Background Synovial sarcoma is a high-grade malignant tumor of soft tissue, characterized by the specific chromosomal translocation t(X;18), and its resultant SYT-SSX fusion gene. Despite intensive multimodality therapy, the majority of metastatic or relapsed diseases still remain incurable, thus suggesting a need for new therapeutic options. We previously demonstrated the antigenicity of SYT-SSX gene-derived peptides by in vitro analyses. The present study was designed to evaluate in vivo immunological property of a SYT-SSX junction peptide in selected patients with synovial sarcoma.
Background
Synovial sarcoma is a high-grade malignant tumor of soft tissue, characterized by the specific chromosomal translocation t(X;18), and its resultant SYT-SSX fusion gene. Despite intensive multimodality therapy, the majority of metastatic or relapsed diseases still remain incurable, thus suggesting a need for new therapeutic options. We previously demonstrated the antigenicity of SYT-SSX gene-derived peptides by in vitro analyses. The present study was designed to evaluate in vivo immunological property of a SYT-SSX junction peptide in selected patients with synovial sarcoma.Methods A 9-mer peptide (SYT-SSX B: GYDQIMPKK) spanning the SYT-SSX fusion region was synthesized. Eligible patients were those (i) who have histologically and genetically confirmed, unresectable synovial sarcoma (SYT-SSX1 or SYT-SSX2 positive), (ii) HLA-A*2402 positive, (iii) between 20 and 70 years old, (iv) ECOG performance status between 0 and 3, and (v) who gave informed consent. Vaccinations with SYT-SSX B peptide (0.1 mg or 1.0 mg) were given subcutaneously six times at 14-day intervals. These patients were evaluated for DTH skin test, adverse events, tumor size, tetramer staining, and peptide-specific CTL induction.
Methods
A 9-mer peptide (SYT-SSX B: GYDQIMPKK) spanning the SYT-SSX fusion region was synthesized. Eligible patients were those (i) who have histologically and genetically confirmed, unresectable synovial sarcoma (SYT-SSX1 or SYT-SSX2 positive), (ii) HLA-A*2402 positive, (iii) between 20 and 70 years old, (iv) ECOG performance status between 0 and 3, and (v) who gave informed consent. Vaccinations with SYT-SSX B peptide (0.1 mg or 1.0 mg) were given subcutaneously six times at 14-day intervals. These patients were evaluated for DTH skin test, adverse events, tumor size, tetramer staining, and peptide-specific CTL induction.Results A total of 16 vaccinations were carried out in six patients. The results were (i) no serious adverse effects or DTH reactions, (ii) suppression of tumor progression in one patient, (iii) increases in the frequency of peptide-specific CTLs in three patients and a decrease in one patient, and (iv) successful induction of peptide-specific CTLs from four patients.
Results
A total of 16 vaccinations were carried out in six patients. The results were (i) no serious adverse effects or DTH reactions, (ii) suppression of tumor progression in one patient, (iii) increases in the frequency of peptide-specific CTLs in three patients and a decrease in one patient, and (iv) successful induction of peptide-specific CTLs from four patients.Conclusions Our findings indicate the safety of the SYT-SSX junction peptide in the use of vaccination and also give support to the property of the peptide to evoke in vivo immunological responses. Modification of both the peptide itself and the related protocol is required to further improve the therapeutic efficacy.
Conclusions
Our findings indicate the safety of the SYT-SSX junction peptide in the use of vaccination and also give support to the property of the peptide to evoke in vivo immunological responses. Modification of both the peptide itself and the related protocol is required to further improve the therapeutic efficacy.

Background Synovial sarcoma is a relatively rare, high-grade malignant tumor of soft tissue, characterized by biphasic or monophasic histology, specific chromosomal translocation t(X;18), and its resultant SYT-SSX fusion gene[ 1 ]. This tumor affects mostly adolescents and young adults. The 5-year survival rates of patients with this localized disease have ranged from 66% to 80% in the current literature [ 2 - 5 ]. However, the majority of metastatic or relapsed diseases still remain incurable despite intensive multimodality therapy. Therefore there is a need for additional new therapeutic options other than conventional surgery, radiotherapy, and chemotherapy. Vaccination of tumor antigenic peptide serves as a commonly accepted method in anti-cancer immunotherapy[ 6 , 7 ]. This is based on the rationale that T cells recognize antigenic peptide in the context of MHC molecules on the tumor cell or antigen presenting cells through the T cell receptor, which elicits subsequent anti-tumor immune responses. Identification of antigenic peptides recognized T cells enabled us to apply vaccination trials to a variety of tumors, except bone and soft tissue sarcomas[ 8 ]. Currently tumor specific chromosomal translocations are defined in leukemias, lymphomas, and sarcomas[ 9 , 10 ]. The fusion regions of translocation products are specifically expressed by its corresponding tumors, thereby serving as targets of great potential for tumor specific therapies, including immunotherapy[ 11 , 12 ]. We[ 13 , 14 ] found that SYT-SSX fusion gene-derived peptides can be recognized by circulating CD8+ T cells in patients with synovial sarcoma and elicit HLA-restricted, tumor specific cytotoxic responses by in vitro stimulations. In this study, we conducted a phase I pilot trial of vaccination of a SYT-SSX-derived junction peptide in elected synovial sarcoma patients.
Background
Synovial sarcoma is a relatively rare, high-grade malignant tumor of soft tissue, characterized by biphasic or monophasic histology, specific chromosomal translocation t(X;18), and its resultant SYT-SSX fusion gene[ 1 ]. This tumor affects mostly adolescents and young adults. The 5-year survival rates of patients with this localized disease have ranged from 66% to 80% in the current literature [ 2 - 5 ]. However, the majority of metastatic or relapsed diseases still remain incurable despite intensive multimodality therapy. Therefore there is a need for additional new therapeutic options other than conventional surgery, radiotherapy, and chemotherapy.Vaccination of tumor antigenic peptide serves as a commonly accepted method in anti-cancer immunotherapy[ 6 , 7 ]. This is based on the rationale that T cells recognize antigenic peptide in the context of MHC molecules on the tumor cell or antigen presenting cells through the T cell receptor, which elicits subsequent anti-tumor immune responses. Identification of antigenic peptides recognized T cells enabled us to apply vaccination trials to a variety of tumors, except bone and soft tissue sarcomas[ 8 ].Currently tumor specific chromosomal translocations are defined in leukemias, lymphomas, and sarcomas[ 9 , 10 ]. The fusion regions of translocation products are specifically expressed by its corresponding tumors, thereby serving as targets of great potential for tumor specific therapies, including immunotherapy[ 11 , 12 ]. We[ 13 , 14 ] found that SYT-SSX fusion gene-derived peptides can be recognized by circulating CD8+ T cells in patients with synovial sarcoma and elicit HLA-restricted, tumor specific cytotoxic responses by in vitro stimulations. In this study, we conducted a phase I pilot trial of vaccination of a SYT-SSX-derived junction peptide in elected synovial sarcoma patients.Methods Peptide A 9-mer peptide (SYT-SSX B: GYDQIMPKK) spanning the SYT-SSX fusion region was synthesized under good manufacturing practice (GMP) conditions by Multiple Peptide Systems (San Diego, CA). The identity of the peptide was confirmed by mass spectral analysis, and was shown to have more than 98% purity when assessed by high pressure liquid chromatography analysis. The peptide was delivered us in the form of a freeze-dried, sterile white powder. It was dissolved in 1.0 ml of physiological saline (Otsuka Pharmaceutical Co., Ltd., Tokyo, Japan) and stored at -80°C until just before usage. The affinity of the peptide to HLA-A24 molecules and its antigenicity were determined in previous studies[ 13 , 14 ]. Eligibility The study protocol was approved by the Clinical Institutional Ethical Review Board of the Medical Institute of Bioregulation, Sapporo Medical University, Japan. Eligible patients were those (i) who have histologically and genetically confirmed, unresectable synovial sarcoma (SYT-SSX1 or SYT-SSX2 positive), (ii) HLA-A*2402 positive, (iii) between 20 and 70 years old, (iv) ECOG performance status between 0 and 3, and (v) who gave informed consent. Exclusion criteria included (i) prior chemotherapy, steroid therapy, or other immunotherapy within the past 4 weeks, (ii) presence of other cancers that might influence the prognosis, (iii) immunodeficiency or a history of splenectomy, (iv) severe cardiac insufficiency, acute infection, or hematopoietic failure, (v) ongoing breast-feeding, (vi) unsuitability for the trial based on the clinical judgment of the doctors involved. This study was carried out at the Department of Orthopaedic Surgery, Sapporo Medical University Hospital from June 2003 until the end of September 2004. Vaccination schedule Vaccinations with SYT-SSX B peptide were administered subcutaneously into the upper arm six times at 14-day intervals. In order to set up a dose-escalation trial, the patients were separated into the two groups. Each group included three patients. Those from group 1 received 0.1 mg and group 2 participants received 1.0 mg. Delayed-type hypersensitivity (DTH) skin test Delayed-type hypersensitivity (DTH) skin test was performed at each vaccination. The peptide (10 μg) solution in physiological saline (0.1 ml) or physiological saline alone (0.1 ml) were separately injected intradermally into the forearm. A positive reaction was defined as a diameter of erythema of more than 4 mm, 48 hr after the injection. Toxicity evaluation Patients were examined closely for signs of toxicity during and after vaccination. Adverse events were recorded using the National Cancer Institute Common Toxicity Criteria (NCI-CTC). Clinical response evaluation Physical examinations and hematological examinations were monitored before and after each vaccination. Tumor size was evaluated by computed tomography (CT) scans before treatment, and again after three vaccinations, and then at the end of the study period. A complete response (CR) was defined as complete disappearance of all measurable diseases. A partial response (PR) was defined as a >= 50% decrease from the baseline in size of all measurable lesions (sum of products of maximal perpendicular diameters) lasting for a period of at least 4 weeks. Progressive disease (PD) was defined as an increase in the sum of the bi-dimensional measurements of all known disease sites by at least 25% or by the appearance of new lesions. No change (NC) was defined as the absence of matched criteria for CR, PR, or PD. Tetramer staining HLA-A24/peptide tetramers (HLA-A24/B, HLA-A24/R49.2, and HLA-A24/HIV) were previously constructed [ 13 - 15 ]. Flowcytometric analysis was performed by taking peripheral blood mononuclear cells (PBMCs) from patients. PBMCs were taken at pre-vaccination and again one week after 1 st , 3 rd , and 6 th  vaccination. Cells were stained with PE-labeled tetramers at 37°C for 20 min and a FITC-conjugated anti-CD8 mAb (Becton Dickinson) at 4°C for 30 min. Analysis of stained PBMCs was performed using FACScan (Becton Dickinson) and CellQuest software (Becton Dickinson). The frequency of CTL precursors was calculated as the number of tetramer positive cells / the number of CD8 +  cells. CTL induction Cytotoxic T lymphocytes (CTLs) were induced from the PBMCs of patients using SYT-SSX B peptides according to the method described before[ 13 , 14 ]. The cytotoxic activity was evaluated by 6-h  51 Cr release assay[ 13 ]. As target cells, synovial sarcoma cell lines (Fuji, HS-SY-II, and SW982), an erythroleukemia cell line (K562), and a T-B Lymphoblast hybrid transfected with HLA-A*2402 (T2-A*2402) were used. Fuji and HS-SY-II were both HLA-A24 and SYT-SSX positive lines. SW982 and K562 were both HLA-A24 and SYT-SSX negative lines used as controls. T2-A*2402 cells were used to determine peptide-specific cytotoxicity by pulsation with SYT-SSX B or HIV peptide before labeling. The stimulated CD8 +  T cells were mixed with the labeled target cells. After a 6-h incubation period at 37°C, the release of the  51 Cr label was measured by collecting the supernatant, followed by quantification in an automated gamma counter. The percentage of specific cytotoxicity was calculated as the percentage of specific  51 Cr release: [(experimental  51 Cr release - spontaneous  51 Cr release) / (maximum  51 Cr release - spontaneous  51 Cr release)] × 100. Maximum  51 Cr release was measured by incubating the labelled target cells with 2% NP-40, instead of the stimulated CD8 +  T cells. CTL induction was determined as successful when specific cyotoxicity of 10% or more was achieved on Fuji, HS-SY-II, and SYT-SSX B peptide-pulsed T2-A*2402 cells.
Methods
Peptide A 9-mer peptide (SYT-SSX B: GYDQIMPKK) spanning the SYT-SSX fusion region was synthesized under good manufacturing practice (GMP) conditions by Multiple Peptide Systems (San Diego, CA). The identity of the peptide was confirmed by mass spectral analysis, and was shown to have more than 98% purity when assessed by high pressure liquid chromatography analysis. The peptide was delivered us in the form of a freeze-dried, sterile white powder. It was dissolved in 1.0 ml of physiological saline (Otsuka Pharmaceutical Co., Ltd., Tokyo, Japan) and stored at -80°C until just before usage. The affinity of the peptide to HLA-A24 molecules and its antigenicity were determined in previous studies[ 13 , 14 ].
Peptide
A 9-mer peptide (SYT-SSX B: GYDQIMPKK) spanning the SYT-SSX fusion region was synthesized under good manufacturing practice (GMP) conditions by Multiple Peptide Systems (San Diego, CA). The identity of the peptide was confirmed by mass spectral analysis, and was shown to have more than 98% purity when assessed by high pressure liquid chromatography analysis. The peptide was delivered us in the form of a freeze-dried, sterile white powder. It was dissolved in 1.0 ml of physiological saline (Otsuka Pharmaceutical Co., Ltd., Tokyo, Japan) and stored at -80°C until just before usage. The affinity of the peptide to HLA-A24 molecules and its antigenicity were determined in previous studies[ 13 , 14 ].Eligibility The study protocol was approved by the Clinical Institutional Ethical Review Board of the Medical Institute of Bioregulation, Sapporo Medical University, Japan. Eligible patients were those (i) who have histologically and genetically confirmed, unresectable synovial sarcoma (SYT-SSX1 or SYT-SSX2 positive), (ii) HLA-A*2402 positive, (iii) between 20 and 70 years old, (iv) ECOG performance status between 0 and 3, and (v) who gave informed consent. Exclusion criteria included (i) prior chemotherapy, steroid therapy, or other immunotherapy within the past 4 weeks, (ii) presence of other cancers that might influence the prognosis, (iii) immunodeficiency or a history of splenectomy, (iv) severe cardiac insufficiency, acute infection, or hematopoietic failure, (v) ongoing breast-feeding, (vi) unsuitability for the trial based on the clinical judgment of the doctors involved. This study was carried out at the Department of Orthopaedic Surgery, Sapporo Medical University Hospital from June 2003 until the end of September 2004.
Eligibility
The study protocol was approved by the Clinical Institutional Ethical Review Board of the Medical Institute of Bioregulation, Sapporo Medical University, Japan. Eligible patients were those (i) who have histologically and genetically confirmed, unresectable synovial sarcoma (SYT-SSX1 or SYT-SSX2 positive), (ii) HLA-A*2402 positive, (iii) between 20 and 70 years old, (iv) ECOG performance status between 0 and 3, and (v) who gave informed consent. Exclusion criteria included (i) prior chemotherapy, steroid therapy, or other immunotherapy within the past 4 weeks, (ii) presence of other cancers that might influence the prognosis, (iii) immunodeficiency or a history of splenectomy, (iv) severe cardiac insufficiency, acute infection, or hematopoietic failure, (v) ongoing breast-feeding, (vi) unsuitability for the trial based on the clinical judgment of the doctors involved. This study was carried out at the Department of Orthopaedic Surgery, Sapporo Medical University Hospital from June 2003 until the end of September 2004.Vaccination schedule Vaccinations with SYT-SSX B peptide were administered subcutaneously into the upper arm six times at 14-day intervals. In order to set up a dose-escalation trial, the patients were separated into the two groups. Each group included three patients. Those from group 1 received 0.1 mg and group 2 participants received 1.0 mg.
Vaccination schedule
Vaccinations with SYT-SSX B peptide were administered subcutaneously into the upper arm six times at 14-day intervals. In order to set up a dose-escalation trial, the patients were separated into the two groups. Each group included three patients. Those from group 1 received 0.1 mg and group 2 participants received 1.0 mg.Delayed-type hypersensitivity (DTH) skin test Delayed-type hypersensitivity (DTH) skin test was performed at each vaccination. The peptide (10 μg) solution in physiological saline (0.1 ml) or physiological saline alone (0.1 ml) were separately injected intradermally into the forearm. A positive reaction was defined as a diameter of erythema of more than 4 mm, 48 hr after the injection.
Delayed-type hypersensitivity (DTH) skin test
Delayed-type hypersensitivity (DTH) skin test was performed at each vaccination. The peptide (10 μg) solution in physiological saline (0.1 ml) or physiological saline alone (0.1 ml) were separately injected intradermally into the forearm. A positive reaction was defined as a diameter of erythema of more than 4 mm, 48 hr after the injection.Toxicity evaluation Patients were examined closely for signs of toxicity during and after vaccination. Adverse events were recorded using the National Cancer Institute Common Toxicity Criteria (NCI-CTC).
Toxicity evaluation
Patients were examined closely for signs of toxicity during and after vaccination. Adverse events were recorded using the National Cancer Institute Common Toxicity Criteria (NCI-CTC).Clinical response evaluation Physical examinations and hematological examinations were monitored before and after each vaccination. Tumor size was evaluated by computed tomography (CT) scans before treatment, and again after three vaccinations, and then at the end of the study period. A complete response (CR) was defined as complete disappearance of all measurable diseases. A partial response (PR) was defined as a >= 50% decrease from the baseline in size of all measurable lesions (sum of products of maximal perpendicular diameters) lasting for a period of at least 4 weeks. Progressive disease (PD) was defined as an increase in the sum of the bi-dimensional measurements of all known disease sites by at least 25% or by the appearance of new lesions. No change (NC) was defined as the absence of matched criteria for CR, PR, or PD.
Clinical response evaluation
Physical examinations and hematological examinations were monitored before and after each vaccination. Tumor size was evaluated by computed tomography (CT) scans before treatment, and again after three vaccinations, and then at the end of the study period. A complete response (CR) was defined as complete disappearance of all measurable diseases. A partial response (PR) was defined as a >= 50% decrease from the baseline in size of all measurable lesions (sum of products of maximal perpendicular diameters) lasting for a period of at least 4 weeks. Progressive disease (PD) was defined as an increase in the sum of the bi-dimensional measurements of all known disease sites by at least 25% or by the appearance of new lesions. No change (NC) was defined as the absence of matched criteria for CR, PR, or PD.Tetramer staining HLA-A24/peptide tetramers (HLA-A24/B, HLA-A24/R49.2, and HLA-A24/HIV) were previously constructed [ 13 - 15 ]. Flowcytometric analysis was performed by taking peripheral blood mononuclear cells (PBMCs) from patients. PBMCs were taken at pre-vaccination and again one week after 1 st , 3 rd , and 6 th  vaccination. Cells were stained with PE-labeled tetramers at 37°C for 20 min and a FITC-conjugated anti-CD8 mAb (Becton Dickinson) at 4°C for 30 min. Analysis of stained PBMCs was performed using FACScan (Becton Dickinson) and CellQuest software (Becton Dickinson). The frequency of CTL precursors was calculated as the number of tetramer positive cells / the number of CD8 +  cells.
Tetramer staining
HLA-A24/peptide tetramers (HLA-A24/B, HLA-A24/R49.2, and HLA-A24/HIV) were previously constructed [ 13 - 15 ]. Flowcytometric analysis was performed by taking peripheral blood mononuclear cells (PBMCs) from patients. PBMCs were taken at pre-vaccination and again one week after 1 st , 3 rd , and 6 th  vaccination. Cells were stained with PE-labeled tetramers at 37°C for 20 min and a FITC-conjugated anti-CD8 mAb (Becton Dickinson) at 4°C for 30 min. Analysis of stained PBMCs was performed using FACScan (Becton Dickinson) and CellQuest software (Becton Dickinson). The frequency of CTL precursors was calculated as the number of tetramer positive cells / the number of CD8 +  cells.CTL induction Cytotoxic T lymphocytes (CTLs) were induced from the PBMCs of patients using SYT-SSX B peptides according to the method described before[ 13 , 14 ]. The cytotoxic activity was evaluated by 6-h  51 Cr release assay[ 13 ]. As target cells, synovial sarcoma cell lines (Fuji, HS-SY-II, and SW982), an erythroleukemia cell line (K562), and a T-B Lymphoblast hybrid transfected with HLA-A*2402 (T2-A*2402) were used. Fuji and HS-SY-II were both HLA-A24 and SYT-SSX positive lines. SW982 and K562 were both HLA-A24 and SYT-SSX negative lines used as controls. T2-A*2402 cells were used to determine peptide-specific cytotoxicity by pulsation with SYT-SSX B or HIV peptide before labeling. The stimulated CD8 +  T cells were mixed with the labeled target cells. After a 6-h incubation period at 37°C, the release of the  51 Cr label was measured by collecting the supernatant, followed by quantification in an automated gamma counter. The percentage of specific cytotoxicity was calculated as the percentage of specific  51 Cr release: [(experimental  51 Cr release - spontaneous  51 Cr release) / (maximum  51 Cr release - spontaneous  51 Cr release)] × 100. Maximum  51 Cr release was measured by incubating the labelled target cells with 2% NP-40, instead of the stimulated CD8 +  T cells. CTL induction was determined as successful when specific cyotoxicity of 10% or more was achieved on Fuji, HS-SY-II, and SYT-SSX B peptide-pulsed T2-A*2402 cells.
CTL induction
Cytotoxic T lymphocytes (CTLs) were induced from the PBMCs of patients using SYT-SSX B peptides according to the method described before[ 13 , 14 ]. The cytotoxic activity was evaluated by 6-h  51 Cr release assay[ 13 ]. As target cells, synovial sarcoma cell lines (Fuji, HS-SY-II, and SW982), an erythroleukemia cell line (K562), and a T-B Lymphoblast hybrid transfected with HLA-A*2402 (T2-A*2402) were used. Fuji and HS-SY-II were both HLA-A24 and SYT-SSX positive lines. SW982 and K562 were both HLA-A24 and SYT-SSX negative lines used as controls. T2-A*2402 cells were used to determine peptide-specific cytotoxicity by pulsation with SYT-SSX B or HIV peptide before labeling. The stimulated CD8 +  T cells were mixed with the labeled target cells. After a 6-h incubation period at 37°C, the release of the  51 Cr label was measured by collecting the supernatant, followed by quantification in an automated gamma counter. The percentage of specific cytotoxicity was calculated as the percentage of specific  51 Cr release: [(experimental  51 Cr release - spontaneous  51 Cr release) / (maximum  51 Cr release - spontaneous  51 Cr release)] × 100. Maximum  51 Cr release was measured by incubating the labelled target cells with 2% NP-40, instead of the stimulated CD8 +  T cells. CTL induction was determined as successful when specific cyotoxicity of 10% or more was achieved on Fuji, HS-SY-II, and SYT-SSX B peptide-pulsed T2-A*2402 cells.Results Patient profiles Six patients were enrolled in the study (Table  1 ). There were four men and two women with an average age of 34.7 years old (range 21–69 years). All patients had multiple metastatic lesions of the lung. A six-time vaccination schedule was completed in three patients, while the remaining three discontinued the vaccination regimen because of rapid disease progression. None of the treatment interruptions were due to the adverse effects of the vaccination. Table 1 Profiles of participants and clinical resoponses Patient no. Age Gender Dose of peptide (mg) Number of vaccination Adverse events DTH skin test Evaluation of CT images 1 69 M 0.1 1 - - PD 2 32 M 0.1 3 - - PD 3 21 F 0.1 6 - - PD 4 21 M 1.0 6 - - PD 5 39 F 1.0 6 Fever - NC 6 26 M 1.0 4 - - PD PD: progressive disease, NC: no change Safety and DTH skin test One patient (case 5) experienced slight fever (grade 1) after the first vaccination. No other adverse events were observed during vaccination. DTH skin test was performed at each vaccination and assessed 48 hr later. Reactions were determined as negative in all patients. Clinical response Recognized disease progression occurred in five out of six patients during the vaccination period (Table  1 , Fig.  1 ). In contrast, one patient (case 5) showed no such rapid progression (Table  1 , Fig.  2 ). These patients, except in case 1, had received systemic multidrug chemotherapy from one to four months before enrolling on this study. Figure 1 CT scan image of the lung of case 3 patient. A: Before vaccination (May 15, 2003). B: After the third vaccination (June 18, 2003). Rapid growth of the metastatic tumors and pleural effusion were seen. Figure 2 CT scan image of the lung of case 5 patient. A: Before vaccination (July 8, 2003). B: After the first vaccination (July 22, 2003). C: After the third vaccination (August 19, 2003). D: After the sixth vaccination (September 16, 2003). The metastatic tumors appeared to be dormant after the first vaccination. Tetramer analysis and CTL induction Peptide-specific immunological responses were evaluated in five patients by using HLA-A24/peptide tetramer analysis and in vitro CTL induction. As determined by flowcytometric analysis using HLA/peptide tetramers (Table  2 ), frequencies of CTLs specific for SYT-SSX B peptide were shown to be at background levels (less than 0.1%) in three patients prior to vaccination. Those frequencies increased after the first (cases 4 and 6) and the third vaccination (case 2) (Fig.  3 ). In the remaining two patients (cases 3 and 5), SYT-SSX B peptide-specific CTLs existed beyond the background levels before vaccination. Of these, B peptide-specific CTL frequencies increased slightly in case 2 upon a series of vaccinations. On the contrary, the CTL frequencies in peripheral blood decreased to the background level after the third vaccination in case 5, whose metastatic diseases remained stable during the vaccination period. For comparison, tetramers with irrelevant peptides were constructed as internal controls and utilized in four patients. Notably, CTL frequencies reacting to those irrelevant tetramers remained under the background level during the course of vaccinations in all patients. Table 2 HLA-A24/peptide tetramer analysis case Pre-vaccination After 1st vac. After 3rd vac. After 6th vac. 2 0.02/N.D 0.02/N.D 3.05 /N.D N.D 3 0.42 /0.02* 0.49 /0.02 0.52 /0.02 0.62 /0.01 4 0.06/0.01 0.41 /0.00 0.36 /0.01 0.47 /0.01 5 0.50 /0.06 0.52 /0.01 0.09/0.00 0.03/0.02 6 0.02/0.01 0.15 /0.01 0.08/0.00 N.D N.D: not determined, *B peptide tetramer / Control peptide tetramer HLA-A24/R49.2 pepitde tetramer was used as control in the case 3 patient. HLA-A24/HIV pepitde tetramer was used as control in the other patients. Figure 3 Frequency of CTLs analyzed by HLA-A24/peptide tetramers in the case 4 patient. Frequencies of each analysis were described in Table 2. Table  3  depicts the results of CTL induction by in vitro stimulations with SYT-SSX B peptide. Before vaccination, CTLs specific for SYT-SSX B peptide were successfully induced from one patient (case 2) who showed a high frequency of CTL precursors. After the first or third vaccination, CTLs were induced from four of five patients. Figure  4  represents the results of cytotoxicity assay. As shown, CTLs induced from the case 4 patient exhibited cytotoxic activities against T2-A*2402 cells pulsed with SYT-SSX B peptide, and synovial sarcoma cell lines expressing HLA-A24 and SYT-SSX (Fuji and HS-SY-II) in various effecter/target ratios examined. In contrast, the cytotoxity was less than 10% against T2-A*2402 cells without peptide pulsation, those pulsed with irrelevant HIV peptide, and tumor cells lacking HLA-A24 and SYT-SSX (SW982 and K562). These findings suggest that induction of peptide-specific immune responses in patients with synovial sarcoma, who received the SYT-SSX junction peptide vaccine. Table 3 Induction of peptide specific CTLs case Pre-vaccination After 1st vac. After 3rd vac. After 6th vac. 2 Failure Failure Success N.D 3 Success Success Success Failure 4 Failure Success Success N.D 5 Failure Success Failure Failure 6 Failure Failure Failure N.D N.D: not determined Figure 4 Cytotoxicity of CTLs induced from the case 4 patient. T2-P(-): T2-A*2402 cells without peptide pulsation, T2-B: T2-A*2402 cells pulsed with SYT-SSX B peptide, T2-HIV: T2-A*2402 cells pulsed with HIV peptide. Specific cytotoxicity was observed against T2-A*2402 cells pulsed with SYT-SSX B peptide, and synovial sarcoma cell lines expressing HLA-A24 and SYT-SSX (Fuji and HS-SY-II). E/T: Effecter cells/target cells ratio.
Results
Patient profiles Six patients were enrolled in the study (Table  1 ). There were four men and two women with an average age of 34.7 years old (range 21–69 years). All patients had multiple metastatic lesions of the lung. A six-time vaccination schedule was completed in three patients, while the remaining three discontinued the vaccination regimen because of rapid disease progression. None of the treatment interruptions were due to the adverse effects of the vaccination. Table 1 Profiles of participants and clinical resoponses Patient no. Age Gender Dose of peptide (mg) Number of vaccination Adverse events DTH skin test Evaluation of CT images 1 69 M 0.1 1 - - PD 2 32 M 0.1 3 - - PD 3 21 F 0.1 6 - - PD 4 21 M 1.0 6 - - PD 5 39 F 1.0 6 Fever - NC 6 26 M 1.0 4 - - PD PD: progressive disease, NC: no change
Patient profiles
Six patients were enrolled in the study (Table  1 ). There were four men and two women with an average age of 34.7 years old (range 21–69 years). All patients had multiple metastatic lesions of the lung. A six-time vaccination schedule was completed in three patients, while the remaining three discontinued the vaccination regimen because of rapid disease progression. None of the treatment interruptions were due to the adverse effects of the vaccination.Profiles of participants and clinical resoponsesPD: progressive disease, NC: no changeSafety and DTH skin test One patient (case 5) experienced slight fever (grade 1) after the first vaccination. No other adverse events were observed during vaccination. DTH skin test was performed at each vaccination and assessed 48 hr later. Reactions were determined as negative in all patients.
Safety and DTH skin test
One patient (case 5) experienced slight fever (grade 1) after the first vaccination. No other adverse events were observed during vaccination. DTH skin test was performed at each vaccination and assessed 48 hr later. Reactions were determined as negative in all patients.Clinical response Recognized disease progression occurred in five out of six patients during the vaccination period (Table  1 , Fig.  1 ). In contrast, one patient (case 5) showed no such rapid progression (Table  1 , Fig.  2 ). These patients, except in case 1, had received systemic multidrug chemotherapy from one to four months before enrolling on this study. Figure 1 CT scan image of the lung of case 3 patient. A: Before vaccination (May 15, 2003). B: After the third vaccination (June 18, 2003). Rapid growth of the metastatic tumors and pleural effusion were seen. Figure 2 CT scan image of the lung of case 5 patient. A: Before vaccination (July 8, 2003). B: After the first vaccination (July 22, 2003). C: After the third vaccination (August 19, 2003). D: After the sixth vaccination (September 16, 2003). The metastatic tumors appeared to be dormant after the first vaccination.
Clinical response
Recognized disease progression occurred in five out of six patients during the vaccination period (Table  1 , Fig.  1 ). In contrast, one patient (case 5) showed no such rapid progression (Table  1 , Fig.  2 ). These patients, except in case 1, had received systemic multidrug chemotherapy from one to four months before enrolling on this study.Tetramer analysis and CTL induction Peptide-specific immunological responses were evaluated in five patients by using HLA-A24/peptide tetramer analysis and in vitro CTL induction. As determined by flowcytometric analysis using HLA/peptide tetramers (Table  2 ), frequencies of CTLs specific for SYT-SSX B peptide were shown to be at background levels (less than 0.1%) in three patients prior to vaccination. Those frequencies increased after the first (cases 4 and 6) and the third vaccination (case 2) (Fig.  3 ). In the remaining two patients (cases 3 and 5), SYT-SSX B peptide-specific CTLs existed beyond the background levels before vaccination. Of these, B peptide-specific CTL frequencies increased slightly in case 2 upon a series of vaccinations. On the contrary, the CTL frequencies in peripheral blood decreased to the background level after the third vaccination in case 5, whose metastatic diseases remained stable during the vaccination period. For comparison, tetramers with irrelevant peptides were constructed as internal controls and utilized in four patients. Notably, CTL frequencies reacting to those irrelevant tetramers remained under the background level during the course of vaccinations in all patients. Table 2 HLA-A24/peptide tetramer analysis case Pre-vaccination After 1st vac. After 3rd vac. After 6th vac. 2 0.02/N.D 0.02/N.D 3.05 /N.D N.D 3 0.42 /0.02* 0.49 /0.02 0.52 /0.02 0.62 /0.01 4 0.06/0.01 0.41 /0.00 0.36 /0.01 0.47 /0.01 5 0.50 /0.06 0.52 /0.01 0.09/0.00 0.03/0.02 6 0.02/0.01 0.15 /0.01 0.08/0.00 N.D N.D: not determined, *B peptide tetramer / Control peptide tetramer HLA-A24/R49.2 pepitde tetramer was used as control in the case 3 patient. HLA-A24/HIV pepitde tetramer was used as control in the other patients. Figure 3 Frequency of CTLs analyzed by HLA-A24/peptide tetramers in the case 4 patient. Frequencies of each analysis were described in Table 2. Table  3  depicts the results of CTL induction by in vitro stimulations with SYT-SSX B peptide. Before vaccination, CTLs specific for SYT-SSX B peptide were successfully induced from one patient (case 2) who showed a high frequency of CTL precursors. After the first or third vaccination, CTLs were induced from four of five patients. Figure  4  represents the results of cytotoxicity assay. As shown, CTLs induced from the case 4 patient exhibited cytotoxic activities against T2-A*2402 cells pulsed with SYT-SSX B peptide, and synovial sarcoma cell lines expressing HLA-A24 and SYT-SSX (Fuji and HS-SY-II) in various effecter/target ratios examined. In contrast, the cytotoxity was less than 10% against T2-A*2402 cells without peptide pulsation, those pulsed with irrelevant HIV peptide, and tumor cells lacking HLA-A24 and SYT-SSX (SW982 and K562). These findings suggest that induction of peptide-specific immune responses in patients with synovial sarcoma, who received the SYT-SSX junction peptide vaccine. Table 3 Induction of peptide specific CTLs case Pre-vaccination After 1st vac. After 3rd vac. After 6th vac. 2 Failure Failure Success N.D 3 Success Success Success Failure 4 Failure Success Success N.D 5 Failure Success Failure Failure 6 Failure Failure Failure N.D N.D: not determined Figure 4 Cytotoxicity of CTLs induced from the case 4 patient. T2-P(-): T2-A*2402 cells without peptide pulsation, T2-B: T2-A*2402 cells pulsed with SYT-SSX B peptide, T2-HIV: T2-A*2402 cells pulsed with HIV peptide. Specific cytotoxicity was observed against T2-A*2402 cells pulsed with SYT-SSX B peptide, and synovial sarcoma cell lines expressing HLA-A24 and SYT-SSX (Fuji and HS-SY-II). E/T: Effecter cells/target cells ratio.
Tetramer analysis and CTL induction
Peptide-specific immunological responses were evaluated in five patients by using HLA-A24/peptide tetramer analysis and in vitro CTL induction. As determined by flowcytometric analysis using HLA/peptide tetramers (Table  2 ), frequencies of CTLs specific for SYT-SSX B peptide were shown to be at background levels (less than 0.1%) in three patients prior to vaccination. Those frequencies increased after the first (cases 4 and 6) and the third vaccination (case 2) (Fig.  3 ). In the remaining two patients (cases 3 and 5), SYT-SSX B peptide-specific CTLs existed beyond the background levels before vaccination. Of these, B peptide-specific CTL frequencies increased slightly in case 2 upon a series of vaccinations. On the contrary, the CTL frequencies in peripheral blood decreased to the background level after the third vaccination in case 5, whose metastatic diseases remained stable during the vaccination period. For comparison, tetramers with irrelevant peptides were constructed as internal controls and utilized in four patients. Notably, CTL frequencies reacting to those irrelevant tetramers remained under the background level during the course of vaccinations in all patients.HLA-A24/peptide tetramer analysis3.050.420.490.520.620.410.360.470.500.520.15N.D: not determined, *B peptide tetramer / Control peptide tetramerHLA-A24/R49.2 pepitde tetramer was used as control in the case 3 patient.HLA-A24/HIV pepitde tetramer was used as control in the other patients.Table  3  depicts the results of CTL induction by in vitro stimulations with SYT-SSX B peptide. Before vaccination, CTLs specific for SYT-SSX B peptide were successfully induced from one patient (case 2) who showed a high frequency of CTL precursors. After the first or third vaccination, CTLs were induced from four of five patients. Figure  4  represents the results of cytotoxicity assay. As shown, CTLs induced from the case 4 patient exhibited cytotoxic activities against T2-A*2402 cells pulsed with SYT-SSX B peptide, and synovial sarcoma cell lines expressing HLA-A24 and SYT-SSX (Fuji and HS-SY-II) in various effecter/target ratios examined. In contrast, the cytotoxity was less than 10% against T2-A*2402 cells without peptide pulsation, those pulsed with irrelevant HIV peptide, and tumor cells lacking HLA-A24 and SYT-SSX (SW982 and K562). These findings suggest that induction of peptide-specific immune responses in patients with synovial sarcoma, who received the SYT-SSX junction peptide vaccine.Induction of peptide specific CTLsSuccessSuccessSuccessSuccessSuccessSuccessSuccessN.D: not determinedDiscussion The present study was designed to evaluate the in vivo immunological property of a 9-mer SYT-SSX junction peptide in patients with disseminated synovial sarcoma. A total of 16 vaccinations of the peptide in six patients revealed (i) no serious adverse effects in any case, (ii) suppression of tumor progression in one patient, (iii) increases in the frequency of peptide-specific CTLs in three patients and a decrease in one patient, and (iv) successful induction of peptide-specific CTLs from four patients. These findings suggest that the SYT-SSX junction peptide is safe to use as a vaccine and also has the property to evoke in vivo immunological responses. With respect to the clinical efficacy, none of the enrolled patients showed signs of tumor remission. However, in the case 5 patient, tumors showed some dormancy during the vaccination period, in comparison to the other five patients in whom tumors grew rapidly from the early phase of the regimen. The case 5 patient had received one cycle of systemic chemotherapy four months before enrollment, making the possibility of residual chemotherapeutic efficacy unlikely. The other five patients, except in case 1, had received systemic chemotherapy one to two months before perticipation. Notably, frequency of circulating peptide-specific CTLs decreased in the case 5 patient, while CTL frequencies remained unchanged or increased significantly in the other four patients examined. Decrease in circulating CTLs in the case 5 patient may have resulted from accumulation of CTLs at the tumor sites, although biopsy of the tumors was not performed. Vaccination trials of fusion gene-derived peptides have been reported with BCR-ABL in 12 patients with chronic myelogenous leukemia[ 16 ], EWS-FLI1 in 12 patients with Ewing's sarcoma[ 17 ], and PAX3-FKHR in four patients with alveolar rhabdomyosarcoma[ 17 ]. In addition, Matsuzaki et al.[ 18 ] reported a case of a synovial sarcoma patient who were treated with autologous dendritic cells pulsed with a mixture of SYT-SSX junction peptides (8–16 mer). In these studies, tumor remission was noted only in one patient with Ewing's sarcoma where IL-2 was concomitantly administered. These findings together with the results of our present trial have indicated the limited therapeutic efficacy of natural junction peptides. In this regard, we discovered improved in vitro immunogenicity of the SYT-SSX junction peptide by the substitution of an HLA-A24 anchor residue (position 9)[ 14 ]. Clinical study of this anchor-substituted peptide is currently underway. Besides modification of the peptide itself, concurrent use of adjuvants and cytokines, and adoptive T cell or/and dendritic transfer should further improve the therapeutic efficacy[ 8 , 19 ]. Also, it is important to determine the appropriate timing of vaccination and proper endpoints of clinical studies. To monitor the immunological responses, we used HLA/peptide tetramer and in vitro CTL induction. Other monitoring procedures such as ELISPOT assay should provide further information. Nevertheless, the use of an internal control tetramer with HIV peptide added strength in this present comparative analysis. As shown in Table  2  and Fig.  4 , reactivity of circulating T cells to HIV peptide tetramer remained under the background level throughout the vaccination period. Due to the rarity and highly malignant nature of synovial sarcoma, three out of six patients failed to complete the six-time vaccination regimen. Such difficulty in continuation of vaccination has also been found in a trial of patients with Ewing's sarcoma and alveolar rhabdomyosarcoma[ 17 ]. Another limitation in the current study is lack of analysis on immunological significance of SYT-SSX variants (SYT-SSX1 and SYT-SSX2). This question should be addressed in a larger scale analysis.
Discussion
The present study was designed to evaluate the in vivo immunological property of a 9-mer SYT-SSX junction peptide in patients with disseminated synovial sarcoma. A total of 16 vaccinations of the peptide in six patients revealed (i) no serious adverse effects in any case, (ii) suppression of tumor progression in one patient, (iii) increases in the frequency of peptide-specific CTLs in three patients and a decrease in one patient, and (iv) successful induction of peptide-specific CTLs from four patients. These findings suggest that the SYT-SSX junction peptide is safe to use as a vaccine and also has the property to evoke in vivo immunological responses.With respect to the clinical efficacy, none of the enrolled patients showed signs of tumor remission. However, in the case 5 patient, tumors showed some dormancy during the vaccination period, in comparison to the other five patients in whom tumors grew rapidly from the early phase of the regimen. The case 5 patient had received one cycle of systemic chemotherapy four months before enrollment, making the possibility of residual chemotherapeutic efficacy unlikely. The other five patients, except in case 1, had received systemic chemotherapy one to two months before perticipation. Notably, frequency of circulating peptide-specific CTLs decreased in the case 5 patient, while CTL frequencies remained unchanged or increased significantly in the other four patients examined. Decrease in circulating CTLs in the case 5 patient may have resulted from accumulation of CTLs at the tumor sites, although biopsy of the tumors was not performed.Vaccination trials of fusion gene-derived peptides have been reported with BCR-ABL in 12 patients with chronic myelogenous leukemia[ 16 ], EWS-FLI1 in 12 patients with Ewing's sarcoma[ 17 ], and PAX3-FKHR in four patients with alveolar rhabdomyosarcoma[ 17 ]. In addition, Matsuzaki et al.[ 18 ] reported a case of a synovial sarcoma patient who were treated with autologous dendritic cells pulsed with a mixture of SYT-SSX junction peptides (8–16 mer). In these studies, tumor remission was noted only in one patient with Ewing's sarcoma where IL-2 was concomitantly administered. These findings together with the results of our present trial have indicated the limited therapeutic efficacy of natural junction peptides. In this regard, we discovered improved in vitro immunogenicity of the SYT-SSX junction peptide by the substitution of an HLA-A24 anchor residue (position 9)[ 14 ]. Clinical study of this anchor-substituted peptide is currently underway. Besides modification of the peptide itself, concurrent use of adjuvants and cytokines, and adoptive T cell or/and dendritic transfer should further improve the therapeutic efficacy[ 8 , 19 ]. Also, it is important to determine the appropriate timing of vaccination and proper endpoints of clinical studies.To monitor the immunological responses, we used HLA/peptide tetramer and in vitro CTL induction. Other monitoring procedures such as ELISPOT assay should provide further information. Nevertheless, the use of an internal control tetramer with HIV peptide added strength in this present comparative analysis. As shown in Table  2  and Fig.  4 , reactivity of circulating T cells to HIV peptide tetramer remained under the background level throughout the vaccination period.Due to the rarity and highly malignant nature of synovial sarcoma, three out of six patients failed to complete the six-time vaccination regimen. Such difficulty in continuation of vaccination has also been found in a trial of patients with Ewing's sarcoma and alveolar rhabdomyosarcoma[ 17 ]. Another limitation in the current study is lack of analysis on immunological significance of SYT-SSX variants (SYT-SSX1 and SYT-SSX2). This question should be addressed in a larger scale analysis.Conclusions This is the first clinical trial of SYT-SSX fusion gene-derived peptide in patients with synovial sarcoma. The present trial demonstrated the safety and immunogenic property of the peptide. Modification of both the peptide itself and the related protocol is required to further improve the therapeutic efficacy.
Conclusions
This is the first clinical trial of SYT-SSX fusion gene-derived peptide in patients with synovial sarcoma. The present trial demonstrated the safety and immunogenic property of the peptide. Modification of both the peptide itself and the related protocol is required to further improve the therapeutic efficacy.Abbreviations CTL; cytotoxic T lymphocyte, HLA; human leukocyte antigen, MHC; major histocompatibility complex, PBMC; peripheral blood mononuclear cell
Abbreviations
CTL; cytotoxic T lymphocyte, HLA; human leukocyte antigen, MHC; major histocompatibility complex, PBMC; peripheral blood mononuclear cellCompeting interests The author(s) declare that they have no competing interests.
Competing interests
The author(s) declare that they have no competing interests.Authors' contributions SK1 and YS carried out vaccinations and clinical evaluations. KI, TT, SK1,2, HA and KS carried out monitoring procedures. TW, SN, HS, HI, TT, HH, TI, ST, NS, and TY made substantial contributions to the design of the study.
Authors' contributions
SK1 and YS carried out vaccinations and clinical evaluations. KI, TT, SK1,2, HA and KS carried out monitoring procedures. TW, SN, HS, HI, TT, HH, TI, ST, NS, and TY made substantial contributions to the design of the study.


In response to the Blackburn and Rowley essay on the President's Council on Bioethics, several thought-provoking opinions on ethical challenges in biomedical research are expressed by prominent stakeholders

In the very thoughtful essay “Reason as Our Guide” by Drs.  Elizabeth Blackburn and Janet Rowley (2004) , the authors highlight a key concern with the reports published by the President's Council on Bioethics—the lack of credible scientific information being passed on to policy makers.Blackburn and Rowley point out many areas of the report “Monitoring Stem Cell Research” that needed correction from a scientific standpoint. While it is impossible to include every suggestion in a report that seeks to draw consensus from a large panel of members, in a heated, political debate like that surrounding embryonic stem cell research and therapeutic cloning, providing the most accurate and complete scientific information to policy makers is crucial. Unfortunately, with the recent dismissal of Dr. Blackburn from the Council, there will now be one less voice for scientific research and for the potential the research holds for curing disease and alleviating the suffering of millions.Speaking for the Coalition for the Advancement of Medical Research, our concern is not only the small number of researchers on the Council and lack of complete scientific data being shared with policy makers, but the absence of patient representation on the Council itself. With the exception of public comment periods, patient organizations have no voice in the work of the Council as it discusses issues that profoundly impact them. Now, with one less member standing up for research and thus patients, our concern grows even stronger.The Blackburn and Rowley essay also correctly points out that there is more published work on adult stem cell research because of a “paucity of funding for research using embryonic stem cells.” Despite this lack of federal and private funding, advances continue to be made—but just think of the advances we could have had if only there were a supportive federal policy that encouraged embryonic stem cell research instead of stifling it. We hope—in light of scientific advances made over the past several years and the strong support of the scientific community, including the National Institutes of Health, the Health and Human Services Department, and the National Academy of Sciences—that the President will reevaluate the current federal policy for stem cell research and consider easing the restrictions.We commend Drs. Blackburn and Rowley for trying to set the record straight in their essay, and applaud their efforts to stand up for medical research, which has the potential to benefit us all.


Background All living organisms emit spontaneous low-level bioluminescence, which can be increased in response to stress. Methods for imaging this ultra-weak luminescence have previously been limited by the sensitivity of the detection systems used.
Background
All living organisms emit spontaneous low-level bioluminescence, which can be increased in response to stress. Methods for imaging this ultra-weak luminescence have previously been limited by the sensitivity of the detection systems used.Results We developed a novel configuration of a cooled charge-coupled device (CCD) for 2-dimensional imaging of light emission from biological material. In this study, we imaged photon emission from plant leaves. The equipment allowed short integration times for image acquisition, providing high resolution spatial and temporal information on bioluminescence. We were able to carry out time course imaging of both delayed chlorophyll fluorescence from whole leaves, and of low level wound-induced luminescence that we showed to be localised to sites of tissue damage. We found that wound-induced luminescence was chlorophyll-dependent and was enhanced at higher temperatures.
Results
We developed a novel configuration of a cooled charge-coupled device (CCD) for 2-dimensional imaging of light emission from biological material. In this study, we imaged photon emission from plant leaves. The equipment allowed short integration times for image acquisition, providing high resolution spatial and temporal information on bioluminescence. We were able to carry out time course imaging of both delayed chlorophyll fluorescence from whole leaves, and of low level wound-induced luminescence that we showed to be localised to sites of tissue damage. We found that wound-induced luminescence was chlorophyll-dependent and was enhanced at higher temperatures.Conclusions The data gathered on plant bioluminescence illustrate that the equipment described here represents an improvement in 2-dimensional luminescence imaging technology. Using this system, we identify chlorophyll as the origin of wound-induced luminescence from leaves.
Conclusions
The data gathered on plant bioluminescence illustrate that the equipment described here represents an improvement in 2-dimensional luminescence imaging technology. Using this system, we identify chlorophyll as the origin of wound-induced luminescence from leaves.

Background It is well documented that essentially all living systems spontaneously generate and emit very low levels of light (reviewed in [ 1 ]). This ultra-weak bioluminescence is generally characterized by emission of photons (sometimes termed "biophotons") at an intensity less than 10 -14  W.cm -2  (< 1000 photons.sec -1 .cm -2 ). This is in contrast to the more widely known bioluminescence from animals that visibly glow, such as certain species of jellyfish, fireflies and beetles, which employ fluorescent proteins and/or luciferase enzymes to catalyse reactions that result in chemiluminescence. Ultra-weak bioluminescence is generally considered to result from oxidative chemistry occurring within cells [ 1 , 2 ], though in most cases, the source of observed emissions has not been identified. Nevertheless, changes in emission levels in response to stress – particularly oxidative stress – have been observed in many systems, including bacteria [ 3 ], plants ( e.g . [ 2 , 4 - 6 ]) and animals [ 7 , 8 ]. For this reason, measurement of ultra-weak bioluminescence may be a useful non-invasive technique to monitor rapid perturbations to cellular activity and for early detection of diseased and damaged cells. In plants, increases in spontaneous low-level luminescence have been observed in response to pathogen infection [ 5 ], salt stress [ 9 ], osmotic stress [ 10 ] and mechanical damage or wounding [ 2 , 4 , 6 , 11 ]. It is suggested that luminescence is produced by singlet oxygen and excited carbonyl species generated as a result of lipid peroxidation reactions [ 2 , 11 ]. Lipid peroxidation in wounded and pathogen-infected plant tissues is a common consequence of the generation of ROS, which also act as signals to induce plant defence responses [ 12 ]. Quantitative measurements of such ultra-weak photon emissions are normally obtained using sensitive photomultiplier tubes as photon counting devices. However, more recently, 2-dimensional photomultiplier tubes and cooled charge-coupled device (CCD) cameras have also been employed as a means of imaging the spatial distribution of light emission from diseased and damaged plant tissues [ 2 - 6 , 9 ]. The clearest images were obtained by Chen  et al ., [ 2 ], using a micro-channel plate coupled to a cooled CCD to image light captured through a lens. However, the acquisition time required for what is a relatively weak signal, was 1 hour, preventing a detailed temporal investigation of stress-induced bioluminescence. Here, we present a novel configuration of a cooled CCD that enables high sensitivity, high resolution 2-dimensional imaging with short integration times. We demonstrate the utility of the system to image and quantify delayed chlorophyll fluorescence and wound-induced luminescence from plant leaves.
Background
It is well documented that essentially all living systems spontaneously generate and emit very low levels of light (reviewed in [ 1 ]). This ultra-weak bioluminescence is generally characterized by emission of photons (sometimes termed "biophotons") at an intensity less than 10 -14  W.cm -2  (< 1000 photons.sec -1 .cm -2 ). This is in contrast to the more widely known bioluminescence from animals that visibly glow, such as certain species of jellyfish, fireflies and beetles, which employ fluorescent proteins and/or luciferase enzymes to catalyse reactions that result in chemiluminescence. Ultra-weak bioluminescence is generally considered to result from oxidative chemistry occurring within cells [ 1 , 2 ], though in most cases, the source of observed emissions has not been identified. Nevertheless, changes in emission levels in response to stress – particularly oxidative stress – have been observed in many systems, including bacteria [ 3 ], plants ( e.g . [ 2 , 4 - 6 ]) and animals [ 7 , 8 ]. For this reason, measurement of ultra-weak bioluminescence may be a useful non-invasive technique to monitor rapid perturbations to cellular activity and for early detection of diseased and damaged cells.e.gIn plants, increases in spontaneous low-level luminescence have been observed in response to pathogen infection [ 5 ], salt stress [ 9 ], osmotic stress [ 10 ] and mechanical damage or wounding [ 2 , 4 , 6 , 11 ]. It is suggested that luminescence is produced by singlet oxygen and excited carbonyl species generated as a result of lipid peroxidation reactions [ 2 , 11 ]. Lipid peroxidation in wounded and pathogen-infected plant tissues is a common consequence of the generation of ROS, which also act as signals to induce plant defence responses [ 12 ].Quantitative measurements of such ultra-weak photon emissions are normally obtained using sensitive photomultiplier tubes as photon counting devices. However, more recently, 2-dimensional photomultiplier tubes and cooled charge-coupled device (CCD) cameras have also been employed as a means of imaging the spatial distribution of light emission from diseased and damaged plant tissues [ 2 - 6 , 9 ]. The clearest images were obtained by Chen  et al ., [ 2 ], using a micro-channel plate coupled to a cooled CCD to image light captured through a lens. However, the acquisition time required for what is a relatively weak signal, was 1 hour, preventing a detailed temporal investigation of stress-induced bioluminescence.et alHere, we present a novel configuration of a cooled CCD that enables high sensitivity, high resolution 2-dimensional imaging with short integration times. We demonstrate the utility of the system to image and quantify delayed chlorophyll fluorescence and wound-induced luminescence from plant leaves.Results and Discussion Construction of a luminescence imager The experimental imaging set-up, shown in Figure  1 , essentially consists of a glass sample stage coupled to a cooled CCD with fibre optics. This configuration enables maximal capture of light emitted from one side of a flat sample placed on the stage by avoiding the inevitable light dispersal associated with lens-based systems. The system is useful for imaging bioluminescence from flat samples such as plant leaves or cells grown transparent culture vessels or on solid supports such as microscope slides or membranes. By analysing the data captured from the CCD, it is possible to estimate the number of photons detected per unit area over a very wide dynamic range. We routinely measured emission levels in the region of 1000 photons/sec/mm 2  from wounded leaves (see below). In addition to its high sensitivity, the imager is significantly less expensive than equipment based on photomultiplier tubes. Imaging delayed chlorophyll fluorescence Delayed chlorophyll fluorescence is a well known phenomenon that is the result of emission of photons from excited chlorophyll molecules following transfer of leaves to the dark. It is generally measured using devices such as photomultiplier tubes and in principle, can be detected by existing lens-based CCD imaging systems. We first tested the spatial and temporal resolution of our system by imaging delayed chlorophyll fluorescence from leaves of  Arabidopsis thaliana  and  Tradescantia albiflora  (Figure  2 ). The image of a leaf from a variegated  T. albiflora  plant clearly indicates a dependence on chlorophyll for the emission to occur, since white, non-pigmented regions of the leaf do not emit light (compare Figure  2B  and  2C ). Bright images could be obtained with relatively short exposure times of 1–5 minutes. Delayed chlorophyll fluorescence rapidly subsided between 5 and 10 minutes after transfer to dark. The absolute level of delayed chlorophyll fluorescence in  Arabidopsis  leaves varied greatly, generally appearing to be lower later in the day. Spatial and temporal resolution of ultra-weak luminescence in wounded leaves Delayed chlorophyll fluorescence produces relatively strong light emission compared to typical levels of ultra-weak bioluminescence. To test the sensitivity of our equipment, we wanted to try to image these lower level emissions. Several previous reports had indicated that mechanical wounding increases ultra-weak luminescence from plant leaves, but studies on the temporal and spatial nature of this phenomenon are limited. We used a haemostat to inflict crushing wounds on  Arabidopsis  leaves and imaged them over a time course by making successive 5-minute exposures. In many experiments, no wound-induced luminescence could be seen in the first 5 minutes after transfer to the imager, since it was masked by delayed chlorophyll fluorescence. After this initial period however, photon emission was much more intense around the wounded tissue. In other experiments, where delayed chlorophyll fluorescence was lower, signals could be seen even in the first exposure. Results from such an experiment are shown in Figure  3  and in an accompanying extended animation (Additional File  1 ). In the first image, which includes an unwounded control leaf and two leaves each wounded twice across the lamina, striking wound-induced luminescence can be seen above a general background of delayed chlorophyll fluorescence (Figure  3A ). In the two subsequent 5 min exposures, chlorophyll fluorescence has subsided, whilst the wound-induced luminescence remains high (Figure  3B,3C ). The serrated pattern of the haemostat surface is clearly reflected in the pattern of luminescence from the leaf, with areas of greater damage exhibiting maximal photon emission. In general, we observed that induced luminescence is strongest immediately after wounding and then gradually decays over a period of an hour or more. Origin of wound-induced luminescence In order to understand more about the origin of the luminescence emitted by wounded leaves, we used coloured filters to investigate its spectral characteristics. The results are shown in Figure  4 . A red filter (LEE 182), which transmits visible light of wavelengths greater than 600 nm, was effectively transparent with respect to the light emitted from wound sites, whereas blue and green filters (LEE 183 and LEE 735), which absorb light in the region of 550 – 750 nm, caused a significant reduction in signal reaching the detector when placed between the wounded leaf and the sample stage. Interestingly, a different blue filter (LEE 118), which transmits light above 700 nm, did not significantly affect luminescence (Figure  4 ). These results suggest that light emitted from wounded  Arabidopsis  leaves has a wavelength between 700 and 750 nm,  i.e . the red region of the visible spectrum. This is consistent with typical fluorescence from chlorophyll (730 nm). To test whether chlorophyll might be the source of wound-induced photon emission, we used the carotenoid biosynthesis inhibitor, norflurazon, to generate photobleached leaves lacking chlorophyll [ 13 ]. Compared with equivalent leaves from control plants, these white leaves failed to luminesce following wounding, except in remaining chlorophyll-containing areas (Figure  5 ). Together, these results demonstrate that light emission from wounded leaves is chlorophyll-dependent. Since luminescence occurs in the dark, and extends for times well beyond delayed chlorophyll fluorescence, we conclude that excitation energy produced by some wound-induced process is transferred to chlorophyll and emitted as light. One possible source of such energy might be products of oxidative damage, such as excited triplet carbonyls produced during lipid peroxidation [ 14 ]. Temperature-dependence of wound-induced luminescence That delayed chlorophyll fluorescence exhibits temperature-dependence has been shown previously in several systems [ 15 ]. Since we determined that chlorophyll is the major source of light emission from wounded leaves, we investigated whether wound-induced luminescence might also be affected by temperature. Application of heat to the sample via a foil heater in the lid of the sample chamber significantly increased the intensity of luminescence over the tested temperature range. Without applied heat, the temperature of the sample stage was approximately 7°C, when only low levels of luminescence could be detected. Luminescence increased significantly upon activation of the heater, resulting in an increase in the temperature of the sample stage to something in the region of 20°C. This phenomenon is illustrated in Figure  6 , and in an accompanying extended animation (see Additional file  2 ). Furthermore, we found that heating could activate luminescence in leaves wounded up to 3 hours before imaging. For example, Figure  7  shows a series of images from an experiment in which leaves were wounded at different times before imaging. Without heating, only the leaf wounded immediately before imaging showed detectable wound-induced luminescence, and this emission decayed to very low levels within 20 min. After 45 mins, heating was applied to the sample chamber, and within 10 minutes, wounds applied immediately, 15 mins and 1 hour before the start of imaging were clearly visible, with a faint emission also detectable from a wound applied to the leaf 2 hours before the start of imaging (therefore the image shown here (t = 65) was captured over 3 hours after wounding this particular leaf). This phenomenon is reminiscent of the light emission measured during thermoluminometry [ 14 ]. Thermoluminescence is a chlorophyll-dependent light emission observed at high temperatures (maxima at 70–90°C and 120–140°C), which, like ultra-weak bioluminescence, is attributed to singlet oxygen and triplet carbonyls produced by lipid peroxidation during oxidative stress [ 14 ]. Coupled with the results above, these data suggest that temperature increases the rate of decay of excited triplet carbonyls, and that the energy released is transferred to chlorophyll and emitted as light. There may also be an additional temperature-dependent component in the rate of emission of light from excited chlorophyll, as observed for delayed fluorescence [ 15 ].
Results and Discussion
Construction of a luminescence imager The experimental imaging set-up, shown in Figure  1 , essentially consists of a glass sample stage coupled to a cooled CCD with fibre optics. This configuration enables maximal capture of light emitted from one side of a flat sample placed on the stage by avoiding the inevitable light dispersal associated with lens-based systems. The system is useful for imaging bioluminescence from flat samples such as plant leaves or cells grown transparent culture vessels or on solid supports such as microscope slides or membranes. By analysing the data captured from the CCD, it is possible to estimate the number of photons detected per unit area over a very wide dynamic range. We routinely measured emission levels in the region of 1000 photons/sec/mm 2  from wounded leaves (see below). In addition to its high sensitivity, the imager is significantly less expensive than equipment based on photomultiplier tubes.
Construction of a luminescence imager
The experimental imaging set-up, shown in Figure  1 , essentially consists of a glass sample stage coupled to a cooled CCD with fibre optics. This configuration enables maximal capture of light emitted from one side of a flat sample placed on the stage by avoiding the inevitable light dispersal associated with lens-based systems. The system is useful for imaging bioluminescence from flat samples such as plant leaves or cells grown transparent culture vessels or on solid supports such as microscope slides or membranes. By analysing the data captured from the CCD, it is possible to estimate the number of photons detected per unit area over a very wide dynamic range. We routinely measured emission levels in the region of 1000 photons/sec/mm 2  from wounded leaves (see below). In addition to its high sensitivity, the imager is significantly less expensive than equipment based on photomultiplier tubes.Imaging delayed chlorophyll fluorescence Delayed chlorophyll fluorescence is a well known phenomenon that is the result of emission of photons from excited chlorophyll molecules following transfer of leaves to the dark. It is generally measured using devices such as photomultiplier tubes and in principle, can be detected by existing lens-based CCD imaging systems. We first tested the spatial and temporal resolution of our system by imaging delayed chlorophyll fluorescence from leaves of  Arabidopsis thaliana  and  Tradescantia albiflora  (Figure  2 ). The image of a leaf from a variegated  T. albiflora  plant clearly indicates a dependence on chlorophyll for the emission to occur, since white, non-pigmented regions of the leaf do not emit light (compare Figure  2B  and  2C ). Bright images could be obtained with relatively short exposure times of 1–5 minutes. Delayed chlorophyll fluorescence rapidly subsided between 5 and 10 minutes after transfer to dark. The absolute level of delayed chlorophyll fluorescence in  Arabidopsis  leaves varied greatly, generally appearing to be lower later in the day.
Imaging delayed chlorophyll fluorescence
Delayed chlorophyll fluorescence is a well known phenomenon that is the result of emission of photons from excited chlorophyll molecules following transfer of leaves to the dark. It is generally measured using devices such as photomultiplier tubes and in principle, can be detected by existing lens-based CCD imaging systems. We first tested the spatial and temporal resolution of our system by imaging delayed chlorophyll fluorescence from leaves of  Arabidopsis thaliana  and  Tradescantia albiflora  (Figure  2 ). The image of a leaf from a variegated  T. albiflora  plant clearly indicates a dependence on chlorophyll for the emission to occur, since white, non-pigmented regions of the leaf do not emit light (compare Figure  2B  and  2C ). Bright images could be obtained with relatively short exposure times of 1–5 minutes. Delayed chlorophyll fluorescence rapidly subsided between 5 and 10 minutes after transfer to dark. The absolute level of delayed chlorophyll fluorescence in  Arabidopsis  leaves varied greatly, generally appearing to be lower later in the day.Arabidopsis thalianaTradescantia albifloraT. albifloraArabidopsisSpatial and temporal resolution of ultra-weak luminescence in wounded leaves Delayed chlorophyll fluorescence produces relatively strong light emission compared to typical levels of ultra-weak bioluminescence. To test the sensitivity of our equipment, we wanted to try to image these lower level emissions. Several previous reports had indicated that mechanical wounding increases ultra-weak luminescence from plant leaves, but studies on the temporal and spatial nature of this phenomenon are limited. We used a haemostat to inflict crushing wounds on  Arabidopsis  leaves and imaged them over a time course by making successive 5-minute exposures. In many experiments, no wound-induced luminescence could be seen in the first 5 minutes after transfer to the imager, since it was masked by delayed chlorophyll fluorescence. After this initial period however, photon emission was much more intense around the wounded tissue. In other experiments, where delayed chlorophyll fluorescence was lower, signals could be seen even in the first exposure. Results from such an experiment are shown in Figure  3  and in an accompanying extended animation (Additional File  1 ). In the first image, which includes an unwounded control leaf and two leaves each wounded twice across the lamina, striking wound-induced luminescence can be seen above a general background of delayed chlorophyll fluorescence (Figure  3A ). In the two subsequent 5 min exposures, chlorophyll fluorescence has subsided, whilst the wound-induced luminescence remains high (Figure  3B,3C ). The serrated pattern of the haemostat surface is clearly reflected in the pattern of luminescence from the leaf, with areas of greater damage exhibiting maximal photon emission. In general, we observed that induced luminescence is strongest immediately after wounding and then gradually decays over a period of an hour or more.
Spatial and temporal resolution of ultra-weak luminescence in wounded leaves
Delayed chlorophyll fluorescence produces relatively strong light emission compared to typical levels of ultra-weak bioluminescence. To test the sensitivity of our equipment, we wanted to try to image these lower level emissions. Several previous reports had indicated that mechanical wounding increases ultra-weak luminescence from plant leaves, but studies on the temporal and spatial nature of this phenomenon are limited. We used a haemostat to inflict crushing wounds on  Arabidopsis  leaves and imaged them over a time course by making successive 5-minute exposures. In many experiments, no wound-induced luminescence could be seen in the first 5 minutes after transfer to the imager, since it was masked by delayed chlorophyll fluorescence. After this initial period however, photon emission was much more intense around the wounded tissue. In other experiments, where delayed chlorophyll fluorescence was lower, signals could be seen even in the first exposure. Results from such an experiment are shown in Figure  3  and in an accompanying extended animation (Additional File  1 ). In the first image, which includes an unwounded control leaf and two leaves each wounded twice across the lamina, striking wound-induced luminescence can be seen above a general background of delayed chlorophyll fluorescence (Figure  3A ). In the two subsequent 5 min exposures, chlorophyll fluorescence has subsided, whilst the wound-induced luminescence remains high (Figure  3B,3C ). The serrated pattern of the haemostat surface is clearly reflected in the pattern of luminescence from the leaf, with areas of greater damage exhibiting maximal photon emission. In general, we observed that induced luminescence is strongest immediately after wounding and then gradually decays over a period of an hour or more.ArabidopsisOrigin of wound-induced luminescence In order to understand more about the origin of the luminescence emitted by wounded leaves, we used coloured filters to investigate its spectral characteristics. The results are shown in Figure  4 . A red filter (LEE 182), which transmits visible light of wavelengths greater than 600 nm, was effectively transparent with respect to the light emitted from wound sites, whereas blue and green filters (LEE 183 and LEE 735), which absorb light in the region of 550 – 750 nm, caused a significant reduction in signal reaching the detector when placed between the wounded leaf and the sample stage. Interestingly, a different blue filter (LEE 118), which transmits light above 700 nm, did not significantly affect luminescence (Figure  4 ). These results suggest that light emitted from wounded  Arabidopsis  leaves has a wavelength between 700 and 750 nm,  i.e . the red region of the visible spectrum. This is consistent with typical fluorescence from chlorophyll (730 nm). To test whether chlorophyll might be the source of wound-induced photon emission, we used the carotenoid biosynthesis inhibitor, norflurazon, to generate photobleached leaves lacking chlorophyll [ 13 ]. Compared with equivalent leaves from control plants, these white leaves failed to luminesce following wounding, except in remaining chlorophyll-containing areas (Figure  5 ). Together, these results demonstrate that light emission from wounded leaves is chlorophyll-dependent. Since luminescence occurs in the dark, and extends for times well beyond delayed chlorophyll fluorescence, we conclude that excitation energy produced by some wound-induced process is transferred to chlorophyll and emitted as light. One possible source of such energy might be products of oxidative damage, such as excited triplet carbonyls produced during lipid peroxidation [ 14 ].
Origin of wound-induced luminescence
In order to understand more about the origin of the luminescence emitted by wounded leaves, we used coloured filters to investigate its spectral characteristics. The results are shown in Figure  4 . A red filter (LEE 182), which transmits visible light of wavelengths greater than 600 nm, was effectively transparent with respect to the light emitted from wound sites, whereas blue and green filters (LEE 183 and LEE 735), which absorb light in the region of 550 – 750 nm, caused a significant reduction in signal reaching the detector when placed between the wounded leaf and the sample stage. Interestingly, a different blue filter (LEE 118), which transmits light above 700 nm, did not significantly affect luminescence (Figure  4 ). These results suggest that light emitted from wounded  Arabidopsis  leaves has a wavelength between 700 and 750 nm,  i.e . the red region of the visible spectrum. This is consistent with typical fluorescence from chlorophyll (730 nm). To test whether chlorophyll might be the source of wound-induced photon emission, we used the carotenoid biosynthesis inhibitor, norflurazon, to generate photobleached leaves lacking chlorophyll [ 13 ]. Compared with equivalent leaves from control plants, these white leaves failed to luminesce following wounding, except in remaining chlorophyll-containing areas (Figure  5 ). Together, these results demonstrate that light emission from wounded leaves is chlorophyll-dependent. Since luminescence occurs in the dark, and extends for times well beyond delayed chlorophyll fluorescence, we conclude that excitation energy produced by some wound-induced process is transferred to chlorophyll and emitted as light. One possible source of such energy might be products of oxidative damage, such as excited triplet carbonyls produced during lipid peroxidation [ 14 ].Arabidopsisi.eTemperature-dependence of wound-induced luminescence That delayed chlorophyll fluorescence exhibits temperature-dependence has been shown previously in several systems [ 15 ]. Since we determined that chlorophyll is the major source of light emission from wounded leaves, we investigated whether wound-induced luminescence might also be affected by temperature. Application of heat to the sample via a foil heater in the lid of the sample chamber significantly increased the intensity of luminescence over the tested temperature range. Without applied heat, the temperature of the sample stage was approximately 7°C, when only low levels of luminescence could be detected. Luminescence increased significantly upon activation of the heater, resulting in an increase in the temperature of the sample stage to something in the region of 20°C. This phenomenon is illustrated in Figure  6 , and in an accompanying extended animation (see Additional file  2 ). Furthermore, we found that heating could activate luminescence in leaves wounded up to 3 hours before imaging. For example, Figure  7  shows a series of images from an experiment in which leaves were wounded at different times before imaging. Without heating, only the leaf wounded immediately before imaging showed detectable wound-induced luminescence, and this emission decayed to very low levels within 20 min. After 45 mins, heating was applied to the sample chamber, and within 10 minutes, wounds applied immediately, 15 mins and 1 hour before the start of imaging were clearly visible, with a faint emission also detectable from a wound applied to the leaf 2 hours before the start of imaging (therefore the image shown here (t = 65) was captured over 3 hours after wounding this particular leaf). This phenomenon is reminiscent of the light emission measured during thermoluminometry [ 14 ]. Thermoluminescence is a chlorophyll-dependent light emission observed at high temperatures (maxima at 70–90°C and 120–140°C), which, like ultra-weak bioluminescence, is attributed to singlet oxygen and triplet carbonyls produced by lipid peroxidation during oxidative stress [ 14 ]. Coupled with the results above, these data suggest that temperature increases the rate of decay of excited triplet carbonyls, and that the energy released is transferred to chlorophyll and emitted as light. There may also be an additional temperature-dependent component in the rate of emission of light from excited chlorophyll, as observed for delayed fluorescence [ 15 ].
Temperature-dependence of wound-induced luminescence
That delayed chlorophyll fluorescence exhibits temperature-dependence has been shown previously in several systems [ 15 ]. Since we determined that chlorophyll is the major source of light emission from wounded leaves, we investigated whether wound-induced luminescence might also be affected by temperature. Application of heat to the sample via a foil heater in the lid of the sample chamber significantly increased the intensity of luminescence over the tested temperature range. Without applied heat, the temperature of the sample stage was approximately 7°C, when only low levels of luminescence could be detected. Luminescence increased significantly upon activation of the heater, resulting in an increase in the temperature of the sample stage to something in the region of 20°C. This phenomenon is illustrated in Figure  6 , and in an accompanying extended animation (see Additional file  2 ).Furthermore, we found that heating could activate luminescence in leaves wounded up to 3 hours before imaging. For example, Figure  7  shows a series of images from an experiment in which leaves were wounded at different times before imaging. Without heating, only the leaf wounded immediately before imaging showed detectable wound-induced luminescence, and this emission decayed to very low levels within 20 min. After 45 mins, heating was applied to the sample chamber, and within 10 minutes, wounds applied immediately, 15 mins and 1 hour before the start of imaging were clearly visible, with a faint emission also detectable from a wound applied to the leaf 2 hours before the start of imaging (therefore the image shown here (t = 65) was captured over 3 hours after wounding this particular leaf). This phenomenon is reminiscent of the light emission measured during thermoluminometry [ 14 ]. Thermoluminescence is a chlorophyll-dependent light emission observed at high temperatures (maxima at 70–90°C and 120–140°C), which, like ultra-weak bioluminescence, is attributed to singlet oxygen and triplet carbonyls produced by lipid peroxidation during oxidative stress [ 14 ]. Coupled with the results above, these data suggest that temperature increases the rate of decay of excited triplet carbonyls, and that the energy released is transferred to chlorophyll and emitted as light. There may also be an additional temperature-dependent component in the rate of emission of light from excited chlorophyll, as observed for delayed fluorescence [ 15 ].Conclusions The novel configuration of cooled CCD that we have used in this study to capture 2-dimensional images of plant leaves provides excellent temporal and spatial resolution of bioluminescence/chemiluminescence processes in biological materials, which are important markers of various forms of stress and disease. Using this system, we are able to define the origin of wound-induced luminescence from plant leaves as chlorophyll, and suggest that this arises via the temperature-dependent release of energy from excited triplet carbonyls produced by oxidative stress.
Conclusions
The novel configuration of cooled CCD that we have used in this study to capture 2-dimensional images of plant leaves provides excellent temporal and spatial resolution of bioluminescence/chemiluminescence processes in biological materials, which are important markers of various forms of stress and disease. Using this system, we are able to define the origin of wound-induced luminescence from plant leaves as chlorophyll, and suggest that this arises via the temperature-dependent release of energy from excited triplet carbonyls produced by oxidative stress.Methods Plant material Plants of  Arabidopsis thaliana , Columbia ecotype, were grown in soil in a glasshouse at 22°C with a 16-hour photoperiod. Wounding was performed by crushing leaves with a haemostat. To generate leaves lacking chlorophyll,  Arabidopsis  plants grown for 3 weeks under 16 h light/8 h dark, were watered with 0.1 mM norflurazon (Sigma Aldrich PS1044) and grown under constant illumination at 120 μmol/m 2 /sec. Under these conditions, newly emerging leaf tissue was white. Leaves with white regions were taken 15 days after the start of norflurazon treatment, and used for luminescence imaging. Two dimensional luminescence imaging apparatus The imager used is a two-dimensional imaging system based on a slow-scan 'scientific grade' Silicon Photo Area-Detector [SPAD] (Integrated SPAD Imaging System – PiXx*ell 1A, Biolumonics Ltd), which consists of a cooled ultra-sensitive area photo-detector within a stainless steel vacuum vessel and associated image acquisition electronics. The input image is coupled to the detector with optical fibres (numerical aperture = 1). The detector contains 222,336 pixels, and in the experiments presented here, ran at an operating temperature of -58°C. The combination of the proprietary low read-out noise electronics, cooling of the detector and the optical fibre coupling results in a sensitive imaging system capable of recording the faintest luminescences at high resolution, (40 μm to 200 μm depending on geometry of sample and integration time). The spectral absorption response of the SPAD ranges from the near UV to IR, thus including the entire visible spectrum up to the band-gap energy barrier of Silicon (1.1 eV)  i.e . at approx 1100 nm; however, for ultimate sensitivity the detector output is monochrome,  i.e . there is no wavelength selectivity in the present instrument. Further information is available on request from Michel Flor-Henry, Biolumonics Ltd. Filters Coloured filters were obtained from Lee Filters (Andover, UK). We used lighting effect filters LEE 182 – "light red," LEE 118 – "light blue," LEE 183 – "moonlight blue" and LEE 735 – "velvet green." Spectral characteristics of the filters can be viewed at  , though readers should note that the transmittance spectra provided with the filters covers the range 300–800 nm, rather than the more limited 400–700 nm spectra shown on the web site. Filters were placed between the leaf and the sample stage prior to imaging.
Methods
Plant material Plants of  Arabidopsis thaliana , Columbia ecotype, were grown in soil in a glasshouse at 22°C with a 16-hour photoperiod. Wounding was performed by crushing leaves with a haemostat. To generate leaves lacking chlorophyll,  Arabidopsis  plants grown for 3 weeks under 16 h light/8 h dark, were watered with 0.1 mM norflurazon (Sigma Aldrich PS1044) and grown under constant illumination at 120 μmol/m 2 /sec. Under these conditions, newly emerging leaf tissue was white. Leaves with white regions were taken 15 days after the start of norflurazon treatment, and used for luminescence imaging.
Plant material
Plants of  Arabidopsis thaliana , Columbia ecotype, were grown in soil in a glasshouse at 22°C with a 16-hour photoperiod. Wounding was performed by crushing leaves with a haemostat. To generate leaves lacking chlorophyll,  Arabidopsis  plants grown for 3 weeks under 16 h light/8 h dark, were watered with 0.1 mM norflurazon (Sigma Aldrich PS1044) and grown under constant illumination at 120 μmol/m 2 /sec. Under these conditions, newly emerging leaf tissue was white. Leaves with white regions were taken 15 days after the start of norflurazon treatment, and used for luminescence imaging.Arabidopsis thalianaArabidopsisTwo dimensional luminescence imaging apparatus The imager used is a two-dimensional imaging system based on a slow-scan 'scientific grade' Silicon Photo Area-Detector [SPAD] (Integrated SPAD Imaging System – PiXx*ell 1A, Biolumonics Ltd), which consists of a cooled ultra-sensitive area photo-detector within a stainless steel vacuum vessel and associated image acquisition electronics. The input image is coupled to the detector with optical fibres (numerical aperture = 1). The detector contains 222,336 pixels, and in the experiments presented here, ran at an operating temperature of -58°C. The combination of the proprietary low read-out noise electronics, cooling of the detector and the optical fibre coupling results in a sensitive imaging system capable of recording the faintest luminescences at high resolution, (40 μm to 200 μm depending on geometry of sample and integration time). The spectral absorption response of the SPAD ranges from the near UV to IR, thus including the entire visible spectrum up to the band-gap energy barrier of Silicon (1.1 eV)  i.e . at approx 1100 nm; however, for ultimate sensitivity the detector output is monochrome,  i.e . there is no wavelength selectivity in the present instrument. Further information is available on request from Michel Flor-Henry, Biolumonics Ltd.
Two dimensional luminescence imaging apparatus
The imager used is a two-dimensional imaging system based on a slow-scan 'scientific grade' Silicon Photo Area-Detector [SPAD] (Integrated SPAD Imaging System – PiXx*ell 1A, Biolumonics Ltd), which consists of a cooled ultra-sensitive area photo-detector within a stainless steel vacuum vessel and associated image acquisition electronics. The input image is coupled to the detector with optical fibres (numerical aperture = 1). The detector contains 222,336 pixels, and in the experiments presented here, ran at an operating temperature of -58°C. The combination of the proprietary low read-out noise electronics, cooling of the detector and the optical fibre coupling results in a sensitive imaging system capable of recording the faintest luminescences at high resolution, (40 μm to 200 μm depending on geometry of sample and integration time). The spectral absorption response of the SPAD ranges from the near UV to IR, thus including the entire visible spectrum up to the band-gap energy barrier of Silicon (1.1 eV)  i.e . at approx 1100 nm; however, for ultimate sensitivity the detector output is monochrome,  i.e . there is no wavelength selectivity in the present instrument. Further information is available on request from Michel Flor-Henry, Biolumonics Ltd.i.ei.eFilters Coloured filters were obtained from Lee Filters (Andover, UK). We used lighting effect filters LEE 182 – "light red," LEE 118 – "light blue," LEE 183 – "moonlight blue" and LEE 735 – "velvet green." Spectral characteristics of the filters can be viewed at  , though readers should note that the transmittance spectra provided with the filters covers the range 300–800 nm, rather than the more limited 400–700 nm spectra shown on the web site. Filters were placed between the leaf and the sample stage prior to imaging.
Filters
Coloured filters were obtained from Lee Filters (Andover, UK). We used lighting effect filters LEE 182 – "light red," LEE 118 – "light blue," LEE 183 – "moonlight blue" and LEE 735 – "velvet green." Spectral characteristics of the filters can be viewed at  , though readers should note that the transmittance spectra provided with the filters covers the range 300–800 nm, rather than the more limited 400–700 nm spectra shown on the web site. Filters were placed between the leaf and the sample stage prior to imaging.Authors' contributions MF-H designed the PiXx*ell 1A luminescence imager. MF-H and MRR conceived of the current study, participated in its design and coordination, carried out experiments and wrote the manuscript. MF-H performed data analysis. TCM performed the norflurazon experiment, and both TCM and GLdB participated in some experimental design and execution. All authors read and approved the final manuscript.
Authors' contributions
MF-H designed the PiXx*ell 1A luminescence imager. MF-H and MRR conceived of the current study, participated in its design and coordination, carried out experiments and wrote the manuscript. MF-H performed data analysis. TCM performed the norflurazon experiment, and both TCM and GLdB participated in some experimental design and execution. All authors read and approved the final manuscript.Supplementary Material Additional File 1 Animated gif file showing images from five sequential 5-minute exposures of one control leaf (bottom) and four wounded  Arabidopsis  leaves. Click here for file Additional File 2 Animated gif file showing images from seven sequential 5-minute exposures of two control leaves (bottom) and four wounded  Arabidopsis  leaves. Images were initially captured from samples on a cold imaging stage, but the heater in the lid of the sample stage was switched on when indicated. Click here for file
Supplementary Material
Additional File 1
Animated gif file showing images from five sequential 5-minute exposures of one control leaf (bottom) and four wounded  Arabidopsis  leaves.ArabidopsisClick here for fileAdditional File 2
Animated gif file showing images from seven sequential 5-minute exposures of two control leaves (bottom) and four wounded  Arabidopsis  leaves. Images were initially captured from samples on a cold imaging stage, but the heater in the lid of the sample stage was switched on when indicated.ArabidopsisClick here for file