[show abstract][hide abstract] ABSTRACT: The movement from the subjects during in vivo confocal Raman spectral measurements could change the measurement volume, leading to non-specific signals and inaccurate interpretation of the acquired spectrum. Here we introduce a generally applicable method that includes (1) developing a multimodal system to achieve real-time monitoring of every spectral measurement with reflectance confocal microscopy (RCM) and multiphoton microscopy (MPM) imaging; (2) performing region-of-interest measurement by scanning an area of the tissue during spectral acquisition. The developed method has been validated by measuring different micro-structures of in vivo human skin. Our results demonstrated great consistency between RCM images and confocal Raman spectra. The superior quality of the images and spectra allows us to derive blood flow velocity and blood glucose level. We believe this method is valuable for realizing accurate microscopic spectral measurement and have great potential to be adapted into clinic to achieve non-invasive measurement of important biological parameters.
[show abstract][hide abstract] ABSTRACT: Here we present a method for improving Raman spectroscopy signal-to-noise ratio (SNR) based on fluorescence photobleaching. Good SNR is essential to obtain biochemical information about biological tissues. Subtracting high levels of tissue autofluorescence background is a major challenge in extracting weak Raman signals. We found that pre-exposure to laser light significantly reduces tissue autofluorescence, but minimally affects Raman signals, allowing subsequent acquisition of high-SNR Raman spectra. We demonstrated this method with in vivo Raman spectral measurements of human skin. This method will benefit clinical skin Raman measurements of body sites with high autofluorescence background such as the forehead and nose.
Photodiagnosis and photodynamic therapy 12/2012; 9(4):299-302.
[show abstract][hide abstract] ABSTRACT: There are increased interests on using multiphoton imaging and spectroscopy for skin tissue characterization and diagnosis. However, most studies have been done with just a few excitation wavelengths. Our objective is to perform a systematic study of the two-photon fluorescence (TPF) properties of skin fluorophores, normal skin, and diseased skin tissues. A nonlinear excitation-emission-matrix (EEM) spectroscopy system with multiphoton imaging guidance was constructed. A tunable femtosecond laser was used to vary excitation wavelengths from 730 to 920 nm for EEM data acquisition. EEM measurements were performed on excised fresh normal skin tissues, seborrheic keratosis tissue samples, and skin fluorophores including: NADH, FAD, keratin, melanin, collagen, and elastin. We found that in the stratum corneum and upper epidermis of normal skin, the cells have large sizes and the TPF originates from keratin. In the lower epidermis, cells are smaller and TPF is dominated by NADH contributions. In the dermis, TPF is dominated by elastin components. The depth resolved EEM measurements also demonstrated that keratin structure has intruded into the middle sublayers of the epidermal part of the seborrheic keratosis lesion. These results suggest that the imaging guided TPF EEM spectroscopy provides useful information for the development of multiphoton clinical devices for skin disease diagnosis.
Journal of Biomedical Optics 07/2012; 17(7):077004. · 2.88 Impact Factor
[show abstract][hide abstract] ABSTRACT: Skin cancer is the most common type of cancer in humans. Current techniques for identifying normal and neoplastic tissues are either destructive or not sensitive and specific enough. Raman spectroscopy and confocal imaging may obviate many limitations of existing methods by providing noninvasive, high-resolution, and real-time morphological and biochemical analysis of living tissues and cells.
We conducted micro-Raman spectroscopy studies on HaCaT cells, melanocytes (MC) and their malignant counterparts squamous cell carcinoma (SCC) and melanoma (MM) cells, respectively. Reflectance confocal imaging is used as guidance for the spectral measurements.
Significant differences were found between the spectra of HaCaT cells and SCC cells, MC cells and MM cells, as well as all normal cells (HaCaT and MC) and all tumor cells (SCC and MM). Approximately 90% sensitivity and specificity was achieved for all the separations that we performed.
Our results demonstrated the robust capability of confocal Raman spectroscopy in separating different cell lines. The acquired Raman spectra of major types of skin cells and their malignant counterparts will be useful for the interpretation of Raman spectra from in vivo skin. We believe it will eventually help diagnosis of skin cancer and other skin disease in clinical dermatology.
Photodermatology Photoimmunology and Photomedicine 06/2012; 28(3):147-52. · 1.52 Impact Factor
[show abstract][hide abstract] ABSTRACT: The spectral dependence of SHG intensity in biological tissues is an
optical property that is not fully understood so far. In this paper, we
will investigate this problem in detail through experiments. Through
examining different biology tissues, it is found that SHG intensity
drops down from shorter to longer wavelength from 375 nm to 460 nm. By
comparing these curves with 1/λn dependence, the n is found to
vary from 4.5 to 8.5. These patterns can not be fully explained by
scattering properties of collagen. Other factors such as direct
generation of SHG may have contribution to these wavelength dependence
patterns, which needs further investigation.
[show abstract][hide abstract] ABSTRACT: Raman spectroscopy is a minimally-invasive optical technique with great potential for in vivo cancer detection and disease diagnosis. However, there is no systematic study of the Raman spectra from different organs to date. We measured and characterized the Raman spectra eighteen naïve mouse organs in a broad frequency range of 700 to 3100 cm⁻¹. The peaks of generic proteins and lipids appeared in Raman spectra of all organs. Some organs like bone, teeth, brain and lung had unique Raman peaks. The autofluorescence was strong in liver, spleen, heart, and kidney. These results suggest that organ specific Raman probe design and specific data processing strategies are required in order to get the most useful information.
[show abstract][hide abstract] ABSTRACT: We present a multiphoton microscopy instrument specially designed for in vivo dermatological use that is capable of imaging human skin at 27 frames per second with 256 pixels × 256 pixels resolution without the use of exogenous contrast agents. Imaging at fast frame rates is critical to reducing image blurring due to patient motion and to providing practically short clinical measurement times. Second harmonic generation and two-photon fluorescence images and videos acquired at optimized wavelengths are presented showing cellular and tissue structures from the skin surface down to the reticular dermis.
[show abstract][hide abstract] ABSTRACT: There has been a dramatic increase in photothermal therapy as a minimally invasive treatment modality for cancer treatment due to the development of novel nanomaterials as the light absorption agents. Single-wall carbon nanotubes (SWNTs) with strong optical absorption in the broad visible and near IR offer unique advantages for photothermal cancer therapy. A broad range of wavelengths can be used for the treatment with SWNTs, whereas conventional photothermal therapeutic agent is designed to absorb light only near one selected wavelength. The objective of this study is to validate the hypothesis that intratumoral injected SWNTs can absorb 785 nm near IR laser light and generate significant local hyperthermia to destroy tumors.
SCCVII tumor in C3H/HeN mice was exposed to 785-nm laser after intratumoral injection of SWNTs with different light and SWNTs dose combinations. The temperatures of the tumor with laser irradiation were monitored. In vivo and ex vivo Raman spectra in different organs were obtained with a rapid Raman system. Tumor responses (tumor volume and mouse survival) were documented daily after treatment up to day 45 to assess the effectiveness of the treatment.
The temperature within the tumors increased in a light- and SWNTs-dose dependent manner. Squamous cell carcinomas can be eradicated at a moderate light irradiance and fluence (200 mW/cm² and 120 J/cm²). This light dose is also comparable to those used with photodynamic therapy. Tissue Raman spectroscopy measurements revealed that SWNTs remained localized in the tumor even 3 months after injection but was not found in other organs.
This animal study represents a significant step forward towards the goal of advancing SWNTs based photothermal cancer therapy into clinical applications.
Lasers in Surgery and Medicine 11/2010; 42(9):638-48. · 2.46 Impact Factor
[show abstract][hide abstract] ABSTRACT: Single-wall carbon nanotubes (SWNTs) are a new type of nanomaterial with strong optical absorption. SWNTs also have intense Raman signals that facilitate convenient monitoring of their location within tissue thereby enabling noninvasive pharmacokinetic study. We hypothesize that SWNTs can absorb 785-nm laser light and generate significant local hyperthermia to destroy cancer cells and eradicate tumors. In this study a 785-nm diode laser is used for both Raman excitation and photothermal therapy. SWNTs are made water-soluble by functionalizing with polyethylene glycol (PEG) and administrated by intratumoral injection. C3H/HeN mice were injected subcutaneously with 2 million mouse squamous cell carcinoma (SCCVII) cells to create the tumor model. We conducted experiments with 100 mice divided into 10 different groups: control, SWNT only, 100 mW/cm2 laser irradiation only, 200 mW/cm2 laser irradiation only, and 6 treatment groups with different drug and light dose combinations (SWNTs 0.1, 0.5. 1 mg/ml, laser 100 and 200 mW/cm2). The treatment time was 10 minutes. The temperatures of the tumors irradiated by laser were monitored by an IR thermometer. Mice survival was observed for 45 days. The study revealed that the temperature within the tumors increased in a light- and drug-dose dependent manner. The optimized light and drug dose combinations (1 mg/ml + 120 J/cm2) resulted in tumor temperature elevation of 18.5°C and successful eradication of the tumors. This light dose is moderate and is as low as 1/10 of other published studies using nanomaterials. The Raman spectroscopy measurements suggest that SWNTs persisted within the tumor tissue for months.
[show abstract][hide abstract] ABSTRACT: We present the construction of a new multimodal, multiphoton spectroscopic and imaging instrument for in vivo patient use. Utilizing a tunable femtosecond laser, we are able to simultaneously acquire two-photon excited fluorescence, second harmonic generation, and confocal reflectance images at half video rate, while concurrently acquiring two-photon excited fluorescence and second harmonic generation spectra.
[show abstract][hide abstract] ABSTRACT: Ten groups of mice were exposed to 785 nm laser after intratumoral injection of single-wall carbon nanotubes (SWNTs). The temperature within the tumors increased in light- and drug-dose dependent manner; optimized light and drug dose combinations resulted in eradication of skin tumors. SWNTs persisted within the tumor tissue for months.
[show abstract][hide abstract] ABSTRACT: We report various technologies developed in our lab for in vivo Raman spectroscopy and related clinical applications. This includes macroscopic probes and endoscopy catheters that interrogate millimeter-scale tissue volumes for skin and lung cancer detection and a confocal microscopy system for depth-resolved Raman measurements of the skin in vivo.
[show abstract][hide abstract] ABSTRACT: BACKGROUND/PURPOSES: Understanding the two-photon excitation spectral characteristics and microscopic morphology of cutaneous collagen and elastic tissue components is important for applying multiphoton microscopy (MPM) in basic skin biology research and for clinical diagnosis.
We developed a system for two-photon excitation spectral measurements at various excitation wavelengths. The microscopic morphology was studied using a commercial multiphoton microscope.
We obtained two-photon excitation fluorescence (TPEF) excitation-emission matrices (EEM), for the first time, of purified collagen and elastin samples, as well as in situ collagen and elastic fibers within excised human dermis. The EEM of the dermis was found to be similar to that of elastin. The excitation spectra for second harmonic generation (SHG) from purified collagen and excised dermis were also studied and were found to have similar spectral shapes.
This study, using the EEM spectroscopic approach, confirmed a previous imaging study inference that in the dermis, TPEF predominantly originates from elastic fibers, while SHG originates solely from collagen fibers. The EEM data and SHG excitation spectra obtained in this study can be used to guide the selection of excitation wavelengths for MPM applications in basic skin biology research and for clinical diagnosis.
Skin Research and Technology 11/2009; 15(4):418-26. · 1.41 Impact Factor
[show abstract][hide abstract] ABSTRACT: As applied to microscopy, the confocal technique is a powerful method that can non-invasively provide depth-resolved information on tissue because of its optical sectioning capability. Raman spectroscopy is a non-invasive optical technique that is very sensitive to the structure and conformation of biochemical constitutes. Combining these two distinct techniques can non-invasively provide depth-resolved biochemical information of the skin in vivo. In this paper, we present a novel confocal Raman spectrometer and preliminary results on in vivo Raman spectral measurements of mouse tumor model. A total of 255 Raman spectra were taken from 15 mice in vivo. We found that Raman spectra of different sub-layers in mouse skin differ significantly. Obvious spectral changes for the dermis were also observed between normal skin and skin immediately overlying subcutaneous tumor.