The team of Ye Jian of Shanghai Jiaotong University achieved the detection of Raman optical signal penetrating 14 cm muscle tissue within the safe dose of light

Non-invasive detection of tumor lesions in vivo is essential
for clinical oncology diagnosis and treatment.
Medical imaging techniques such as computed tomography, magnetic resonance or positron emission computed tomography can diagnose deep lesions in the body, but they are more commonly used for preoperative examinations
due to long collection time, expensive instruments or large radiation doses.
In contrast, optical detection and imaging methods have the advantages of real-time, high sensitivity, non-ionizing radiation, convenient collection, etc.
, combined with exogenous contrast agents can provide accurate information about the structure, function and molecules of organisms, which is an excellent tool
for tumor diagnosis.
However, the further development of existing tumor optical detection technology also faces a bottleneck: the depth of tissue penetration is low, and deep lesions
cannot be detected.
Due to the strong scattering and absorption of photons by biological tissues (Figure 1), the limited penetration depth of light in biological tissues has been a great challenge
in this field.
For example, the average free path of transmission of muscle tissue in the near-infrared region is only 1~2 mm, and the tissue penetration depth of fluorescence imaging technology widely used is usually only a few millimeters
.
Clinical results showed that molecular imaging based on indocyanine green could not detect pulmonary nodules more than 1.
3 cm from the pleural depth, which was prone to false negatives
.

Figure 1.
Scattering and absorption of photons by biological tissues

Surface-enhanced Raman spectroscopy (SERS) greatly enhances the Raman signal of molecules near metal nanoparticles, and has the advantages of high specificity and sensitivity, which is very suitable for biological spectroscopy
.
In order to obtain a higher detection depth, a spatial shift Raman spectroscopy device
with a certain spatial shift between the light source and the detector has been reported.
It takes advantage of the high scattering properties of biological tissue, where photons from deep layers have a greater lateral shift
when they reach the surface.
Spatially shifted Raman spectroscopy suppresses the background signal on the surface, thus improving the signal-to-noise ratio
from the deep signal.
A special form of it is transmission Raman spectroscopy, which places a laser and a Raman detector on either side
of the sample.
Transmission Raman spectroscopy has been reported to enable non-invasive detection
with high tissue penetration.

Nevertheless, the state of the art in transmission Raman spectroscopy has not met the needs of
practical biomedical applications.
First, the detection depth or tissue thickness of transmission Raman spectroscopy currently reported in the literature is still much lower than the thickness value
associated with the human body.
For example, humans have an abdominal dorsal distance of more than 10 cm
.
However, the feasibility of using transmission Raman spectroscopy to penetrate in vitro tissues or live animals more than 10 cm thick has not been confirmed
to date.
Second, the propagation process of photons in transmission Raman detection and how measurement factors determine the signal is unclear
.
The transmitted Raman signal is not only affected by the tissue scattering coefficient and absorption coefficient, but also may be related to
the brightness, lesion burial depth, total tissue thickness and other factors of SERS nanoprobes.
It is crucial
to assess the relationship between these determinants.
Third, the safety of lasers is a long-term concern in the clinical transformation of optical
modalities.
The photosafety of clinical lasers is usually assessed by the maximum permissible amount of irradiation, i.
e.
the highest level of laser radiation with a negligible risk of damage to exposed body
surfaces.
However, most current in vivo SERS studies use laser doses well above light-safe dose limits, which largely hinders the clinical translation
of SERS technology.

Figure 2.
Schematic diagram of in vivo non-invasive imaging of deep tumors using transmission device and ultra-bright SERS contrast agent and theoretical calculation of transmission Raman spectroscopy signal in vivo mice

In order to solve the above important problems in this field, the team of Jian Ye from the School of Biomedical Engineering of Shanghai Jiao Tong University first studied the influence of experimental parameters (tissue thickness, SERS nanoprobe position, nanoprobe brightness, laser power and beam size) on the detection depth of transmission Raman spectroscopy (Figure 2)
from the theoretical modeling and calculation of Raman photon propagation during transmission Raman spectroscopy.
Theoretical calculations show that there is an asymmetrical U-shaped relationship between the transmitted Raman signal and the buried depth of the signal source, indicating that the signal is weakest when the lesion is located in the middle of the tissue, and the detection of transmitted Raman signal is the most challenging
.
Increasing the brightness of SERS nanoprobes is the most direct and effective way to
increase the detection depth/transmission tissue thickness.
In addition, the increase in beam size has little effect
on the transmission Raman detection intensity of deep lesions.
Therefore, larger laser beam sizes can be used to reduce power density
.

Figure 3.
In vitro transmission Raman spectroscopy detection for diffused beam illumination

Based on these findings, the team designed and fabricated ultra-bright SERS nanoprobes combined with a homemade transmission Raman device to develop a Raman detection/imaging system
.
The system offers the following advantages: depth detection capability, using ultra-bright SERS nanoprobes down to the level of single-particle detection; Clinical photosafety, the laser power density of the sample surface is below the safe light dose threshold
.

Using this system, the team successfully achieved detection of SERS nanoprobes embedded in them through 14 cm thick tissue in vitro within a safe light dose (Figure 3), with a depth of penetration of approximately 97%
compared to the currently reported transmission Raman spectroscopy detection study.
Further, the team achieved in vivo noninvasive imaging of deep SERS nanoprobes in unshaved live mice 1.
5 cm thick within a safe light dose (Figure 4), compared to conventional backscattered Raman imaging
.
This work provides new insights into the development of transmission Raman spectroscopy in vivo non-invasive biomedical examinations, demonstrating that transmission Raman spectroscopy has the potential to become a viable tool
for future clinical cancer diagnosis.

Figure 4.
Safe transmission Raman spectroscopy detection of noninvasive light in living mice

The study, "In Vivo Surface-Enhanced Transmission Raman Spectroscopy Under Maximum Permissible Exposure: Toward Photosafe Detection of Deep-Seated Tumors," was recently published in Small Methods (2022).
The first author of the paper is Yumin Zhang, a doctoral student at the School of Biomedical Engineering, Shanghai Jiao Tong University, and assistant professor Lin Li and Professor Ye Jian of the School of Biomedical Engineering, Shanghai Jiao Tong University are co-corresponding authors
.
This work was also strongly supported
by the team of Miao Peng, an associate researcher at the School of Biomedical Engineering, Shanghai Jiao Tong University.
The research was supported
by the National Natural Science Foundation of China, Shanghai Municipal Science and Technology Commission, Shanghai Jiao Tong University, Shanghai Key Laboratory of Gynecologic Oncology, Shanghai Education Development Foundation, and Shanghai Municipal Education Commission's "Chenguang Program".

Link to paper: https://doi.
org/10.
1002/smtd.
202201334