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Keywords:

  • bronchoscopy;
  • confocal endomicroscopy;
  • narrow-band imaging;
  • optical coherence tomography;
  • Raman spectroscopy

ABSTRACT

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. PRINCIPLES OF OPTICAL IMAGING
  5. AFB
  6. NBI
  7. OPTICAL COHERENCE TOMOGRAPHY
  8. CONFOCAL ENDOMICROSCOPY AND ENDOCYTOSCOPY
  9. LASER RAMAN SPECTROSCOPY
  10. SUMMARY AND FUTURE DIRECTIONS
  11. ACKNOWLEDGEMENT
  12. REFERENCES

Bronchoscopy is a minimally invasive method for diagnosis of diseases of the airways and the lung parenchyma. Standard bronchoscopy uses the reflectance/scattering properties of white light from tissue to examine the macroscopic appearance of airways. It does not exploit the full spectrum of the optical properties of bronchial tissues. Advances in optical imaging such as optical coherence tomography (OCT), confocal endomicroscopy, autofluorescence imaging and laser Raman spectroscopy are at the forefront to allow in vivo high-resolution probing of the microscopic structure, biochemical compositions and even molecular alterations in disease states. OCT can visualize cellular and extracellular structures at and below the tissue surface with near histological resolution, as well as to provide three-dimensional imaging of the airways. Cellular and subcellular imaging can be achieved using confocal endomicroscopy or endocytoscopy. Contrast associated with light absorption by haemoglobin can be used to highlight changes in microvascular structures in the subepithelium using narrow-band imaging. Blood vessels in the peribronchial space can be displayed using Doppler OCT. Biochemical compositions can be analysed with laser Raman spectroscopy, autofluorescence or multispectral imaging. Clinically, autofluorescence and narrow-band imaging have been found to be useful for localization of preneoplastic and neoplastic bronchial lesions. OCT can differentiate carcinoma in situ versus microinvasive cancer. Endoscopic optical imaging is a promising technology that can expand the horizon for studying the pathogenesis and progression of airway diseases such as COPD and asthma, as well as to evaluate the effect of novel therapy.


INTRODUCTION

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. PRINCIPLES OF OPTICAL IMAGING
  5. AFB
  6. NBI
  7. OPTICAL COHERENCE TOMOGRAPHY
  8. CONFOCAL ENDOMICROSCOPY AND ENDOCYTOSCOPY
  9. LASER RAMAN SPECTROSCOPY
  10. SUMMARY AND FUTURE DIRECTIONS
  11. ACKNOWLEDGEMENT
  12. REFERENCES

To understand the biological processes in airway diseases such as asthma, COPD and bronchial cancers, it is crucial to have non-invasive or minimally invasive means to image structures in the airways down to the cellular or subcellular level in vivo. Quantitative CT is an emerging radiological tool that allows for direct visualization of airways and lung parenchyma non-invasively, and with minimal discomfort to the patients.1,2 CT imaging studies have reported that changes in airway wall thickness correlate with disease severity.3 Studies in asthma have shown that CT measurements of airway wall dimensions correlate with bronchial biopsy measurements of airway wall thickness.4 Although CT provides airway images up to and including the fourth or fifth generation, resolution beyond this airway generation (in-plane 0.5 mm/out-of-plane 1 mm thickness) is inadequate.5 More importantly, CT cannot measure morphological changes in different layers of the airway wall because the tissue layers are below the resolution of CT. Likewise, CT cannot detect preinvasive or microinvasive bronchial cancers in the central airways, as these lesions are below the resolution of CT. However, bronchoscopic optical imaging can provide micron-scale resolution of tissue structures including the microvasculature. Biochemical changes in airway can also be analysed using optical methods. In this review, we summarize the principles of optical imaging and the recent advances in bronchoscopic imaging applying these concepts, and discuss the future directions.

PRINCIPLES OF OPTICAL IMAGING

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. PRINCIPLES OF OPTICAL IMAGING
  5. AFB
  6. NBI
  7. OPTICAL COHERENCE TOMOGRAPHY
  8. CONFOCAL ENDOMICROSCOPY AND ENDOCYTOSCOPY
  9. LASER RAMAN SPECTROSCOPY
  10. SUMMARY AND FUTURE DIRECTIONS
  11. ACKNOWLEDGEMENT
  12. REFERENCES

When the bronchial surface is illuminated by light, the light can be reflected from the surface (specular reflection), be absorbed, induce autofluorescence, or travel into the bronchial tissue and backscatter at the same wavelength as the incident light (elastic scattering). The light can also be scattered at a different wavelength (inelastic or Raman scattering) by exciting molecular vibrations.6 White-light bronchoscopy (WLB), the simplest and most commonly used bronchoscopic imaging method, makes use of the specular reflection, backscattering and absorption properties of broadband visible light from ∼400 nm to 700 nm to define the structural features of the bronchial surface to discriminate between normal and abnormal tissues. To highlight the vasculature, narrow-band blue light centred at 415 nm (400 nm to 430 nm) and green light centred at 540 nm (530–550 nm) corresponding to the maximal haemoglobin absorption peaks can be used. The blue light highlights the superficial capillaries, while the green light can penetrate deeper to highlight the larger blood vessels in the submucosa. The narrow bandwidths reduce the scattering of light from other wavelengths that are present in a broad spectrum white-light and enable enhanced visualization of blood vessels. This is called narrow-band imaging (NBI).7–9 NBI provides more detailed images of the microvasculature in preneoplastic and neoplastic lesions reflective of the altered angiogenesis process. Autofluorescence bronchoscopy (AFB) makes use of fluorescence and absorption properties to provide information about the biochemical composition and metabolic state of endogenous fluorophores in bronchial tissues.10 Most endogenous fluorophores are associated with the tissue matrix or are involved in cellular metabolic processes. The most important fluorophores are structural proteins, such as collagen and elastin, and those involved in cellular metabolism such as nicotinamide adenine dinucleotide (NADH) and flavins. Other fluorophores include the aromatic amino acids, various porphyrins and lipopigments. The fluorescence properties of bronchial tissue is determined by the distribution of fluorophores, their distinct excitation and emission spectra, their metabolic state, the tissue architecture and the wavelength-dependent light attenuation due to the concentration, as well as distribution of non-fluorescent chromophores such as haemoglobin.6 Upon illumination by violet or blue light (380–460 nm), normal bronchial tissues fluoresce strongly in the green (480–520 nm). In disease states such as lung cancer, as the bronchial epithelium changes from normal to dysplasia and then to carcinoma in situ (CIS) and invasive cancer, there is a progressive decrease in green autofluorescence but proportionately less decrease in the red fluorescence intensity. These differences between normal, preneoplastic and neoplastic tissues are due to breakdown of stromal collagen cross-links, increase in cellular metabolic activity leading to increases in NADH, flavin adenine dinucleotide coenzymes, increased absorption of the excitation violet/blue light by haemoglobin due to angiogenesis, as well as changes in the light-scattering process from an increase in nuclear size, cellular density and distribution of the cells associated with lung cancer development. Exploitation of the full spectrum of the optical properties of bronchial tissues leads to development of better endoscopic imaging tools for clinical applications and for research.

AFB

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. PRINCIPLES OF OPTICAL IMAGING
  5. AFB
  6. NBI
  7. OPTICAL COHERENCE TOMOGRAPHY
  8. CONFOCAL ENDOMICROSCOPY AND ENDOCYTOSCOPY
  9. LASER RAMAN SPECTROSCOPY
  10. SUMMARY AND FUTURE DIRECTIONS
  11. ACKNOWLEDGEMENT
  12. REFERENCES

The excitation wavelength that produces the highest tumour to normal tissue intensity and chromatic contrast is 405 nm.11,12 The fluorescence differences between 480 nm and 700 nm in normal, preneoplastic and preneoplastic tissues serve as the basis for the design of several autofluorescence endoscopic imaging devices for localization of early lung cancer in the bronchial tree.10 Commercially available AFB devices make use of a combination of autofluorescence and reflectance imaging to optimize the image quality. AFB allows rapid scanning of large areas of the bronchial surface for subtle abnormalities that are not visible to white-light examination. Since its first introduction in 1991,13,14 several improvements have been made in the device design. Most devices now use a filtered lamp for illumination and non-image intensified sensors for imaging.15–19 Switching between white-light and fluorescence examination no longer requires plugging the light guide into different light sources but rather by pressing a switch button. Instead of mounting the camera onto the eyepiece of a fibre-optic bronchoscope, the charged coupled device (CCD) sensor at the tip of a videoendoscope is used for imaging.16,20 Small amounts of reflected light (blue, green or near infrared) is employed to form a reflectance image that is then used to enhance the chromatic contrast and to normalize the green autofluorescence image to correct for non-uniformity caused by optical and geometrical factors, such as variable distances and angles between the endoscope tip to the bronchial surface. Depending on the type of reflected light used to combine with the fluorescence image for display, abnormal areas appear brownish red, red, purple or magenta, while normal areas appear green or light blue.14–20 Some devices allow simultaneous display of the white-light and fluorescence images.20,21

In addition to multiple single-centre studies,10 there are two randomized trials22,23 and three large multicentre trials15,24,25 comparing WLB and AFB. The studies showed an improvement in the detection rate of high-grade dysplasia, CIS and microinvasive cancer with AFB compared with WLB. In general, there is a twofold improvement in the relative sensitivity with AFB. The specificity of AFB is low (∼60% vs ∼90% for WLB) due to false positive fluorescence with inflammation, mucous gland hyperplasia and interobserver error. The specificity of AFB can be improved to 80% by quantifying the red to green fluorescence (R/G) ratio of the target lesion during the bronchoscopic procedure.26 Combining the R/G ratios with the visual score improved the specificity further to 88%. Similar results were found in a multicentre trial, where the R/G ratios were hidden from the bronchoscopists when making the visual classification of the bronchial mucosal changes.15 Quantitative imaging decreases intraobserver and interobserver variation.

NBI

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. PRINCIPLES OF OPTICAL IMAGING
  5. AFB
  6. NBI
  7. OPTICAL COHERENCE TOMOGRAPHY
  8. CONFOCAL ENDOMICROSCOPY AND ENDOCYTOSCOPY
  9. LASER RAMAN SPECTROSCOPY
  10. SUMMARY AND FUTURE DIRECTIONS
  11. ACKNOWLEDGEMENT
  12. REFERENCES

NBI is now classified as an image-enhanced endoscopy technology.27,28 It is widely used in gastrointestinal endoscopy for classification of lesions.29,30 Its place in routine bronchoscopic examination has not been established. The advantage of NBI over other techniques is its ability to enhance fine superficial microvessel patterns. Angiogenesis is a relatively early event during lung cancer pathogenesis.31 A new morphological entity called angiogenic squamous dysplasia (ASD) was identified in the large central airways by fluorescence bronchoscopy, whereby collections of capillary blood vessels were closely juxtaposed to and projected into dysplastic bronchial epithelium.32,33 NBI can target the angiogenic features of neoplasia to improve the detection of preinvasive lesions and to differentiate these lesions from invasive carcinoma. Shibuya et al.34 used NBI in conjunction with high-magnification bronchovideoscopy to observe 67 sites of abnormal fluorescence in 48 patients with sputum cytology specimens suspicious or positive for malignancy. Dotted vessels among the increased capillary density and complex vascular networks of tortuous vessels were observed in 18 abnormal fluorescence sites, of which 14 (78%) exhibited ASD. Of the 49 sites with abnormal fluorescence but no dotted vessels, 48 (98%) were without ASD. There was a significant association between the frequency of dotted vessels found by NBI and pathologically confirmed ASD (P = 0.002). More recently, NBI with a high-resolution videobronchoscope was investigated in patients with squamous cell carcinoma, CIS or ASD.35 Capillary blood vessel and tumour-vessel diameters were compared between 11 ASD, 5 CIS, 5 microinvasive carcinomas and 10 invasive carcinomas using image cytometry. There is a progressive pattern of neovascularization correlating with the progression of invasiveness. Some dotted vessels, increased vessel growth and complex networks of tortuous vessels of various sizes were observed with ASD. With CIS, dotted vessels and small spiral or corkscrew-type tumour vessels were observed. Prominent spiral or corkscrew-type tumour vessels of various sizes and grades were visible in microinvasive or invasive lung cancer.35 A prospective study compares the performance of WLB followed by either autofluorescence imaging (AFI) or NBI as determined by a randomized code in the diagnosis of intraepithelial neoplasia.36 Ninety-eight biopsies in 57 patients were evaluated: 18 lesions from 17 patients were either CIS or high-grade dysplasia (moderate to severe), and 80 lesions from 17 patients were squamous metaplasia or mild dysplasia. The sensitivity of WLB to detect high-grade dysplasia or CIS was 0.18, and the specificity was 0.88. The relative sensitivity of WLB + AFI versus WLB alone was 3.7 compared with 3.0 with WLB + NBI, but the difference was not statistically significant. The relative specificities of WLB + AFI and WLB + NBI were 0.5 and 1.0, respectively. WLB + NBI showed a significantly higher specificity compared with WLB + AFI (P < 0.001). The number of angiogenic dysplastic lesions in this study was not reported.

A question that has not been addressed adequately is how NBI compares with high-definition white-light endoscopy that makes use of CCD with markedly higher pixel densities and high-definition images. The issue of training to recognize vascular patterns versus abnormal autofluorescence needs to be investigated further as well.

OPTICAL COHERENCE TOMOGRAPHY

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. PRINCIPLES OF OPTICAL IMAGING
  5. AFB
  6. NBI
  7. OPTICAL COHERENCE TOMOGRAPHY
  8. CONFOCAL ENDOMICROSCOPY AND ENDOCYTOSCOPY
  9. LASER RAMAN SPECTROSCOPY
  10. SUMMARY AND FUTURE DIRECTIONS
  11. ACKNOWLEDGEMENT
  12. REFERENCES

Optical coherence tomography (OCT) is an optical imaging method that can offer near histological resolution for visualizing cellular and extracellular structures at and below the tissue surface.37–41 OCT is similar to ultrasound, but properties of light waves instead of sound waves is used for imaging. Optical interferometry is used to detect the light that is scattered or reflected by the tissue to generate a one-dimensional tissue profile along the light direction. By scanning the light beam over the tissue, two-dimensional images or three-dimensional volumetric images can be recorded. The image contrast is the backscattered light from interfaces at different depths in the tissue due to the heterogeneity of optical refractive indices from different tissue compositions and densities. Changes in the extracellular matrix can be readily seen due to the strong backscattering properties of collagen and elastin. The imaging procedure is performed using fibre-optic probes that can be miniaturized to enable imaging of airways down to the terminal bronchiole. These probes can be inserted down the instrument channel during standard bronchoscopic examination under conscious sedation.

OCT axial and lateral resolutions range from approximately 5 µm to 30 µm depending on imaging conditions, typically more than an order of magnitude better than ultrasound. The increase in resolution with OCT compared with ultrasound is offset by a corresponding loss in imaging depth, typically 2–3 mm in tissue for OCT. However, this combination of resolution and imaging depth is ideal for examining preneoplastic changes originating in epithelial tissues or airway diseases involving smaller airways. Additionally, unlike ultrasound, light does not require a liquid coupling medium and thus is more compatible with airway imaging. There are no associated risks from the weak near-infrared light sources that are used for OCT.

OCT probes that scan in a circumferential manner are ideally suited to imaging airways because they produce cross-sectional images of the airways. This type of scanning is usually accomplished using optics at the distal end of the probe to direct the imaging light approximately perpendicular to the fibre-optic axis. By rotating the optical fibre, the imaging light traces a circular path. If the probe is also pulled back while spinning, a helical scan trace is followed, allowing a cylindrical volume of tissue to be imaged (Fig. 1a). To minimize motion artefacts due to various sources of motion (e.g. patient movement, cardiac motion, breathing, bronchoscopist motion), fast OCT imaging systems are generally desired.

image

Figure 1. Cross-sectional and three-dimensional optical coherence tomography image of a terminal bronchiole with adjacent alveoli from a smoker without (a) and with emphysema with enlarged air spaces and destroyed alveolar walls (b).

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A recently commercialized OCT device that can be adapted for lung exploration (Lightlabs C7XR, St. Jude Medical, Inc., St. Paul, MN, USA) can produce images of the respiratory tract with an axial resolution of 12–15 µm and a penetration depth of up to 2.5 mm. The system uses a small optical probe with an outer diameter of 0.9 mm. The imaging speed is such that several centimetres of airway can be imaged in a few seconds.

Tsuboi et al.40 collected OCT images of normal bronchus, primary tumours and alveoli from seven human lung cancer lobectomy specimens. OCT images were also collected in vivo from five patients. OCT imaging revealed a layered bronchial wall structure in normal bronchus that is lost in lung cancer. In peripheral lung, air-containing alveoli are imaged by OCT as a honeycomb structure beyond the bronchial wall. Lam et al.41 investigated the ability of OCT to discern the pathology of lung lesions identified by AFB in a group of high-risk smokers. A total of 281 OCT images were taken from 148 participants (histopathology from the imaged locations consisted of 145 normal/hyperplasia, 61 metaplasia, 39 mild dysplasia, 10 moderate dysplasia, 6 severe dysplasia, 7 CIS, and 13 invasive carcinomas). Normal or hyperplasia is characterized by one or two cell layers above a highly scattering basement membrane and upper submucosa. As the epithelium changes from normal/hyperplasia to metaplasia, various grades of dysplasia and CIS, the thickness of the epithelial layer increases. Quantitative measurement of the epithelial thickness showed that invasive carcinoma was significantly thicker than CIS (P = 0.004) and dysplasia was significantly thicker than metaplasia or hyperplasia (P = 0.002). The nuclei became more readily visible in high-grade dysplasia or CIS. The basement membrane was still intact in CIS but became discontinuous or no longer visible with invasive cancer (Fig. 2).

image

Figure 2. Optical coherence tomography image of a carcinoma in situ with focal microinvasion (arrow).

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OCT is also useful for assessment of COPD. COPD is characterized by irreversible airflow obstruction, in most cases caused by inhalation of toxic particles, such as cigarette smoke.42,43 It is well known that in susceptible hosts, chronic exposure to these particles causes an inflammatory response that remodels the wall of the small airways.44,45 Destruction of alveolar walls and loss of lung elasticity are also characteristic features of COPD. A recent ex vivo study using micro-CT showed that narrowing and disappearance of small conducting airways occurs prior to the onset of emphysematous destruction and that these changes can explain the increased peripheral airways resistance reported in COPD.46 Clinical CT using an acceptable dose of radiation provides airway images up to and including the fourth or fifth generation. Unfortunately, the resolution of CT is not adequate to image critical events that begin at the seventh branching generation46 nor can it measure morphological changes in different layers of the airway wall. OCT can overcome this limitation with small optical probes that can image airways as small as terminal bronchioles with high resolution. Coxson et al.47 compared OCT measurements with CT scans and lung function in COPD patients. In 44 current and former smokers, OCT imaging was used to measure the airway dimensions in specific bronchial segments. These data were compared with CT measurements of the exact same airway using a three-dimensional reconstruction of the airway tree (Pulmonary Workstation 2.0, VIDA Diagnostics, Inc., Iowa City, IA, USA). A strong correlation between CT and OCT measurements of lumen and wall area was observed. The correlation between FEV1% predicted and CT- and OCT-measured wall area (as percentage of the total area) of fifth generation airways was very strong, but the slope of the relationship was much steeper using OCT than using CT, indicating greater sensitivity of OCT in detecting changes in wall measurements that relate to FEV1. They concluded that OCT is more sensitive for discriminating the changes in the more distal airways of subjects with a range of expiratory airflow obstruction compared with CT. This result is not entirely unexpected, as measurements at this level of lung are at the limit of CT resolution. In addition to airway wall remodelling, alveolar wall destruction in COPD can also be clearly visualized using OCT with the emphysematous alveoli appearing as large voids compared with the small alveoli seen in those with normal lung function (Fig. 1b).

OCT systems have been developed to provide functional information in addition to morphometric measurements. Doppler OCT provides quantitative information on blood flow in the microvasculature and larger blood vessels.48,49Figure 3 is an example of Doppler OCT in a smoker with COPD compared with a smoker without COPD showing remodelling of the airway and adjacent blood vessel. Remodelling of the pulmonary muscular arteries, as well as precapillary vessels occurs with increasing severity of COPD.50 Doppler OCT is a potentially useful method to study remodelling of the pulmonary vasculature in COPD, in addition to the remodelling process in the airway wall. Polarization sensitive OCT (PS-OCT) is another OCT variant that is capable of identifying and analysing tissue components such as collagen and cartilage based on their intrinsic birefringence. Although not currently realized in the lung, fibre-based PS-OCT has been developed.51 OCT shows promise as a non-invasive, high-resolution diagnostic technique for the lung.

image

Figure 3. Colour Doppler optical coherence tomography (OCT) images from the airways of (left) a subject with normal lung function and (right) a subject with moderate COPD, FEV1/FVC = 61% and FEV1 = 60% predicted. The detected vasculature using colour Doppler OCT imaging (coloured) has been overlaid onto the structural OCT images (greyscale). Velocity aliasing is apparent from the presence of adjacent positive and negative velocity pixels. Note that the airway remodelling indicated by the airway wall thickening in the COPD subject. Fiducial markers are spaced 0.25-mm apart, assuming air as the imaging medium.

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CONFOCAL ENDOMICROSCOPY AND ENDOCYTOSCOPY

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. PRINCIPLES OF OPTICAL IMAGING
  5. AFB
  6. NBI
  7. OPTICAL COHERENCE TOMOGRAPHY
  8. CONFOCAL ENDOMICROSCOPY AND ENDOCYTOSCOPY
  9. LASER RAMAN SPECTROSCOPY
  10. SUMMARY AND FUTURE DIRECTIONS
  11. ACKNOWLEDGEMENT
  12. REFERENCES

To achieve micron resolution to visualize cellular or extracellular details, confocal laser endomicroscopy or endocytoscopy has been developed. Confocal laser endomicroscopy uses a laser illumination source that is focused into a spot in the tissue. The light emitted from the focal point is imaged to a pinhole to reject out-of-focus emitted light.52,53 In this way, bulk tissue can be optically sectioned to image a single cellular plane. Although confocal microscopy can be done in either fluorescence or reflectance mode, currently available confocal endomicroscopes uses fluorescence mode because of technical issues performing reflectance imaging through fibre-optic probes. The currently commercialized device (Cellvizio, Mauna Kea Technologies, Paris, France) has a flexible probe with an outer diameter of 1 mm that can be inserted through the working channel of the bronchoscope to the tissue surface. The probe is a coherent fibre image bundle made of thousands of individual fibres. Each individual fibre is in essence its own pinhole, thereby assuring the confocal properties of the system. Two different wave lengths are available: 488 nm is used for autofluorescence microimaging of the respiratory tract,54 while 660 nm excitation can be used for epithelial cell imaging after topical application of a contrast agent such as methylene blue.55 Imaging is done at 12 frames/s with a circular diameter field of view of 600 µm, a lateral resolution of 3.5 µm and a depth of 0–50 µm.56 Using an excitation wavelength of 488 nm, fine details of the alveolar wall can be seen. A good correlation was found between the number of cigarettes smoked per day and the number of alveolar macrophages observed in vivo.56 In the central airways, the basement membrane structure can be clearly seen. However, the epithelial cells are not visible because of the low autofluorescence. A topical contrast agent such as crystal violet or methylene blue that binds to cytoplasmic structure or DNA is needed to enhance the fluorescence.57 A potential application is for imaging peripheral lung lesions before deciding upon a biopsy in conjunction with virtual or navigational bronchoscopy.55

A different approach to microscopic imaging of the airways is to use an endocytoscopy system (XEC-300F, Olympus Optical Corp., Tokyo, Japan).58 The tip of the endoscope contained an optical magnifying lens and a CCD for imaging. The system has a spatial resolution of 4.2 µm and a depth of view of 0–30 µm. Using 0.5% methylene blue as a topical stain, normal ciliated bronchial epithelial cells, bronchial squamous dysplastic cells and malignant squamous carcinoma cells could be seen similar to conventional cytology.58 The size of the bronchoscope does not allow examination of smaller airways or alveoli.

While having the advantage of higher resolution compares with OCT, confocal endomicroscopy or endocytoscopy is limited by the smaller field of view and the requirement of contrast agents. Some of these contrast agents such as methylene blue dye is known to induce oxidative damage of DNA when exposed to white light.59 The risk of exposure to methylene blue is not known. An alternative to dye is to use targeted agents directed to specific receptors in the cell membrane. Confocal endomicroscopy coupled with a targeted agent has been investigated in the gastrointestinal tract.60–62 A similar approach has not been investigated in the respiratory tract.

An additional disadvantage of endomicroscopic and endocytoscopic approaches is that currently available probes require end-on contact with the tissue. This is not a practical geometry for examining small airways or mid-airway lesions. Also, as the depth of the imaging plane is fixed with respect to the end window of the probe, the imaged depth into the tissue is not adjustable without inserting an entirely different probe.

LASER RAMAN SPECTROSCOPY

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. PRINCIPLES OF OPTICAL IMAGING
  5. AFB
  6. NBI
  7. OPTICAL COHERENCE TOMOGRAPHY
  8. CONFOCAL ENDOMICROSCOPY AND ENDOCYTOSCOPY
  9. LASER RAMAN SPECTROSCOPY
  10. SUMMARY AND FUTURE DIRECTIONS
  11. ACKNOWLEDGEMENT
  12. REFERENCES

Raman Spectroscopy (RS) utilizes the Raman effect—an inelastic light-scattering process whereby a very small proportion of incident photons are scattered (∼1 in 108) with a corresponding change in frequency. The difference between the incident and scattered frequencies corresponds to the vibration modes of molecules participating in the interaction.63 Raman spectra are depicted by plotting the intensity of the scattered photons as a function of the frequency shift. Raman spectra usually exhibit sharp spectral features that are characteristic for specific molecular structures and tissue types. Thus, Raman spectra can capture a fingerprint of specific molecular species and can therefore be potentially used to provide biochemical information about a given tissue or disease state.64–66 Up until recently, direct in vivo clinical applications of RS have been limited by the ability to measure spectra in a relatively short time period (∼2 s) to minimize motion artefacts and to overcome the much stronger autofluorescence background. This problem can be circumvented to some degree by choosing a high frequency Raman range (1500–3400/cm) that still contains significant biomolecular information for tissue classification.67,68 Recently, Short et al. reported in vivo pilot study using a 1.8-mm fibre-optic catheter passed down the instrument channel of a bronchoscope.69,70 Lesions with moderate dysplasia or worse pathology were found to have relative increases in DNA, haemoglobin, phenylalanine, triolein and a corresponding decrease in collagen I compared with lesions that were mild dysplasia, metaplasia, hyperplasia or normal.70 Moderate dysplasia or worse lesions can be distinguished from lower grade lesions, with a sensitivity of 90% and a specificity of 91%.70

SUMMARY AND FUTURE DIRECTIONS

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. PRINCIPLES OF OPTICAL IMAGING
  5. AFB
  6. NBI
  7. OPTICAL COHERENCE TOMOGRAPHY
  8. CONFOCAL ENDOMICROSCOPY AND ENDOCYTOSCOPY
  9. LASER RAMAN SPECTROSCOPY
  10. SUMMARY AND FUTURE DIRECTIONS
  11. ACKNOWLEDGEMENT
  12. REFERENCES

Standard WLB enables examination of the macroscopic appearance of airways. Advances in optical imaging such as OCT, confocal endomicroscopy, AFI and laser RS move the frontiers to enable probing of the microscopic and biochemical structures of the airways. The differences in spatial resolution and tissue penetration depth of OCT, confocal endomicroscopy/endocystoscopy versus endoscopic ultrasound are summarized in Table 1. A higher spatial resolution is generally associated with a shallower depth of penetration. To study changes in airway diseases such as bronchial cancers, asthma and COPD, the thickness of the epithelium and subepithelial layers superficial to the cartilage is within the probing limits of these optical imaging methods. OCT or confocal endomicroscopy in combination with autofluorescence spectroscopy or RS is an area of active research. Although technically challenging, this approach can integrate morphological and biochemical information at the cellular level. It will be a powerful tool to study the alterations in disease and the effect of treatment. Another promising tool is multispectral imaging. Conventional AFI has been performed by illuminating the bronchial surface at a single excitation wavelength and detecting the emission wavelengths. A new method of fluorescence imaging called selective excitation light fluorescence imaging71 employs multiple excitation wavelengths from a rapidly tuneable light source and detection of multiple emitted wavelength images. From the differences in the excitation—emission spectra of fluorophores—the biochemical composition of the tissue can be analysed. These novel optical imaging techniques hold promise to study airway diseases and the effect of novel therapies.

Table 1.  Comparison of optical imaging techniques with endoscopic ultrasound
Resolution and imaging depthOptical coherence tomographyConfocal endomicroscopy/endocystocopyEndoscopic ultrasound
Lateral resolution5–30 µm1–4 µm200–300 µm
Tissue penetration1.0–2.5 mm0–50 µm10 mm
Contrast agentNoYesNo but requires liquid coupling medium
Three-dimensional imageYesNoNo

REFERENCES

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. PRINCIPLES OF OPTICAL IMAGING
  5. AFB
  6. NBI
  7. OPTICAL COHERENCE TOMOGRAPHY
  8. CONFOCAL ENDOMICROSCOPY AND ENDOCYTOSCOPY
  9. LASER RAMAN SPECTROSCOPY
  10. SUMMARY AND FUTURE DIRECTIONS
  11. ACKNOWLEDGEMENT
  12. REFERENCES
  • 1
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  • 2
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  • 3
    Deveci F, Murat A, Turgut T et al. Airway wall thickness in patients with COPD and healthy current smokers and healthy non-smokers: assessment with high resolution computed tomographic scanning. Respiration 2004; 71: 60210.
  • 4
    Aysola RS, Hoffman EA, Gierada D et al. Airway remodeling measured by multidetector CT is increased in severe asthma and correlates with pathology. Chest 2008; 134: 118391.
  • 5
    Coxson HO. Quantitative computed tomography assessment of airway wall dimensions: current status and potential applications for phenotyping chronic obstructive pulmonary disease. Proc. Am. Thorac. Soc. 2008; 5: 9405.
  • 6
    Wagnieres G, McWilliams A, Lam S. Lung cancer imaging with fluorescence endoscopy. In: Mycek M, Pogue B (eds) Handbook of Biomedical Fluorescence. Marcel Dekker, New York, 2003; 36196.
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    Shibuya K, Hoshino H, Chiyo M et al. High magnification bronchovideoscopy combined with narrow band imaging could detect capillary loos of angiogenic squamous dysplasia in heavy smokers at high risk for lung cancer. Thorax 2003; 58: 98995.
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    Vincent B, Fraig M, Silvestri G. A pilot study of narrow-band imaging compared to white light bronchoscopy for evaluation of normal airways and premalignant and malignant airways disease. Chest 2007; 131: 179488.
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    Gono K, Obi T, Yamaguchi M et al. Appearance of enhanced tissue features in narrow-band endoscopic imaging. J. Biomed. Opt. 2004; 9: 56878.
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    Hung J, Lam S, LeRiche JC et al. Autofluorescence of normal and malignant bronchial tissue. Lasers Surg. Med. 1991; 11: 99105.
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    Zellweger M, Grosjean P, Goujon D et al. In vivo autofluorescence spectroscopy of human bronchial tissue to optimize the detection and imaging of early cancers. J. Biomed. Opt. 2001; 6: 4151.
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