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Coxsackievirus B3 is an important infectious agent of viral myocarditis, pancreatitis and aseptic meningitis, but there are no specific antiviral therapeutic reagents in clinical use. RNA interference-based technology has been developed to prevent the viral infection.
To evaluate the impact of RNA interference on viral replication, cytopathogenicity and animal survival, short hairpin RNAs targeting the viral 2B region (shRNA-2B) expressed by a recombinant vector (pGCL-2B) or a recombinant lentivirus (Lenti-2B) were tansfected in HeLa cells or transduced in mice infected with CVB3.
ShRNA-2B exhibited a significant effect on inhibition of viral production in HeLa cells. Furthermore, shRNA-2B improved mouse survival rate, reduced the viral tissues titers and attenuated tissue damage compared with those of the shRNA-NC treated control group. Lenti-2B displayed more effective role in inhibition of viral replication than pGCL-2B in vivo.
Coxsackievirus B3 2B is an effective target of gene silencing against coxsackievirus B3 infection, suggesting that shRNA-2B is a potential agent for further development into a treatment for enterviral diseases.
Keywords:Coxsackievirus; Myocarditis; shRNA; Lentivirus vector
Coxsackievirus B3 (CVB3) is among the most common and significant causative agents of heart muscle disease in human. CVB3 is a member of the genus Enterovirus, which belongs to the family Picornaviridae. The spectrum of disease caused by these viruses ranges from very mild to life-threatening [1]. In some cases, CVB3 may escape the intestinal tract to produce serious diseases, such as viral myocarditis [2], pancreatitis [3] and aseptic meningitis [4]. It is estimated that at least 50% of the cases of infection-caused heart diseases are attributable to CVB3 infection [5]. However, there is currently no available vaccine and no specific drug to eliminate virus infection.
The CVB3 genome is a single-stranded positive-sense RNA molecule of approximately 7.4 kb. It contains a 5'-untranslated region (5'-UTR), a single long open reading frame, 3'-UTR, and a poly(A) tail. The four capsid proteins (VP1-4) and seven nonstructural proteins (2A, 2B, 2C, 3A, 3B, 3C, 3D) are generated by viral protease-mediated process of the single polyprotein translated from the genomic RNA [6]. CVB3 replication induces a direct cytopathic effect (CPE) on infected cells and direct tissue damage in various animal models [7,8]. Therefore, it is considered that virus elimination at a specific target can be the key therapeutic strategy to treat or attenuate CVB3-related disease.
RNA interference (RNAi) is a mechanism that inhibits specific gene expression by using short double-stranded RNA (dsRNA), which is converted to small interfering RNA (siRNA) as the active agent. RNAi is now employed as a potential therapeutic method against pathogenic viruses [9,10]. It is reported that several human pathogens, including human immunodeficiency virus [11], the hepatitis B [12] and C [13] viruses, influenza A virus [14] and coxsackievirus [15], were inhibited or eliminated by RNAi.
In our previous study, we designed twelve siRNAs targeting distinct regions of the CVB3 genome, and investigated their antiviral activities in HeLa cell [16]. The most effective one is siRNA-2B, targeting viral RNA at 2B region (3753-3771), achieving a 90% reduction of CVB3 replication [17]. In this study, we developed a recombinant plasmid and a lentivirus vector that delivered the short hairpin RNAs (shRNAs) targeting 2B gene (3753-3771) in cell culture system and in an animal model to evaluate the impact of shRNA-2B on CVB3 replication, pathogenicity and survival in vitro and in vivo.
Cell culture, virus, transfection and infection
HeLa and 293T cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin. CVB3M strain was amplified in HeLa cells by infection. This strain was a kind gift from Dr S.A. Huber, University of Vermont, U.S.A. [18]. To assess the validity of shRNA-2B, HeLa cells were seeded into 12-well plates (1 × 105 cells/well) overnight. When reached 50-60% confluency, cells were washed with phosphate buffered saline (PBS) and transfected with plasmid or transduced with lentivirus. At 24 h post transfection or 48 h post transduction, cells were washed and infected with CVB3 (MOI = 0.01) for 1 h. The cells were then overlaid with complete medium and incubated at 37°C with 5% CO2. At different time points post infection, supernatants were collected to determine the virus titers by viral plaque assay. Experiments were carried out 3 times.
Recombinant plasmid and lentivirus generation
pHelper 1.0, pHelper 2.0 and pGCSIL-GFP plasmids were purchased from Shanghai GeneChem Co. Ltd. (Shanghai, China). To construct the recombinant vector, RNAi stem-loop DNA oligos containing the target sequences (GGACTATGTGGAACAGCTT) in the region of CVB3 2B were chemically synthesized, annealed and cloned into the AgeI/EcoRI-digested pGCSIL-GFP, thus generating the plasmid pGCL-2B. The lentiviral vector pGCSIL-GFP contained a U6 promoter to express continuously the shRNA and CMV promoter to express the green fluorescent protein (GFP), which could be used to detect the transfection efficiency of viral packaging and infection. Recombinant lentiviruses were generated by co-transfection of 293T cells with 20 μg pGCL-2B and packaging vectors (15 μg pHelper1.0, 10 μg pHelper2.0) using 100 μl of Lipofectamine 2000™ reagent according to the manufacturer's instructions (Invitrogen, USA). The recombinant lentivirus was designated Lenti-2B. The viral supernatant was harvested 48 h after transfection, passed through 0.45 μm filters and concentrated. The viral titer was determined by infecting 293T cells with serial dilutions of concentrated lentivirus, and then determining the GFP expression of infected cells by fluorescence microscopy 96 h after infection. Therefore, the titer is expressed as "transduction unit (TU)/ml". A scramble siRNA sequence (5'-UUCUCCGAACGUGUCACGU-3') was used to generate the non-silence control plasmid and lentivirus that were designated pGCL-NC and Lenti-NC.
Short hairpin RNAs treatment in vivo
6-week-old BALB/c male mice were injected via caudal vein with plasmid or lentivirus. Entranster™-in vivo transfection reagent (Engreen Biosystem Co, Ltd. China) was used to deliver the plasmids. Control mice were injected with pGCL-NC, Lenti-NC and minimum essential medium (MEM), respectively. After 24 h, all mice were inoculated intraperitoneally with a dose of 8 × 103 plaque-forming units (pfu)/mouse of CVB3 virus. Some mice were observed for survival time. Other mice were euthanized on days 3, 5 and 7 after infected with CVB3 (n = 10 per group). Experiments were carried out 3 times. The animal experiment has been performed with the approval of the ethics committee of Capital Institute of Pediatrics and followed internationally recognized guidelines.
Organ virus titers and histological analysis
Hearts and pancreases from mice were weighted, homogenized in 0.5 ml MEM, and centrifuged for 10 minutes at 1000 rpm. In the supernatants, infectious virus was analyzed by viral plaque assay, and the viral titer in infected tissue was expressed as pfu/g. To assess severity of myocarditis and pancreatitis, paraffin-embedded sections of tissues were stained with hematoxylin-eosin and examined histopathologically for evidence of inflammation and necrosis. The immunohistochemistry staining for CVB3 and GFP were performed using anti-coxsackievirus B3 monoclonal antibody (Chemicon International, USA) and anti-GFP monoclonal antibody (Chemicon International, USA) as the primary antibody following the procedure described previously [19].
Viral plaque assay
HeLa cells were seeded into six-well plates (4 × 105 cells/well) and incubated at 37°C with 5% CO2 for 48 h. When cell confluency reached approximately 90%, cells were washed with PBS to remove the fetal bovine serum and then overlaid with 200 μl of 1:10 diluted supernatant. The cells were incubated at 37°C for 60 min and the supernatants were removed, and washed with PBS. Finally, cells were overlaid with 2 ml of sterilized soft Bacto-agar/MEM (2 × MEM:1.5% Bacto-agar = 1:1). The cells were incubated at 37°C for 72 h, fixed with Carnoy's fixative for 30 min and then stained with 1% crystal violet. The plaques were counted and the viral pfu/ml was calculated [20]. Each sample was tested in triplicate.
All statistical analyses were performed using the SPSS 11.5 computer software program. Survival was analyzed using Log-rank (Mantel-Cox) method. The significance of variability among the experimental groups was determined by Mann–Whitney U test. All differences were considered statistically significant at P < 0.05.
Construction of recombinant plasmid and lentivirus expressing shRNA targeting CVB3
In this study, recombinant plasmid expressing the shRNA targeting 2B sequence was cloned into pGCSIL-GFP and named pGCL-2B. This plasmid was used to pack the infectious lentivirus particles by co-transfection of 293T cells with other packaging plasmids. The lentivirus was named Lenti-2B and its titer was determined by fluorescent microscopy. The titer of the recombinant lentiviruses was approximately 2 × 109 TU/ml.
Inhibition of CVB3 replication in HeLa cells
To evaluate the transfection efficacy of pGCL-2B, HeLa cells were transfected with pGCL-2B at a series of doses and the GFP-expressing cells were visualized through fluorescence microscopy after 24 h. The transfection rate of pGCL-2B was determined by the percent of the area of GFP-expressing cells versus that of whole HeLa cells in one field calculated using Anymicro DSS™ digital shoot system (Beijing Yutianshijiweiye Technology Development Co. Ltd., China). Figure 1A shows that 1.25 μg/well was the lowest dose of pGCL-2B to reach about 60% of cells expressing GFP and was less toxic to the cells. Higher the dose of plasmid used, more vacuolar change occurred in the cells. When HeLa cells were transfected with pGCL-2B of 6.25 μg/well, the cells died after 24 h. Subsequently, pGCL-2B of 1.25 μg/well was used to transfect HeLa cells, and GFP expressions detected at different time. After 48 h, GFP expression rate reached to the peak (Figure 1B). The transfection rate of control plasmid was similar to that of pGCL-2B. We also studied its antiviral efficacy as a function of time and dose in HeLa cells. Compared to the viral titer of pGCL-NC, an approximate 96% decrease in viral titer was detected in these cultures treated with pGCL-2B (1.25 μg/well) at 48 h post challenge. This protective effect was reduced after 60 h and 72 h (Figure 1C). Determination of CVB3 titers 48 h after infection showed a strong antiviral effect of the pGCL-2B transfection with titers reduced 95.83% at the plasmid dose of 1.25 μg/well and 96.46% at the plasmid dose of 1.75 μg/well (Figure 1D). Expectedly, HeLa cells pre-treated with a dose of 1.25 μg/well of pGCL-2B were efficiently protected against CVB3-induced cell lysis at 48 h after infection, while strong CPE occurred in other two groups (Figure 1E).
Figure 1. Evaluation of the antiviral effects of pGCL-2B in HeLa cells.A HeLa cells were pretreated with pGCL-2B at series of doses and the percent of GFP-expressing cells were counted through fluorescence microscopy after 24 h. The results of three independent experiments are expressed as mean ± standard deviation. B HeLa cells were transfected with pGCL-2B (1.25 μg/well), and the transfection rates were counted at different time points. The results of three independent experiments are expressed as mean ± standard deviation. C HeLa cells were transfected with pGCL-2B (1.25 μg/well) or pGCL-NC or MEM only (V), followed by CVB3 infection (MOI = 0.01). The viral titers were measured at 36, 48, 60, 72 h p.i. using supernatants of cell cultures by plaque-forming analysis and the results of three independent experiments are expressed as mean ± standard deviation. Significant differences between pGCL-2B treated (white bars) and pGCL-NC treated samples (striped bars) are indicated (*: P < 0.05) D HeLa cells were transfected with pGCL-2B at 0.75, 1.25 or 1.75 μg/well, respectively, or pGCL-NC or MEM only (V), followed by CVB3 infection (MOI = 0.01). The viral titers were measured at 48 h p.i. using supernatants of cell cultures by plaque-forming analysis and the results of three independent experiments are expressed as mean ± standard deviation. Significant differences between pGCL-2B treated (white bars) and pGCL-NC treated samples (striped bars) are indicated (*: P < 0.05) E HeLa cells were transfected with pGCL-2B (1.25 μg/well) or pGCL-NC or MEM only (Virus), followed by CVB3 infection (MOI = 0.01). After 48 h, GFP-expressing cells were visualized by fluorescence microscopy. LM: light microscopy, FM: fluorescence microscopy.
To evaluate the efficacy of Lenti-2B on coxsackieviral replication, Lenti-2B (1, 10, 100 TU/cell) or Lenti-NC were used to transduce HeLa cells respectively. After 48 h, cells were infected with CVB3 (MOI = 0.01), and then the culture supernatants were collected to determine the CVB3 titers by viral plaque analysis at 48 h post challenge. Lenti-2B at a dose of 10 TU/cell or 100 TU/cell achieved an approximately 84% and 90%, respectively, reduction of CVB3 titer compared with that of control cells treated with Lenti-NC. The antiviral effect was almost absent at the dose of 1 TU/cell because of the low transduction efficacy of the lentivirus (Figure 2A). Lenti-2B also exhibited the protective effect against CPE (Figure 2B). These results indicate that recombinant plasmid and lentivirus expressing shRNA-2B are highly efficient antiviral agents in vitro.
Figure 2. Evaluation of the antiviral effects of Lenti-2B in HeLa cells.A HeLa cells were transduced with Lenti-2B at 1, 10, 100 TU/ml, respectively, or Lenti-NC or MEM only (V), followed by CVB3 infection (MOI = 0.01). The viral titers were measured at 48 h p.i. using supernatants of cell cultures by plaque-forming analysis and the results of three independent experiments are expressed as mean ± standard deviation. Significant differences between Lenti-2B treated (white bars) and Lenti-NC treated samples (striped bars) are indicated (*: P < 0.05) B HeLa cells were transduced with Lenti-2B (10 TU/well) or Lenti-NC or MEM only (Virus), followed by CVB3 infection (MOI = 0.01). After 48 h, GFP-expressing cells were visualized by fluorescence microscopy. LM: light microscopy, FM: fluorescence microscopy.
Inhibition of CVB3 replication in coxsackievirus-induced myocarditis model
To reveal the effect of shRNA-2B on survival, virus replication, and tissue damage, pGCL-2B (20 μg/mouse, 30 μg/mouse or 40 μg/mouse) or Lenti-2B (1 × 107 TU/mouse, 5 × 107 TU/mouse or 1 × 108 TU/mouse) was injected into mice before infection with CVB3 virus. The control vector pGCL-NC or Lenti-NC was applied in the same way. Transfection of pGCL-NC or Lenti-NC had no effect on survival compared with the infected but non-treated animals. All mice died in 9 days after infected with CVB3. In contrast, in the group infected with 1 × 108 TU/mouse Lenti-2B, 50% mice were still alive at day 9 and 40% mice lived at 30 days after challenge with CVB3 (Figure 3A). The pGCL-2B group (40 μg/mouse) also showed a higher survival rate than that of the pGCL-NC control group (30% vs. 0 survival rate, P < 0.01) at day 9 post CVB3 infection (Figure 3B). However, transfection of pGCL-2B or Lenti-2B with lower doses had no difference on survival between treated group and control group. Both pGCL-2B and Lenti-2B improved significantly the life span of mice, suggesting that shRNA-2B could be the potential agent to reduce the mortality caused by viral myocarditis.
Figure 3. Evaluation of the antiviral effects of shRNA-2B on survival rate. BALB/c mice (n = 10) were injected with plasmid (40 μg/mouse) or lentivirus (1 × 108 TU/mouse) through a tail vein, 24 h, post injection, 8 × 103 pfu/mouse of CVB3 was used as the challenge.
To further determine the potency of Lenti-2B, mice were euthanized to directly compare the tissue virus titers by plaque-forming assay at days 3, 5 and 7 post CVB3 infection. The virus titers in the hearts and pancreases of mice infected with Lenti-2B were approximately 4-log10 lower than those in the control group (P < 0.01 versus Lenti-NC group for both organs, n = 5 in each group) (Figure 4). Virus titers were 1-log10 lower in the heart and 2-log10 lower in the pancreas of mice that received pGCL-2B than that in the respective control mice (Figure 5). Furthermore, the virus titer was 103-fold lower in the heart of Lenti-2B group than that of pGCL-2B group, suggesting that lentivirus vector expressed shRNA-2B more effectively than plasmid in vivo (P < 0.01).
Figure 4. Antiviral effects of Lenti-2B on the viral titers in the hearts (A) and pancreases (B) of mice infected with CVB3. BALB/c mice were pretreated with lentiviruses expressing shRNAs under the condition described in the legend to Figure 3, and euthanized at days, 3, 5 and 7 post infection with CVB3 (n = 5). The heart and pancreas tissues were weighted and made a 0.2% (w/v) homogenate in 0.5 ml MEM, followed by the determination of the viral titers. Histogram represents average values of experiments performed with samples obtained from five different mice. Error bars represent mean ± standard deviation. *P < 0.01.
Figure 5. Antiviral effects of pGCL-2B on the viral titers in the hearts (A) and pancreases (B) of mice infected with CVB3. BALB/c mice were pretreated with plasmid expressing shRNAs under the condition described in the legend to Figure 3, and euthanized at days 3, 5 and 7 after infection with CVB3 (n = 5). The heart and pancreas tissues were weighted and made a 0.2% (w/v) homogenate in 0.5 ml MEM, followed by the determination of the viral titers. Histogram represents average values of experiments performed with samples obtained from five different mice. Error bars represent mean ± standard deviation. *P < 0.01.
To confirm the protect effect of Lenti-2B on the hearts and pancreases of CVB3 infected mice, hematoxylin-eosin staining was used to detect inflammation and immunohistochemistry staining was used to detect the distribution of CVB3 viruses or GFP expression in the organs. No, or only slight, myocardial inflammation and CVB3 infection were revealed in the Lenti-2B group; however, there were large extent of necrosis, calcification and serious virus infection in the Lenti-NC group and non-shRNA-treated group (Figure 6).
Figure 6. Histology of myocardial inflammation (A) and pancreatic inflammation (B) at day 7 after CVB3 challenge. BALB/c mice were injected with Lenti-2B (1 × 108 TU/mouse) through the tail vein, at 24 h post injection, 8 × 103 pfu/mouse of CVB3 was used as the challenge, and then euthanized at day 7 post CVB3 infection. The four groups were injected with Lenti-2B or Lenti-NC or MEM but infected with CVB3 (V) or MEM only as normal control. Hematoxylin-eosin staining and immunohistochemical analysis of CVB3 or GFP were performed on the sections prepared from the hearts. Untreated Control shows the control section was treated without the first antibody in IHC staining. Green arrow shows a example of infiltration, red arrows show examples of necrosis and calcification, black arrows show examples of CVB3 infection, purple arrows show examples of GFP-expressing. Magnification, 200×.
Coxsackievirus infection can injure cell directly and damage various tissues, leading to viral heart disease [2], pancreatitis [3], or meningitis [4]. At present, there is no specific antiviral therapy available [21]. RNAi-based antiviral therapy has potential to degrade viral RNA and promote viral clearance [22]. In previous publications, RNAi was applied to inhibit coxsackievirus replication by siRNAs targeting viral 2A [23,24], 3D [25], and VP1 [15,25]. These synthetic siRNAs were tansfected into mammalian cells or delivered into mice via the tail vein by high-volume injections and protected cells or organs from virus-mediated injury. However, the degradation of siRNAs by intracellular nucleases and the method of delivery limited the effect in vivo. To overcome some limitations of siRNAs in antiviral applications, adeno-associated virus-delivered shRNA directed at the RNA polymerase of CVB3 was reported to significantly attenuate the cardiac dysfunction [26] and lentivirus-delivered shRNA against 2C was reported to improve survival rate in an animal mode [27]. It was suggested that shRNA viral vector could be used effectively to prevent coxsackievirus replication in mice.
In this study, RNA interference was employed to target the CVB3 protein 2B region (3753-3771), which is a highly conserved region in most enteroviruses and therefore is an attractive therapeutic target. Among twelve siRNAs we tested, siRNA-2B was proved most highly efficient in the inhibition of viral replication in HeLa cells. In subsequent animal experiments, lentivirus-delivered shRNA-2B was shown to improve the survival rate of 40% at 30 days after challenge. Furthermore, the viral titers in the mouse hearts and pancreas of the Lenti-2B group were significantly reduced compared with those of the control group. The biopsy of hearts and pancreas of mice injected with Lenti-2B also showed less inflammation, suggesting that shRNA-2B effectively inhibits viral replication and reduces the virus-induced myocarditis in the animal model. This study indicated that shRNA-2B can reduce CVB3 progeny production by cleavage of viral genomic RNA, which results in the suppression of viral particle assembly with intact CVB3 RNAs and hampering of the whole translation process of the viral polyprotein. On the other hand, it was reported that 2B protein plays a major role in suppressing apoptotic host cell response by manipulating intracellular Ca2+ homeostasis and, thereby, in extending the life span of the host cell [28]. However, this RNAi by shRNA-2B may enhance host cell death and subsequently limit CVB3 replication. Therefore, shRNA-2B inhibits CVB3 replication not only directly through RNAi but also indirectly through enhancing host cell death.
In recent years, many studies have reported successful inhibition of viral replication in cultured cells or in murine models using transient transfection of synthetic siRNA or plasmid expressing shRNA. However, the antiviral effect of RNAi depends on the delivery efficacy of siRNA or shRNA. We evaluated the effect of shRNA-2B expressed using both plasmid vector and lentiviral vector. The data presented here indicate that the viral titers in HeLa cell supernatants were less in the pGCL-2B group than in the Lenti-2B group. Inversely, the viral titers in mouse heart and pancreas were decreased much more in the Lenti-2B group than in the pGCL-2B group, suggesting that the expression of shRNA-2B with viral vector exhibited higher inhibitory efficiency on viral replication than with plasmid vector in vivo. Thus, lentiviral vector is a promising viral vehicle for delivery of shRNAs and shRNA-2B has great potential to be further developed into an effective therapeutics for the treatment of coxsackievirus and other enterovirus infections.
shRNA-2B significantly reduced the virus titer of CVB3 in HeLa cells, also prolonged the mouse survival span, attenuated the tissue damage and inhibited the viral production in vivo compared with those of the shRNA-NC treated control group, suggesting that shRNA-2B is a potentially therapeutic agent for the treatment of enterviral diseases.
CVB3: Coxsackievirus B3; RNAi: RNA interference; shRNA: Short hairpin RNA; dsRNA: Short double-stranded RNA; siRNA: Small interfering RNA; DMEM: Dulbecco's modified Eagle's medium; PBS: Phosphate buffered saline; GFP: Green fluorescent protein.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
HY carried out most experiments and drafted the manuscript. FH carried out the animal experiments. CW measured virus titers in cells. ZX & JZ evaluated pathology. FW performed the statistical analysis. YZ & ZL conceived of the study, and participated in its design and revised the manuscript. All authors read and approved the final manuscript.
We thank Mrs Lingling Cai and Sha Wu for expert technique assistance. This work was supported by National Natural Science Foundation of China (30772025), Beijing Natural Science Foundation (7102023), and Beijing Training Programme Foundation for the Talents (2011A003034000032).
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A mid-infrared system for optical probing is disclosed that comprises a mid-infrared fiber laser based on cascaded Raman wavelength shifting, a sample volume, and a detector or detection system. The cascaded Raman wavelength shifting process in optical fibers involves the emission of a plurality of optical phonons for at least some of the pump photonics involved in the process. As one example, using the cascaded Raman wavelength shifting process a pump laser wavelength between 1 and 2 μm can be shifted down to between 2.5 to 10 μm. In one embodiment, the mid-infrared fiber laser comprises a pump laser with a wavelength between 1 and 2 μm, one or more stages of cascaded Raman oscillators implemented in fused silica fiber, and one or more stages of cascaded Raman oscillators implemented in mid-infrared fiber that transmits beyond 2 μm. Examples of mid-infrared fibers include chalcogenides, fluorides and tellurite fibers. The output wavelength from the mid-infrared fiber laser is at an exemplary wavelength longer than 2.5 μm. The mid-infrared optical probing system can be used in applications such as semiconductor process control, combustion monitoring for engines, and defense or homeland security applications such as chemical sensing and infrared counter-measures.
1. A mid-infrared laser beam source comprising:
a pump laser to produce a pump signal having a wavelength between 0.8 and 2.5 μm, the pump laser comprising a double-clad fiber with a doped core region coupled to a mode converter and a multiplexer to combine outputs from a plurality of laser diodes to form the pump signal;
a cascaded Raman oscillator apparatus coupled to receive the pump signal and having a plurality of stages each to produce a separate output signal at a different mid-infrared wavelength from each other stage and through a Raman wavelength shifting process, wherein each stage is to shift a separate input signal for the stage to a longer wavelength to produce the separate output signal, and wherein each separate output signal corresponds to a different portion of a mid-infrared spectrum;
a coupler apparatus to produce a signature spectral profile comprising tapped portions of the separate output signals; and
a directional stage to direct the signature spectral profile to an external target.
2. The mid-infrared laser beam source of
3. The mid-infrared laser beam source of
4. The mid-infrared laser beam source of
5. A method of producing an overall output mid-infrared signal having frequency components at separate regions of a mid-infrared spectrum, the method comprising:
generating through a pump laser a pump signal having a wavelength between 0.8 and 2.5 μm, wherein in the pump laser comprises a double-clad fiber with a doped core region coupled to a mode converter and a multiplexer to combine outputs from a plurality of laser diodes to form the pump signal;
supplying the pump signal to a first one of a plurality of cascaded Raman oscillator stages that each produce a separate output signal at a different output wavelength in response to a separate input signal coupled to that stage, wherein each stage applies a Raman wavelength shifting process to the separate input signal coupled to that stage, and where at least one of the stages is capable of producing its separate output signal at a wavelength longer than 2 μm;
supplying the separate output signal from one stage as the separate input signal to another stage; and
each stage producing one of the frequency components such that under continual pumping of the pump signal the combined separate output signals from the stages form the overall output mid-infrared signal.
6. The method of
7. The method of
directing the overall output mid-infrared signal to an external target to provide the predetermined heat signature to the external target.
8. The method of
9. The method of
10. The mid-infrared laser beam source of
11. The method of
This invention generally relates to mid-infrared laser systems and, more particularly, to a system and method for generating mid-infrared light using fiber optic technologies.
Various laser sources exist for producing radiation in the mid-infrared (mid-IR) region. The earliest mid-IR semiconductor lasers, for example, used lead salts operating at cryogenic temperatures to produce output wavelengths ranging from 3 to 30 μm. These lasers were limited to relatively low output power levels and exhibited wide variations in emission characteristics, particularly from thermal cycling. As a result, researchers began focusing on Antimonide based laser sources, quantum cascade (QC) laser sources, and optical parametric oscillators (OPOs) as more viable alternatives.
Although there has been significant mid-IR laser development in the past few years, particularly in QC lasers and OPOs, a number of problems persist. For instance, OPO's are bulky and require a large pump laser and moving parts. QC lasers have relatively low efficiency, requiring strict temperature control and in some instances cryogenic temperatures for high power operation. QC lasers also operate at wavelengths longer than 4.2 μm near room temperature, and thus do not sufficiently cover the entire mid-IR wavelength range.
In contrast, mid-IR sources based on the cascaded Raman shifting process exhibit some distinct advantages, including the maturity of underlying technology, simple modulation and wavelength tuning, and excellent beam quality with M2<1.4 (using single spatial mode in fiber), where the M2 factor is the beam quality factor, or beam propagation factor, defined as the beam parameter product divided by λ/π These mid-IR sources have been implemented in fiber-based systems that offer additional advantages such as robustness, compactness, lightweight, ease of use, room temperature operation over a flexible repetition rate, a wide wavelength range from 1-6.5 μm or longer, and very high efficiency (close to quantum limit). Compared with QC lasers, cascaded Raman shifting sources offer a larger tuning range, better beam quality, room temperature operation with flexible repetition rate, scalability to high powers, and higher efficiency. Compared with OPO's, cascaded Raman shifting sources offer a more mature underlying technology, better beam quality, the advantages of fiber-based systems, and a wider wavelength range with better efficiency.
While cascaded Raman shifting sources have been proposed, the state of the art sources are limited in operation and commercial application. Some have proposed cascaded Raman shifting apparatuses designed to shift a single pump signal from one mid-IR wavelength to a different mid-IR wavelength. Yet, because of the varying attenuation characteristics of Raman shifting materials, no single material can be used to cover the entire mid-IR spectral range, which limits the reach of Raman sources. Furthermore, even where techniques have been developed to provide numerous Raman orders in a single cascaded Raman stage, the output is limited to the fully converted, final Raman order. That is, effective techniques for taping different Raman shifted orders during the entire conversation process have not been attained. Some have proposed using a coupler with a single Raman process stage, but such configurations nonetheless limit the output from a mid-IR laser source and they limit the applications that might otherwise benefit from such a source. This limitation is particular noticeable in applications like countermeasures where a supercontinuum produced laser source may be undesirable over a discrete characteristic spectrum.
In one embodiment, a system for mid-infrared probing comprises a mid-infrared fiber laser coupled to a sample volume and a detector. The mid-infrared fiber laser comprises a pump laser with a pump wavelength between 1 and 2 μm, a wavelength shifter coupled to the pump laser, wherein the wavelength shifter comprises one or more optical fibers, where at least one of the optical fibers is capable of transmitting at an output laser with an optical wavelength longer than 2 μm, and wherein the pump wavelength is shifted to an output wavelength longer than 2.5 μm by a cascaded Raman wavelength shifting process in at least one of the optical fibers. As a result, optical phonons may be emitted for at least some of the pump laser photons involved in the cascaded Raman wavelength shifting process in the one or more optical fibers. The sample volume is capable of transmitting the output wavelength from the mid-infrared fiber laser, and the detector is capable of receiving at least a fraction of the output wavelength after transmitting through at least a portion of the sample volume.
In another embodiment, a method includes generating a pump source having a wavelength between 1 and 2 μm, shifting the pump to an output source having a wavelength longer than 2.5 μm through a cascaded Raman wavelength process in one or more optical fibers, transmitting the output source through at least a portion of the sample volume, and detecting the output source after transmitting through at least the portion of the sample volume. The cascaded Raman wavelength shifting process emits a plurality of optical phonons in response to pump photons involved in the cascaded Raman wavelength shifting process in one or more optical fibers, wherein at least one of the optical fibers is capable of transmitting optical wavelengths longer than 2 μm.
In another embodiment, a mid-infrared light source comprises a pump laser coupled to a first stage cascaded Raman wavelength shifter and a second stage cascaded Raman wavelength shifter. The pump laser is capable of generating more than 10 W of peak power at a pump wavelength between 1 and 2 μm. The first stage cascaded Raman wavelength shifter comprises a first set of one or more cascaded Raman oscillators, each having a fiber grating and a length of fused silica fiber, wherein the first stage shifts the pump wavelength to beyond 2 μm. The second stage cascaded Raman wavelength shifter comprises a second set of one or more cascaded Raman oscillators, each having at least one reflector and a length of a second fiber that transmits light at a wavelength greater than 2 μm, and wherein the second stage cascaded Raman λ shifter shifts the pump wavelength to beyond 2.5 μm.
Various embodiments may be capable of serving as a mid-infrared light source in systems for semiconductor process control, combustion monitoring, and infrared counter-measures. Some embodiments may be capable of generating time averaged output powers of 10 W or more; some embodiments may have more than one output wavelength or be tunable in wavelength; some embodiments may be pumped, producing a pulsed output; some embodiments may produce continuous wave output.
Other technical aspects will be readily apparent to one skilled in the art from the following figures, description and claims.
An example configuration for a fiber laser 100 capable of transmitting laser light in the mid-IR region is illustrated in
For pulsed pump lasers, the peak laser power should be high enough to have efficient Raman wavelength shifting even with open loop (i.e., straight length) fibers. However, for cw pump sources, the efficiency of the Raman process can be enhanced by using oscillators to multi-pass the pump and the various cascaded orders. However, more generally a pulsed or cw pump may be used either with open loop systems or oscillators.
For wavelengths above approximately 2 μm, the coupling fiber 140 and the CRO 130 may be formed of mid-IR fibers made of different mid-IR materials, such as fluoride fibers for an effective range out to ˜6 μm or chalcogenide fibers for an effective range out to ˜10 μm. Other example materials for forming Roman wavelength shifting fibers at longer wavelengths include tellurite (TeO2) fibers.
To propagate light in the mid-IR, long wavelength transmitting glasses typically need heavy atoms dopants. As an example, various mid-IR transmitting chalcogenide glasses include sulfide (S), selenide (Se) and telluride (Te). More generally, a chalcogenide glass may be composed of one or more of the chalcogen elements S, Se and Te with other elements such as germanium (Ge), arsenide (As) and antimony (Sb). It has been shown that these glasses are chemically durable, possess wide glass-forming capabilities, and can be fabricated into relatively low-loss fibers. As an example,
Selenide fibers have been used for Raman amplification in the near-infrared wavelength region. In particular, an As—Se fiber has been used with a 7 μm core diameter and a numerical aperture of 0.45 at a wavelength of 1.56 μm. The peak of the Raman gain coefficient may be ˜700 times larger in As—Se chalcogenide fibers compared with standard single-mode fused-silica fiber. In addition, As—Se fiber can have a much narrower Raman line (˜60 cm−1) than silica fiber (˜250 cm−1). Also, the Raman shift for As—Se fiber can be smaller (˜240 cm−1) than the Raman shift in silica fiber (˜440 cm−1). These differences reflect the resulting phonon energy due to the pump and correspond to the energy shift between the pump light and the signal light for a single Raman order. Moreover, the As—Se fiber loss can be less than 1 dB/m over a wide range from ˜1.5 to ˜9 μm. For most of the mid-IR range between ˜2 to ˜6.5 μm, the loss can be actually closer to 0.5 dB/m. However, there usually is a loss peak near ˜4.5 μm, corresponding to a H—Se resonance. This absorption peak can be reduced with a modification of the glass's composition. If the selenide resonance falls at a wavelength of interest, then alternate chalcogenide fibers can be used instead, such as sulfide-based fibers. Because selenide fibers have been used for Raman amplification and because the fiber properties have been measured, the simulations presented below are based on selenide mid-IR fibers with parameter values based on the measured properties.
One alternate mid-IR fiber material is based on fluoride composition. One of the more mature fluoride fibers is the heavy metal fluoride ZBLAN (ZrF4-BaF2-LaF3-AlF3-NaF). As shown in
In addition, for Raman wavelength shifting, the figure of merit is usually proportional to the Raman gain coefficient divided by the fiber loss. Although the actual gain coefficient can be smaller in fluoride fiber, the ratio of gain to loss may be comparable in the two (fluoride and chalcogenide) fibers, and combined with the larger wavelength shift the two mid-IR fibers can have comparable performance. However, fluoride fibers tend to be more mature technology with higher laser damage thresholds and little evidence of photo-darkening.
Other fiber types that transmit in the mid-IR could also be used, such as fibers based on tellurite glasses, sapphire 420, AgBrCl 440, or hollow glass waveguides 450.
The pump laser 110 in
The core 510 may be formed of a silica (SiO2) single-mode core doped with Yb3+, while the outer core 520 may be formed of a lower index of refraction polymer cladding, and the layer 530 a protective polymer.
If the CPF 500 is an amplifier it would have a seed input that the CPF 500 boosts in power. As a laser, on the other hand, the CPF 500 may have two reflectors formed in the inner core, with at least one of the reflectors being partially transmitting. In the illustrated example, these reflectors are gratings 550 formed directly into the CPF 500, as shown in
Although one example of the pump laser is a cladding pumped fiber laser, other pump lasers may be used instead. For example, the pump laser may include a solid state laser, semiconductor laser diodes, single spatial mode rare-earth doped fiber amplifiers, as well as optical parametric amplifiers or oscillators.
The grating reflectors 720 and 730 are shown has having multiple gratings that depending on the position will reflect various Raman shifted frequencies produced during the overall Raman shifting process. Grating pairs 760A for example correspond to the output wavelength 1480 nm, which is the final Raman shifted step for the CRO 700. The grating pairs 760E reflect 1175 nm within the system 700, which corresponds to the first Raman shifted step producing photons that are to be further shifted by the CRO 700. A grating 770 is also shown and serves as a pump reflector for the CPF 742 so as to prevent unconverted pump energy at the 1117 nm wavelength from exiting the as part of signal 750.
Although two examples of CRO's are given in
To simulate the efficiency of the cascaded Raman process, a set of coupled mode equations can be solved numerically. For example, the governing equations can be the coupled wave equations,
An initial value problem can be solved where the pump at the first wavelength is given, and a spontaneous background power level corresponding to one photon per mode over a bandwidth of ˜10 cm−1 is assumed at all of the Raman cascade order wavelengths. Parameters for the selenide fibers were used in the simulations. Because the lengths for the mid-IR fibers are a few meters or less, it may be assumed that the polarization mode scrambling is negligible and that the pump and signal wavelengths are co-polarized. The coupled equations can be solved by standard numerical integration techniques, such as the Runge-Kutta method. In these initial simulations, only the gain coefficient at the peak of the Raman gain curve is included.
The pump power into the mid-IR fiber is assumed to be 2 GW/cm2, which is approximately the experimentally measured damage threshold in the selenide fibers for pulsed operation. For the test, 15 ns pulses and a 10 KHz repetition rate were assumed, which corresponds to a peak power of Ppeak=770 W and an average power of Pavg=116 mW at the beginning of the chalcogenide fiber. The results are shown in
The efficiency of the cascaded Raman wavelength shifting can be close to the quantum efficiency (i.e., the ratio of the photon energies), less any propagation loss in the fiber, which is not so large for the short lengths involved. Note that the efficiency for the longest wavelength can be degraded by the absorption peak near 4.5 μm. In addition, as
For many applications such as chemical sensing and spectral fingerprinting, the tunability of the mid-IR laser can be a valuable attribute. In one embodiment, the mid-JR laser can be tuned by tuning the wavelength of the pump laser. For example, if the pump laser is a laser diode centered around 1.5 μm, then the mid-IR source can be made tunable by replacing the laser diode with a tunable laser diode. It should be noted that the tuning range is proportional to the change in photon energy. Therefore, a small or moderate wavelength tuning at ˜1.5 μm can lead to a large Δλ in the mid-IR wavelength range. For example, tunable laser diodes are commercially available that tune over a band—i.e., a C-band or conventional band tunable laser diode will tune over 1.53 μm to 1.57 μm. As shown in the table below, this 40 nm energy tuning at the seed signal wavelength can lead to a ˜100 nm tuning around 2.5 μm, a ˜220 nm tuning around 3.5 μm, and a ˜400 nm tuning around 4.6 μm. Consequently, it may only be necessary to temperature or current tune the seed signal laser (i.e., about 5 nm) to obtain a significant tuning in the mid-IR wavelengths.
Another desired attribute of the mid-IR fiber laser may be to increase the time-averaged power from the laser. To scale the average power to higher levels, larger core sizes for the fibers can be used (i.e., the intensity would be fixed at the damage threshold, but the power is the area of the fiber times that intensity). As an example, the cascaded Raman wavelength shifting has been simulated starting from a 1.532 μm pump out to 2.2 μm (2 W output), 3.4 μm (1 W output) and 5 μm (4 W output) μm with 10 ns pulses at 100 kHz repetition rate. Staying within the 2 GW/cm2 damage intensity, the pump power and fiber parameters are provided in the table below. It should be noted that although the larger core size of these fibers means that they are multi-mode fibers, the meter or so lengths involved single spatial mode (i.e., M2<1.4) can be maintained by launching the light properly, avoiding twists and mode scrambling in the fiber, and perhaps using some mode-stripping at the end. Furthermore, the line widths at these output wavelengths should be 2-5 nm or wider. Example configuration numbers are provided in the table below.
Another advantage of the cascaded Raman wavelength shifter approach is that the pulse repetition rate can be varied from cw to 100 kHz or more. When using a cw pump, however, the peak power is low, so resonators must be used to multi-pass the signal to increase the efficiency. As another example of multiple order cascaded Raman wavelength shifting,
In this particular example, a summary of the calculated values for the configuration illustrated in
The pump laser 1410 is coupled to the three stages of CROs, which are used to wavelength shift out to respective output values of ˜2.5 μm (1430), ˜3.5 μm (1450) and ˜4.6 μm (1470). The output signals 1430 and 1450 are partially coupled as an input signal to a subsequent CRO stage and partially tapped to produce an output signal at different mid-IR wavelengths. Couplers 1480 and 1490 are shown, where the final CRO 1460 stage has no coupler.
For an n=3 application of the configuration 1400, the intermediate Raman orders 1500 are illustrated in
In summary, a simple, highly efficient, power scalable mid-IR laser can be implemented in fibers that can have a range of pulse repetition rates and is based on cascaded Raman wavelength shifting in optical fibers. Exemplary fiber types include fused silica fiber, chalcogenide fibers, fluoride fibers, tellurite fibers, or some combination of these fibers. The power limitation can arise from the damage threshold in these fibers, and average powers of several watts can be achieved by increasing the fiber core size. Also, the higher power mid-IR lasers can be pumped using either solid state lasers or cladding-pumped fiber lasers. Moreover, the pulse repetition rate can be varied from cw to 100 KHz or higher. Whereas pulsed pumping can use an open loop fiber for wavelength shifting, in cw pumping the efficiency can be increased by using oscillators. Particular configurations are shown by way of example, but many other embodiments can be used within the scope of the disclosure. For example, a cascade of CROs can be used, with light taped off after each CRO so as to create a multiple wavelength output. In addition, other waveguides can be used instead of fibers, such as semiconductor waveguides, glass waveguides, doped substrate waveguides, hollow core fibers, or photonic crystal fibers.
The mid-IR laser may be particularly beneficial in a number of applications, including semiconductor process control, combustion monitoring, military infrared countermeasures (IRCM) and chemical sensing, and biomedical ablation and imaging. The fiber based mid-IR laser benefits these numerous applications because it can be compact, lightweight, robust, with no moving parts and potentially inexpensive. In addition, the mid-IR laser may be made mostly of commercial off-the-shelf parts, and the fiber integration can mean that no optical bench is required. In the following, some of the applications of the mid-IR laser will be described. There are, of course, many other applications for the mid-IR laser consistent with the scope of the disclosure.
One example of the application of the mid-IR laser is to the semiconductor processing industry, where an important criterion for commercial success is yield. One of the significant contributors to the yield of a processing plant is the etch process, particularly the etching of gates and the etching of holes for contacts (so-called dielectric etching). The mid-IR fiber-based light source can potentially measure in-situ the chemicals in a semiconductor processing chamber as an input to the advanced process control (APC) engine. By monitoring the chamber conditions, advanced process control has the potential to increase the yield and repeatability of the process.
Currently, most commercial semiconductor processing machines operate open loop. For example, the APC treats the semiconductor growth chamber as a black box. The input and output are correlated, and then the output can be measured to guide the APC engine. However, the yield could be improved by having feedback from the chamber that can be used as an input to the APC engine. Two problems have plagued obtaining an appropriate feedback signal: (a) relatively low concentration of molecules in the chamber; and (b) the complexity of the "soup" in the chamber, and clearly identifying species that are signatures of the process state. Many of the previous studies have been hampered by complicated and time consuming signal processing to improve the signal-to-noise ratio. Another alternative is to look at the exhaust hose of the chamber, where there is a relatively high concentration of gas. However, this is done primarily for environmental reasons, and it is usually too far downstream in the processing to benefit APC. The mid-IR laser could potentially enable in-situ process control by providing a feedback signal that can guide the APC engine.
Optical absorption spectroscopy can be used to monitor the etch process in a semiconductor chamber. For example, the poly gate etching can be monitored by measuring the composition of HCl and HBr. Moreover, the contact hole etching or dielectric etching can be characterized by monitoring the species of CxFy in the vacuum chamber. Most of these compounds and molecules have their molecular or vibrational absorption in the mid-infrared region (e.g., 1.7 to 6.5 μm). The mid-IR fiber laser could be a tunable, bright light source to operate over this wavelength range and that can be compatible with use on a semiconductor vacuum chamber.
A compact, lightweight, rugged, tunable, mid-IR laser with 10's to 100's milli-watts of power can be beneficial for optical absorption spectroscopy. Some of the advantages of the fiber-based mid-IR source over other methods of generating mid-IR light include: (a) maturity of underlying technology; (b) simple tuning over wide wavelength range; (c) excellent beam quality with M2<1.4; (d) qualities of fiber-based sources, such as being robust, compact, lightweight and easy to use; (e) room temperature operation over a flexible repetition rate; (f) wide wavelength range from 1-6.5 μm or longer; and (g) simple system with efficiency close to the quantum limit.
For many etch processes, the detailed chemical mechanisms of the process are often not well characterized. As an example, etching of SiO2 contact holes (vias) often suffers from a problem known as etch-stop (the etch process prematurely stops before etching through the desired amount of SiO2). This problem is related to unusually high rates of polymer formation during the etch reaction (some polymer formation is normal and desirable for control of the sidewall shape), but the sparse amount of real-time data on chemical species in the chamber during both normal operation and during etch-stop conditions has limited the detailed scientific study of this problem. The proposed monitoring system based on the fiber mid-IR laser would greatly aid in studying this problem meaning that new scientific studies could be enabled based on real-time monitoring of chemical compositions in the chamber.
Thus, the basic idea of using the mid-IR source is to measure in-situ with high SNR the chemicals in the chamber and to feed this data back to the advance process control engine. However, for such a tool to be commercially viable in the process plant, a number of criteria for the light source would likely need to be satisfied, including: (a) reliability; (b) simple to operate and maintain; (c) room temperature operation; (d) compact; and (e) no modification to the machine and no contaminants. Fortunately, the fiber-based mid-IR light source can potentially meet these criteria.
As an example,
The mid-IR laser 1710 may be any of the lasers systems described herein, and includes the laser source 1760 and wavelength/power detector 1770 which may be used as part of a closed loop feedback control of the source 1760.
Another example of an application of the mid-IR light source can be in combustion monitoring. For instance, most applications relevant to gas dynamic and combustion flows are usually based on absorption by low-molecular weight molecules with well resolved transitions. Example of gases include O2, H2O, CO, NO, NO2, OH, NH3, HF, H2S, and CH4. In current systems, absorption measurements are generally performed on overtone or combination vibration absorption bands. However, typical line strengths for these transitions are usually two or three orders of magnitude below the fundamental vibrational transitions in the mid-IR. Therefore, by using a mid-IR laser to perform the absorption measurements, there is the potential for much higher sensitivity because the vibrational and rotational lines are being measured at their fundamental transitions.
An exemplary system 1800 for performing combustion flow monitoring is illustrated in
More generally, chemical sensors can be implemented using the mid-IR fiber laser for detecting a wide range of atmospheric chemicals, many of which have their vibrational or rotational absorption in the mid-IR. This is one reason that the wavelength range is often referred to as the chemical fingerprint region. Applications of such detection systems can include environmental monitoring, chemical warfare agent detection, pollution control, explosives or weapons detection, etc.
Another potential application of the mid-IR fiber laser is to infrared countermeasures (IRCM). Most heat seeking missiles operate based on finding their target based on the black body radiation see plot 1900 from the hot engine 1910 or the heat associated with the engine plume 1920. The holes in the spectrum 1930, 1940, 1950 arise because the atmosphere does not transmit well at these wavelengths. Therefore, the laser used in IRCM usually has to provide energy in at least three bands: one from approximately 1.5 μm to approximately 2.5 μm, one from approximately 2.7 μm to approximately 3.5 μm, and another from approximately 3.7 μm to approximately 4.5 μm. Furthermore, since these lasers may reside on aircraft or helicopters, they must be lightweight, robust, and operate at room temperature.
A fiber-based mid-IR laser may serve as the light source for an IRCM system. As illustrated in the examples from
Each of the couplers 2006 and 2010 provide a portion of their respective output beams, along with the output beam from the CRO stage 2012 to beam combiner 2014 which is coupled to a directional transmitter stage 2016. That is, each of the couplers 2006 and 2010 have tapped portions which optically decouple a portion of the shifted light from the corresponding CRO to be combined with other shifted output signals. These taped portions may be standard optical couplers, e.g., a branch of a Y-splitter or more generally any evanescently-coupled N dB splitter, where N represents the desired amount of energy (in a decibel form) tapped from the output signal of the corresponding CRO. Under continual pumping by the laser 2002, that is pumping for a long enough time for all CROs to reach full Raman signal conversion and for all couplers to simultaneously output a tapped portions of their respective output signals, the combiner 2014 and the assembly 2016 operate to produce an overall mid-IR signal 2018 of a particular frequency signature depending on the individual output beam wavelengths from the CRO stages 2004, 2008, and 2012. This overall mid-IR signal can take on the characteristic spectrum of a desired object, for example that of a exhaust spectrum from an aircraft or other vehicle. The system 2000 may be adapted to produce a desired overall mid-IR spectral profile by adjusting various stage dependent variables such as the length of fibers forming the stages 2004, 2008, 2012, the number of Raman order shifts in each stage, the conversion efficiency of each stage, the coupling (i.e., output) efficiency of the taps for the various couplers. The overall mid-IR signal 2018 may be transmitted to an external target 2020 that includes an infrared seeking sensor system, as may be used guidance based system. While only three CRO stages are shown, if the infrared seeking system looks for characteristic spectral emissions at N number of different mid-infrared regions of the spectrum, then the system 200 may be formed with N number of CRO stages, each producing a different mid-IR wavelength output signal corresponding to one of the N spectral regions of interest at the external target.
Although the present invention has been described in several embodiments, a myriad of changes, variations, alterations, transformations, and modifications may be suggested to one skilled in the art, and it is intended that the present invention encompass such changes, variations, alterations, transformations, and modifications as falling within the spirit and scope of the appended claims.
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