Advances in optical imaging technologies have a way of revealing structures in new ways, seeing things in a new light, and providing new information about fundamental biological processes such as carcinogenesis. Two of the factors driving these technologies are new laser sources and new ways of pulse-shaping or manipulating light to enhance nonlinear optical imaging processes in tissues. Recent work has focused on using lower repetition-rate ultrafast lasers to pump photonic crystal fibers to generate stable, low-noise, widely coherent supercontinuum that can then be used to excite multiple nonlinear processes in tissues. Simultaneous label-free autofluorescence multi-harmonic (SLAM) microscopy was recently demonstrated to generate two- and three-photon autofluorescence and second- and third-harmonic generation imaging channels simultaneously, enabling structural, molecular, and metabolic imaging in real time. Importantly, this is label-free, as labels may alter the biology. SLAM microscopy was recognized as one of the recipients of the Microscopy Today Innovation Awards for 2019.
Historically, advances in imaging have enabled investigators to visualize things in a novel manner, and therefore ask new questions that were previously not conceivable. Recently the group headed by Stephen Boppart, the same group that developed SLAM microscopy, has taken the technology a step futher. In a publication by Sixian You, Stephen Boppart and others showed that it was possible to visualize extracellular vesicles (EVs) in tissues and in vivo without the use of dyes, probes, or other labels [Reference You1]. The most exciting part is that this may have clinical applications. EVs is a collective term for exosomes, microvesicles, and apoptotic bodies. These cell-derived particles have generated extensive interest as we learn more about how cells use them for inter-cellular communication in states of health and disease, particularly in cancer. It is known that cancer cells rapidly increase the production of EVs early in the disease, well before solid tumors appear. Therefore, it is thought that these EVs are important in the progression and spread of cancer. It is hypothesized that EVs released from cancer cells circulate throughout the body early in carcinogenesis, are taken up by normal cells, and direct them to upregulate their metabolism. This could facilitate the body to host the growing and spreading tumor.
Whereas much remains to be elucidated about EVs, it is exciting that SLAM microscopy is able to detect and visualize the presence and spatial distribution of EVs in tissue specimens and in vivo. The recent paper by You et al., with their multimodal optical imaging approach, identified unique optical signatures in breast cancer. They also showed that these signatures of EVs can be used to differentiate tumor-associated EVs from normal EVs and also indicate the aggressiveness of the tumor (Figure 1). They think that the autofluorescence generated by nicotinamide adenine dinucleotide phosphate (NADPH) within the EVs, as well as the size and structural composition of the EV membrane, in turn generates a strong third-harmonic signal between lipid-aqueous interfaces. You et al. showed that these signatures from the EVs coming from various breast cancer (and normal) cell lines from rat mammary tumors and from human breast cancer specimens indicate the tumor when imaging during surgery. They also found signatures for normal EVs in breast tissues from normal patients.
Figure 1: Label-free SLAM microscopy image of a mammary tumor. The multimodal color channels include: Green – Second Harmonic Generation from collagen; Magenta – Third Harmonic Generation from interfaces, such as between lipid/aqueous compartments; Yellow – Two-Photon Excited Autofluorescence from FAD; Blue – Three-Photon Excited Autofluorescence from NAD(P)H. Extracellular vesicles appear primarily as blue/magenta single-pixel objects due to their NAD(P)H content and size.
Whereas SLAM microscopy has generated stunning and data-rich images, there also exists the potential for using these unique multi-modal optical signatures as biomarkers for both clinical diagnosis and for screening. Ongoing efforts by this team may lead to a way of detecting cancer in urine samples[Reference Boppart2]. This potentially will shed new light on liquid biopsies for cancer!
