Fluorescence molecular imaging visualizes biological processes at molecular level1–3 and provides advantages including nonradioactivity, high detection sensitivity, and relatively low instrumental cost.4,5 Low tissue autofluorescence and absorption in the near-infrared (NIR) window benefit the detection performance of fluorescence molecular imaging.6–8 Therefore, NIR fluorescence imaging serves as a valuable tool9 for research fields including tumor pharmacokinetics,10–12 vascular function,13,14 and tissue ischemia.15–17
Kidneys receive of the cardiac output,18 and the renal anatomy is vascular-rich. Clinically, renal ischemia could be caused by various reasons, including arterial and venous occlusion, thrombosis, and vasculitis.19,20 Renal ischemia often results in serious renal physiology alteration, even leading to severe prognosis. Therefore, in vivo detection and assessment of renal ischemia are important for evaluating renal conditions during ischemia.
For renal physiology studies, previous works have shown that the epidermal growth factor receptor (EGFR) was a valuable target to investigate.21–25 An EGFR-binding fluorescent probe named EGF-IRDye was generated by conjugating an NIR dye (IRDye CW800) with the recombinant human EGF protein (which forms specifically binding with EGFR). After entering blood circulation, the EGF-IRDye probe quickly reaches and accumulates in kidneys. In the present study, this probe was used to indicate renal blood flow characteristics in mouse renal ischemia model.
The limited dynamic range of charged-couple device (CCD)26–32 prevents the imaging system from fully capturing the fluorescence intensity difference of imaging targets that have significant different fluorophore concentrations. Consequently, fluorescence images of renal ischemia model using single exposure times could not fully present the ischemia features, due to the significant fluorescence intensity difference between the normal kidney and the ischemic kidney.
Modifying CCD hardware could help to solve this limitation,33–35 but hardware modification could be costly and time-consuming. Another strategy adopted multiwavelength approaches including optical spectroscopic imaging17,36–39 and autofluorescence imaging40–45 to investigate the renal ischemia physiology. However, these approaches have limitations, including altered fluorophore quantum efficiency,39 weak or absent correlation between fluorescence and ischemia,41 phototoxicity under short-wavelength,43 and restricted clinical application.42,43
The high-dynamic-range (HDR) method is a widely used method in digital photography that could effectively enhance the dynamic range by combining features of images with different exposures.28,46–48 Recently, HDR method has been reported to combine with various imaging modalities and enhance their performance. HDR optical projection tomography (OPT) was developed to resolve details of zebra fish embryos.49 HDR laser-scanning microscopy (LSM) was used to facilitate three-dimensional neural segmentation.50 HDR fluorescence molecular tomography (FMT) was used to detect tumors with different fluorophore concentrations.51 HDR fluorescence laminar optical tomography (FLOT) was used to improve the penetration depth and quantification accuracy of FLOT.52
The significant fluorescence intensity differences between the ischemic kidney and normal kidney could reveal physiological information relevant to renal ischemia, but these differences could not be fully presented due to the limited dynamic range. In our work, HDR method has been employed to increase the dynamic range of the fluorescence imaging and it could be a potential strategy for enhancing renal ischemia assessment. To the best of our knowledge, there are no reports of combining HDR with NIR fluorescence imaging to improve renal ischemia assessment.
In this paper, we developed an HDR fluorescence imaging method to enhance the visualization of physiological relevant information of the renal ischemia model in order to improve the assessment of in vivo renal ischemia. First, the CCD response function was recovered. Then, HDR workflow was validated using images of phantoms with different fluorophore concentrations. Last, HDR images of mouse renal ischemia model (both ex vivo and in vivo) were constructed and compared with original images. The results suggested that our approach constitutes an initial step toward enhancing the assessment of in vivo renal ischemia using HDR method and could serve as a postprocessing imaging augmentation strategy for further studies on organ ischemia detection and evaluation.
Construction of HDR Fluorescence Images
Our approach for constructing HDR images was developed based on a previously described method.46
Recovering CCD response function
A series of fluorescence images with multiple exposure times was collected to recover the CCD response function. The image series was selected to ensure that the fluorescence intensity of each image sufficiently overlapped with each other. Twenty four images were selected to recover the response function accurately.
The fluorescence image series consisted of images, and each image had pixels. The original fluorescence images with exposure time were denoted by , where is the spatial index over pixels, and is the image index in the series. The low dynamic range fluorescence images used to calculate CCD response function were denoted as , which were obtained as46
The scene was assumed static, as the low dynamic range images were collected rapidly that the fluorescence changes could be safely ignored. Therefore, the irradiance remained constant among images with different exposure times.
The film reciprocity equation could be written as follows:4646
Taking natural logarithm on both sides of Eq. (3)46
was redefined as to simplify notation46
The fluorescence intensity values of were finite, therefore recovering only required finite values. The least and the greatest pixel values of were denoted by and , respectively. Therefore, could be recovered by finding values of and values of that minimized the following quadratic objective function:4646 46
In order to properly locate the CCD response curve, another constraint was introduced to ensure the pixel with have the unit exposure, as follows:46
Not all available pixels were needed for solving Eq. (6). In order to keep Eq. (6) as an overdetermined equation, the following condition should be met:46
In our dataset, the fluorescence intensity range and the number of fluorescence images . Therefore at least 698 pixels should be sampled from each image (). In the actual calculation process, 1200 pixels were sampled from each image and used for calculating the CCD response curve. These pixels were chosen from regions with low intensity variation, and they distributed evenly from to .
Phantom study: recovering CCD response function
The fluorescent probe was EGF-conjugated IRDye 800CW (EGF-IRDye; LI-COR Biosciences, Lincoln, Nebraska). A homemade reflectance fluorescence imaging system12,15,16,55 was used in this work for fluorescence molecular imaging (Fig. 1). A 300-W xeon lamp (MAX-302, Asahi Spectra, Torrance, California) was used as the excitation source. The excitation light was filtered through a band-pass filter (XBPA770, Asahi Spectra, Torrance, California) before reaching the object at a power density of . The emitted fluorescence was filtered through an band-pass filter (FBH800-10 Premium Bandpass Filter, Thorlabs, Newton, New Jersey) and then detected by a , electron multiplying CCD (EMCCD) camera (iXon DU-897, Andor Technologies, Belfast, Northern Ireland, United Kingdom) coupled with a 35-mm lens (C3514-M, Pentax, Tokyo, Japan). Each pixel has a size of .
Fluorescence images of a phantom with 24 different exposure times (i.e., 0.01, 0.02, 0.05, 0.10, 0.20, 0.25, 0.30, 0.40, 0.50, 0.60, 0.70, 0.80, 0.90, 1.00, 1.10, 1.20, 1.30 1.40, 1.50, 1.60 1.70 1.80, 1.90, and 2.00 s) were collected to recover the CCD response function. The phantom was a 0.5-ml centrifugal tube (Eppendorf, Hamburg, Germany) filled with 0.2 ml EGF-IRDye dissolved in buffer saline (PBS) (HyClone, Logan, Utah). This image dataset was collected and the fluorophore concentration of phantom remained consistent throughout the whole collection process.
The excitation light was adjusted to ensure that it was focused at the center of the FOV, and then the phantom was placed at the center of the FOV to ensure that the emission fluorescence was captured on the center of the CCD. Note that the source-to-target distance was and remained unchanged throughout the experiments.
Phantom study: validating HDR workflow
Phantoms with different concentrations of EGF-IRDye solution were used to validate the HDR workflow. Three 0.5-ml centrifugal tubes were filled with 1.2, 3.6, and EGF-IRDye solution and denoted as P1, P2, and P3, respectively (0.2 ml in each tube). Fluorescence images were collected with exposure time of 0.8, 1.8, and 2.6 s. Then these fluorescence images were used to construct the HDR result image of the phantoms using Eq. (12).
Animal studies: imaging ex vivo and in vivo renal ischemia
8-week-old female BALB/c-nude mice (each weighted 20 to 22 g) were placed under anesthesia using 2.5% isoflurane–oxygen mixture. A dorsal laparotomy was conducted. Then, the arterial and venous flow of the left kidney (in dorsal view) was simultaneously occluded using cotton thread, resulting in unilateral renal ischemia. The right kidney was not ligated, to serve as the normal control. Animal experiments were conducted with approval from the Ethics Committee of Tsinghua University.
After the renal ischemia model was established, each mouse was intravenously injected a bolus of 1-nmol EGF-IRDye dissolved in 0.3 ml . The dose of EGF-IRDye injected per body weight ranged from 0.045 to , approximately. Then, the mice were divided into two groups randomly (three mice per group). One group was used for in vivo imaging while the kidneys of the other group were harvested for ex vivo imaging.
Fluorescence images were collected 60 min after renal ischemia was induced. The positions of the kidneys (ex vivo study) and mouse (in vivo study) were maintained through the entire imaging process. The exposure times used in the animal studies were 3.2, 4.0, and 4.4 s for ex vivo imaging, and 3.6, 4.2, and 4.6 s for in vivo imaging. The whole imaging process normally takes . In the in vivo imaging, tissue surrounding kidneys was covered with black cloth to diminish the fluorescence from outside the renal ischemia model. HDR fluorescence images were constructed as described in Sec. 2.1.
Relative fluorescence intensity of the phantom
The fluorescence intensity of a phantom () was the average of fluorescence intensities of 10 rectangular region-of-interest (ROI) in 10 positions of the phantom. Then, the relative fluorescence intensity was calculated by normalizing using upper fluorescence intensity limit of the CCD ()
Relative fluorescence intensity of the ischemic kidney
In this study, the relative fluorescence intensity of the ischemic kidney () is defined as the ratio between the fluorescence intensity of the ischemic kidneys and that of the control kidneys, as follows:
Recovered CCD Response Curve from Multiexposure Fluorescence Images
Fluorescence images of a phantom are collected with different exposure times [Fig. 2(a)]. The sampling positions and a representative fluorescence image after sampling are shown [bottom right, Fig. 2(a)]. The original data points are represented by red dots, whereas the fitted response curve is plotted as black line [Fig. 2(b)].
Validation of HDR Workflow
With the exposure time as 0.8 s, fluorescence intensity of P1 is close to the background level, although P2 and P3 can be clearly visualized [Fig. 3(a)]. In order to sufficiently display the details of P1, the exposure time has to be extended. As the exposure time is increased to 1.8 s, P1 becomes distinguishable, but some pixels in P3 begin to reach saturation level [indicated by the white arrow in Fig. 3(b)]. When the exposure time is further extended to 2.6 s, the top part of P1 with peak intensity finally can be distinguished. However, both P2 and P3 suffer from saturation and distortion [white arrows, Fig. 3(c)], due to the increased exposure time.
In the HDR result, the fluorescence intensity of P1 is further increased compared to that of P1 in Fig. 3(c), and the area with peak fluorescence intensity remains visible. Moreover, the saturation of P2 and P3 is diminished. Relative fluorescence intensity values of these three phantoms are extracted as described in Sec. 2 and plotted against the probe concentration [Fig. 3(e)]. The HDR result decreases the relative fluorescence intensity of the phantoms that appear saturated in Fig. 3(c), thus suppresses the saturation. The HDR result also increases the intensity of P1 compared to its original relative intensity even with the longest exposure time. Moreover, the HDR result shows a better linear relationship between the relative fluorescence intensity and probe concentration.
HDR Results of the Mouse Renal Ischemia Model
Ex vivo HDR results
Ischemic and control kidneys are harvested and imaged ex vivo (Fig. 4). In the renal ischemia model, the blood vessels were not completely occluded; therefore, the blood stream was partially remained, resulting in the renal area with better perfusion. The ischemic kidneys are shown on the right side, whereas the control kidneys are on the left. The positions of kidneys are labeled (H = head; T = tail; L = left; R = right). With the exposure time as 3.2 s, the ischemic kidney exhibits low fluorescence intensity, and the control kidney is not sufficiently visualized [Fig. 4(a)]. Increasing the exposure time to 4.0 s increases the overall fluorescence intensity of the ischemic kidney, but the renal area that has better perfusion in the ischemic kidney could not be clearly distinguished [Fig. 4(b)]. When the exposure time is increased to 4.4 s, the relatively better perfused renal area starts to distinguish itself from the rest of the kidney, as the overall fluorescence intensity of the ischemic kidney is further increased. However, large portion of the normal kidney is saturated [the white arrow, Fig. 4(c)].
In the HDR result of the ex vivo study [Fig. 4(d)], the fluorescence intensity of the ischemic kidney is further increased, compared to that of the ischemic kidney in original image with the longest exposure time [Fig. 4(c)]. The area with better perfusion in the ischemic kidney becomes clearly distinguishable. Moreover, the saturation in control kidney is largely reduced. The area close to the center of the normal kidney remains relatively high fluorescence intensity in the HDR result. Then, the HDR result is compared to the original image that has the longest exposure time (4.4 s). The HDR result shows higher relative fluorescence intensity of the ischemic kidneys (0.68, 0.75, and 0.72 for the three mice, respectively) than the original image (0.58, 0.64, and 0.60) [Fig. 4(f)], as well as higher image entropy values (3.20, 3.26, and 3.25, respectively) than the original image (2.45, 2.70, and 2.27) [Fig. 4(g)].
In vivo HDR results
The mouse renal ischemia model was also imaged in vivo (Fig. 5). Mouse body position is labeled (H = head; T = tail; L = left; R = right). When the exposure time is 3.6 s, fluorescence intensity of the ischemic kidney is close to the background level, leaving the fluorescence distribution pattern difficult to detect [Fig. 5(a)]. As the exposure time increases to 4.2 s, the overall fluorescence intensity of the ischemic kidney is elevated, but the relatively better perfused area remains unclear [Fig. 5(b)]. With the exposure time further extends to 4.6 s [Fig. 5(c)], the better perfused area in the ischemic kidney begins to distinguish itself. However, large part of the control kidney is saturated (indicated by the white arrow), which is similar to the saturation in Fig. 4(c).
In the HDR result of the in vivo study [Fig. 5(d)], the fluorescence intensity of the ischemic kidney is increased. Wedge-shaped areas with better blood perfusion in the ischemic kidney could be clearly visualized, suggesting that the HDR result preserves more physiological characteristics than the original images. Moreover, saturation in the control kidney is reduced, and the center area remains to have peak fluorescence intensity. The HDR result is compared to the original image taken with the longest exposure time (4.6 s). The HDR result exhibits higher relative fluorescence intensity of the ischemic kidneys (0.72, 0.73, and 0.72, respectively) compared to the original image (0.61, 0.63, and 0.59) [Fig. 5(f)] and also shows higher image entropy values (2.92, 3.05, and 2.83, respectively) than the original image (2.35, 2.47, and 2.34) [Fig. 5(g)].
The main goal of this study was to enhance the in vivo assessment of renal ischemia by incorporating HDR method with fluorescence imaging. HDR images of both phantoms and mouse renal ischemia model were constructed based on the original fluorescence images taken with multiple exposure time. The results suggested that the HDR preserved physiological characteristics of both ischemic and control kidneys by highlighting the fluorophore distribution details and diminishing saturation caused by limited dynamic range.
Previous works on enhancing renal ischemia assessment include spectroscopic imaging17,36–39 and autofluorescence imaging.40–45 These approaches have improved the assessment of ischemic physiology; however, they have limitations such as interference with imaging process due to hypothermic preservation,36 altered fluorophore quantum efficiency,39 phototoxicity,43 and limited application in clinical settings.42,43
The fluorescence intensity values and the scene exposure have a nonlinear relationship [Fig. 2(b)], due to the nonlinear mapping of CCD.46 Consequently, a suitable exposure time has to be determined according to the fluorescence intensity of the imaging targets in order to avoid saturation. However, if imaging targets in the FOV have large fluorescence intensity differences, it would be difficult to find a proper exposure time to ensure both preserving the details of the low intensity targets and avoiding saturation in the high intensity targets, because the dynamic range of CCD is limited.
This limitation could be improved by the HDR method. HDR has been integrated with several imaging modalities, including OPT,49 LSM,50 FMT,51 and FLOT.52 In these studies, HDR has helped to resolve details of imaging targets49,51 and to enhance the detection capability of the imaging apparatus.50,52
We have adopted a similar strategy. In our work, fluorescence images with different exposure times were mathematically merged by HDR method46,54 to preserve physiological features of targets with large intensity differences and enhance in vivo renal ischemia assessment. It is convenient to collect fluorescence images using different exposure times, because the animal position is fixed during the imaging process. Note that exposure times used in phantom studies were different from those in animal studies because of overall fluorescence intensity differences between these two conditions.
The HDR results of phantom studies effectively reduced the saturation, and further increased the fluorescence level of low intensity targets, maintaining a better linearly relationship between probe concentration and fluorescence intensity [Figs. 3(d)–3(e)]. This showed that HDR method has the potential to overcome dynamic range limitation and display imaging targets more accurately.
In the animal studies, ischemic kidneys and control kidneys had significantly different fluorescence intensity level, but this difference was not fully presented with single exposure time. The HDR results of animal studies suggested that our approach could resolve this issue and enhance assessment of renal ischemia from two aspects. The first one is improving the visualization of ischemic kidneys. The HDR results showed significantly higher relative fluorescence intensity of ischemic kidneys, compared to even the original images with the longest exposure times, both ex vivo [Fig. 4(d) vs. Fig. 4(c)] and in vivo [Fig. 5(d) vs. Fig. 5(c)]. This could be validated by the comparison of relative fluorescence intensity of ischemic kidneys illustrated in Figs. 4(f) and 5(f). In addition, HDR results showed more details of the area with better perfusion in the ischemic kidneys. This could be validated by the increased image entropy values shown in Figs. 4(g) and 5(g). The second aspect is suppressing saturation in control kidneys. Saturation became more severe as the exposure time increased and eventually covered major parts of the control kidneys [Figs. 4(c) and 5(c)]. The HDR results suppressed the saturation and maintained the fluorophore distribution pattern in the control kidneys [Fig. 4(d) and 5(d)].
A number of limitations might have influenced the results. First, the animal ischemia model was not strictly static because of animal respiration and renal blood circulation. This might introduce slight disturbance to the results. Second, the exposure times for three types of images required by HDR (insufficiently exposed, normally exposed and overexposed) were selected empirically. For instance, to reach overexposure, 2.6 seconds was chosen for phantom study while 4.6 seconds was chosen for the in vivo ischemia model because of their difference in fluorescence intensity level. Third, the ischemia status is limited. In addition, the gap between the vascular supply profiles and the cellular mechanisms for renal ischemia needs to be explored. Furthermore, our method needs to be validated using a larger cohort size.
Future work will focus on accelerating data collection to reduce potential disturbance from animal movement. We also intend to investigate the appropriate selection of exposure times based on the overall fluorescence intensity level. More ischemia models, especially the models that closely resemble the clinical practice, need to be established with different occlusion times and used for further evaluating the HDR fluorescence imaging approach. Finally yet importantly, we need to investigate the underlying cellular mechanisms of renal ischemia based on previous studies,58–61 by tracking the concentration and distribution of key molecules in the ischemia mechanisms using specific fluorescent probes, for expanding our understanding of the renal ischemia progress.
The results of our study indicate that the visualization of renal ischemia model could be effectively improved by the HDR-based imaging approach. The HDR results successfully combined features related to renal ischemia that could only be captured with very different exposure times into a single image. HDR method has the potential for contributing to the medical practice, including providing real-time intraoperative imaging results, removing imaging disturbance due to surgical instruments, helping physicians to interpret the biopsy results and improve the efficacy of other modern imaging modalities. This approach also has the potential for providing more quantitative fluorescence estimation, improving clinical renal ischemia detection and assessment, and could serve as a valuable imaging tool for organ function evaluation.
This work was supported by the National Key R&D Program of China (Grant No. 2016YFC0102200), the National Natural Science Foundation of China (Grant Nos. 81471665 and 81561168023), and the Fundamental Research Funds for the Central Universities under Grant No. 2017RC023. We would like to thank Wei Liu from the Department of Biomedical Engineering at School of Medicine, Tsinghua University, for his help with the animal experiments.
Yang Gao received his bachelor’s degree in pharmaceutical sciences from Tsinghua University, Beijing, China, in 2014. He is currently a PhD candidate in the Department of Biomedical Engineering, Tsinghua University, Beijing, China. His research interest is dynamic fluorescence molecular imaging for small animal imaging.
Yuan Zhou received his bachelor’s degree in biomedical engineering from Beijing Jiaotong University, Beijing, China, in 2014. He is currently a PhD candidate in the Department of Biomedical Engineering, Tsinghua University, Beijing, China. His research interest is fluorescence molecular tomography for small animal imaging.
Fei Liu is an associate professor at Beijing Jiaotong University. She received her PhD in biomedical engineering from Tsinghua University in 2013 and was a postdoctoral research scientist from 2013 to 2015 in School of Medicine at Tsinghua University. Her research interest is fluorescence molecular tomography for small animal imaging. She has authored or coauthored over 60 journal papers.
Jianwen Luo is a professor at Tsinghua University. He was enrolled in the Thousand Young Talents Program in 2012, received the excellent young scientists fund from the National Natural Science Foundation of China in 2013, and was supported by the Young Scientists Project of National Key R&D Program of China in 2016. His research interests include ultrasound elasticity imaging and fluorescence molecular imaging. He has published more than 130 journal papers and 50 conference papers.