**4.2 Multimodal imaging**

Every imaging technique has its unique advantages in consort with integral limitations such as insufficient sensitivity or spatial resolution (**Figure 4**) [59]. Even the fluorescence imaging provides high sensitivity but lacks of sufficient resolution. To compensate this drawback, the combination of fluorescence imaging techniques with other modalities, such as FL/MRI has gained attention to enhance the currently used imaging techniques for diagnosis [58]. Multimodal imaging is a combination of two or more imaging techniques to overcome individual limitations. The development of multi-modality imaging with the FL imaging is

#### **Figure 4.**

*Advantages and disadvantages of various imaging techniques.*

to achieve non-invasive imaging at greater depths of penetration, sensitivity and higher resolution required for an accurate diagnosis. Thus, optical imaging assisted multi-modalities has emerged as potent tools, which can improve the detection sensitivity, precise identification and provide more detailed anatomical or biological information of the pathology. Each imaging technique uses different contrast agents with distinctive functional, chemical compositions and sizes. In designing and developing a multi modal contrast agent, the researchers should judiciously forbid the overlay of pros and somewhat counterbalance each modality's limitations to enhance the synergistic effect. Thus, the FL imaging modality with high sensitivity is frequently combined with other imaging modalities with a high spatial resolution modality such as MR, CT, and PA etc. (**Figure 5**). Hence, multimodal contrast agents' development and application are clinically significant for enhanced imagery from desirable imaging modality. Multimodal imaging agents based on fluorescent CDs are the recent cutting-edge technologies where CDs' advantages are maximized. CDs based multimodal imaging agents are prepared by conjugating or incorporating one or more imaging agents into CDs. Here we have discussed various kinds of fluorescent CDs' based multi-modality imaging approaches such as FL/PA, FL/MR and FL/CT imaging.

#### *4.2.1 Fluorescence/photoacoustic imaging*

Photoacoustic (PA) imaging is a non-invasive, hybrid, optical and ultrasound imaging modality. The PA imaging is performed at varying depths with high depth to resolution ratio with rich optical contrast beyond the optical detection limit. The CDs have shown significant PA imaging application because of the NIR absorption, high extinction coefficient, and non-radiative heat generation. Wang, *et al.* synthesized dual-mode FL/PA imaging agent based on CDs [60]. Within the NIR spectrum, the imaging agent exhibited a maximum optical absorption at 710 nm approximately. In *in vivo* PA imaging of mice tumor model, enhancement of signal and clear demarcation of the tumor was observed due to long circulation time and increased tumor accumulation of CDs. Hence, the combination of fluorescence and photoacoustic imaging into a single probe based on CDs enabled deeper tissue penetration for tumor identification. Compared with single optical imaging, the

**Figure 5.** *Carbon dots integrated multi-modal bio imaging.*

dual-mode FL/PA imaging upholds the sensitivity and provides higher-resolution anatomical images. Porphyrin implanted CDs developed by selective pyrolysis with good aqueous dispersibility displayed a strong PA signal at 686 nm in a slightly acidic or neutral environment and somewhat alkaline conditions pH 7–8 the signal was weak [61]. In breast cancer, the sentinel lymph nodes detection, the application of photo acoustic visualization of CDs was reported by Wu, *et al.* [62]. After injection the CDs exhibited rapid signal enhancement and relatively fast clearance from the lymph nodes.
