Monitoring neoadjuvant therapy responses in rectal cancer using multimodal nonlinear optical microscopy.

Most patients with rectal cancer have a better prognosis after receiving neoadjuvant therapy because of its remarkable curative effect. However, no device delivers real-time histopathologic information on treatment response to help clinicians tailor individual therapeutic strategies. We assessed the potential of multimodal nonlinear optical microscopy to monitor therapeutic responses, including tumoral and stromal responses. We found that two-photon excited fluorescence imaging can, without labeling, identify colloid response, inflammatory cell infiltration, vascular proliferation, and tumor regression. It can also directly detect rare residual tumor cells, which may be helpful for distinguishing tumor shrinkage from tumor fragmentation. In addition, second harmonic generation imaging shows the ability to monitor three types of fibrotic responses: mature, intermediate, and immature. We also determined nonlinear spectra, collagen density, and collagen orientation indexes to quantitatively analyze the histopathologic changes induced by neoadjuvant therapy in rectal cancer. Our work demonstrates that nonlinear optical microscopy has the potential to become a label-free, real-time, in vivo medical imaging technique and provides the groundwork for further exploration into the application of nonlinear optical microscopy in a clinical setting.

Multiphoton microscopy for tumor regression grading after neoadjuvant treatment for colorectal carcinoma.

AIM: To evaluate the feasibility of using multiphoton microscopy (MPM) to assess a tumor regression grading (TRG) system. METHODS: Fresh specimens from seven patients with colorectal carcinoma undergoing neoadjuvant radiochemotherapy at the Fujian Medical University Union Hospital were obtained immediately after proctectomy. Specimens were serially sectioned (10 µm thickness) and used for MPM or stained with hematoxylin and eosin for comparison. Sections were imaged by MPM using 810 nm excitation, and images were collected in two wavelength channels corresponding to second-harmonic generation (SHG) and two-photon excited fluorescence (TPEF) signals. The ratio of these signal intensities was used to distinguish fibrosis from normal mucosal and serosal tissues. RESULTS: TRG of specimens assessed by MPM were in complete agreement with histologic grading performed by a consulting pathologist. SHG and TPEF images clearly revealed collagen fibers and fragmented elastic fibers in the muscularis propria specimens following neoadjuvant radiochemotherapy. Additionally, blood vessel hyperplasia was observed as thickening and fibrosis of the intima and media, which was accompanied by minimal inflammatory cell infiltration. Furthermore, the SHG/TPEF ratio in stromal fibrosis (4.15 ± 0.58) was significantly higher than those in the normal submucosal (2.31 ± 0.52) and serosal (1.47 ± 0.10) tissues (P < 0.001 for both). Analysis of emission spectra from cancerous tumor cells revealed two peaks corresponding to nicotinamide adenine dinucleotide hydrogen and flavin adenine dinucleotide signals; the ratio of these values was 1.19 ± 0.02, which is close to a normal metabolic state. CONCLUSION: MPM can be used to perform real-time diagnosis of tumor response after neoadjuvant treatment, and can be applied to evaluate TRG.

[Effects of laser-assisted cochleostomy on inner hair cell ribbon synapse in rats].

OBJECTIVE: To investigate the effects on ribbon synapse of inner hair cells after superpulsed CO2 laser-assisted cochleostomy in SD rats. METHODS: Eighteen SD rats were randomly divided into laser-assisted surgery groups (2 W group and 5 W group), sham-operated group and control group. Ten of those were performed a cochleostomy using superpulsed CO2 laser with a corresponding power. Auditory brainstem responses (ABR) were measured pre-and postoperatively. The ribbon synapses at apical and middle cochlear turns were observed under laser scanning confocal microscope and then were quantified with 3ds Max software. RESULTS: The postoperative ABR thresholds of the 2 W and 5 W groups were larger than the preoperative case (t = -5.65, P < 0.01; t = -4.97, P < 0.01). The synapse number at the middle turn decreased significantly in 5 W group (F = 17.15, P < 0.01), while no significant changes were noted at the apical turn (P > 0.05). There was no statistical difference in 2 W group (P > 0.05). CONCLUSIONS: The superpulsed CO2 laser-assisted cochleostomy with high-power is accompanied by a synaptic injury, while no obvious effects after the low-power laser surgery, which might be a safe strategy to preform cochleostomy.

[Nonlinear spectral imaging of DNA specimens derived from tumor cells based on second harmonic generation].

Second harmonic generation (SHG) is a second-order nonlinear optical process that has symmetry constraints confining signal to regions lacking a center of symmetry. Using SHG microscopy, a variety of tissue structures have noninvasively been imaged by virtue of intrinsic signal generated by structured proteins such as collagen fibrils in connective tissues or the actomyosin lattice of muscle cells. In biochemistry and structure biology, the high-level structures of DNA and protein macro-molecules are similar in constructing mechanism, although DNAs consist of deoxynucleotides and proteins of amino acid residues. The principal purpose of present work is to detect the SHG signal from different DNA samples by spectral imaging technology based on two-photon excited fluorescence (TPEF) and SHG. These DNA samples include the solution of genomic DNA and extracted nuclei, and cultured living cells. Results show that we can obtain the SHG signal from solution of genomic DNA and extracted nuclei in routine condition, but nothing from cultured cell nuclei. After adding a little of absolute ethanol (less than 5% by volume) in culture medium, the SHG signal is detectable in the interested region of nuclei. The findings suggest that the interaction between ethanol and DNA in living cell gives rise to the shift of molecular conformation, and this shift changes some nonlinear optical properties of DNA molecules.