bad Wildbad, Germany) equipped with a sensitive, thermoelectrically cooled slow-scan NighOwlcam CCD device has been employed. The instrument was controlled by the IndiGo™ 2.0.5.0. software. Intensities of light were converted into counts per second (cps) by using the controlling software. The exposure time was kept at 60 sec using a pixel binning of 2 x 2. In the duration of taking the images both the “background correction” and the „cosmic suppression” options were enabled to ensure the elimination of high intensity pixels potentially caused by cosmic radiation. One pot for each treatment (0, 1, 3 and 7 days after cadmium treatment) of the seedlings to be imaged was placed into the dark imaging box and subsequently dark-adapted for 5 min in order to avoid chlorophyll-autofluorescence deriving from electron recoupling of the photosystems.
7
Measuring UPE in Plant Seedlings
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For measuring UPE, the NightShade LB 985 In Vivo Plant Imaging System (Berthold Technologies GmbH & Co. KG, 75323 Bad Wildbad, Germany) equipped with a sensitive, thermoelectrically cooled slow-scan NighOwlcam CCD device has been employed. The instrument was controlled by the IndiGo™ 2.0.5.0. software. Intensities of light were converted into counts per second (cps) by using the controlling software. The exposure time was kept at 60 s using a pixel binning of 2 × 2. During the duration of taking the images both the “background correction” and the “cosmic suppression” options were enabled to ensure the elimination of high-intensity pixels potentially caused by cosmic radiation. One pot for each treatment of the seedlings to be imaged was placed into the dark imaging box. In the first part of the measurement, DF signal was measured immediately after placing the pots in the dark chamber for 10 min. Thereafter, the samples were continued to be kept in dark to provide sufficient time for dark adaptation, and from the 30th minute, for the pots spent in the dark, bioluminescence data was also acquired for 10 min.
8
Luciferase Imaging of Seedling Oscillations
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The Luciferase imaging of whole seedlings and oscillation expression analysis was performed as described 44 . A Lumazon FA imaging system (Nippon Roper) carrying a CCD camera from Princeton Instruments Ltd. (Trenton, NJ, USA) or NightSHADE LB985 in vivo plant imaging system (BERTHOLD TECHNOLOGIES) carrying a deep-cooled slow scan CCD camera from Andor Instruments Ltd. (Belfast, UK) were used for luciferase imaging.
To monitor the pre-branch site numbers, we used 8-day-old DR5:LUC seedlings for pre-branch site quantification. The D-luciferin solution (1 mM) was sprayed gently on the seedlings, and kept for 10min in the dark and imaged in the Lumazon system with a 15-minute exposure time.
For Long-Term Imaging of Luciferase Signal in the root tip, square plates containing 1/2MS medium were sprayed with 1mM D-Luciferin solution (0.01% Tween80) and left to dry in the dark. Then 3-dayold DR5:LUC seedlings were transferred on the plates and imaged immediately with a macro lens every 10 minutes with a 7-minute exposure time for indicated times. The period of the DR5:LUC oscillations was determined based on the number of frames that spaced a DR5:LUC maximum in the OZ of each seedling root, multiplied with the time of each cycle.
To monitor the pre-branch site numbers, we used 8-day-old DR5:LUC seedlings for pre-branch site quantification. The D-luciferin solution (1 mM) was sprayed gently on the seedlings, and kept for 10min in the dark and imaged in the Lumazon system with a 15-minute exposure time.
For Long-Term Imaging of Luciferase Signal in the root tip, square plates containing 1/2MS medium were sprayed with 1mM D-Luciferin solution (0.01% Tween80) and left to dry in the dark. Then 3-dayold DR5:LUC seedlings were transferred on the plates and imaged immediately with a macro lens every 10 minutes with a 7-minute exposure time for indicated times. The period of the DR5:LUC oscillations was determined based on the number of frames that spaced a DR5:LUC maximum in the OZ of each seedling root, multiplied with the time of each cycle.
9
Bioluminescent In Vivo Protein Interaction Assay
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The coding sequences of CA-MxMPK4-1 and MxIQM3 were inserted into the pCambia1300-nLUC and pCambia1300-cLUC vectors, respectively. The subsequent procedures were similar to those of the BiFC assay. The difference was that a CDD imaging system (NightShade LB 985 In Vivo Plant Imaging System; Berthold Technologies USA, LLC, Oak Ridge, TN, USA) was used to observe the N. benthamiana leaves. A volume of 1 mM D-luciferin was sprayed onto the back of leaves and placed in the dark for 7 min before the tissues were observed.
10
Luciferase Complementation Imaging of Brassinosteroid Signaling
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The luciferase complementation imaging assay was performed using an Agrobacterium-mediated transient expression system (Chen et al., 2008) . In brief, the coding sequence of OsBRI1 was ligated in-frame into pCAMBIA-nLuc to construct the OsBRI1-nLuc plasmid, which encoded OsBRI1 fused to the N-terminal part of luciferase. The coding sequences of OsBRI1 and OsBAK1 were each ligated in-frame into pCAMBIA-cLuc to construct cLuc-OsBRI1 and cLuc-OsBAK1 plasmids, encoding the C-terminal part of luciferase fused to OsBRI1 and OsBAK1, respectively. Agrobacterium cultures containing each of the three constructs were diluted to an OD 600 = 0.8 with infiltration buffer (10 mM MES [pH 5.6], 10 mM MgCl 2 , 200 mM acetosyringone), mixed at a 1:1:1 ratio, and then injected into fully expanded leaves of Nicotiana benthamiana. The plants were grown under a 16-h light/8-h dark photoperiod for 48 h at 23 C. The detached leaves were sprayed with 1 mM luciferin (Promega; www.promega.com).
The luminescence signal was captured using the NightSHADE LB 985 in vivo Plant Imaging System (Berthold Technologies; www.berthold.com), and relative luminescence intensity was measured. Quantitative analysis was performed using IndiGo software (Berthold Technologies).
The luminescence signal was captured using the NightSHADE LB 985 in vivo Plant Imaging System (Berthold Technologies; www.berthold.com), and relative luminescence intensity was measured. Quantitative analysis was performed using IndiGo software (Berthold Technologies).
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