Development of multimodal micro-endoscopic system with oblique illumination for simultaneous fluorescence imaging and spectroscopy of oral cancer.

Multimodality of an optical system implies the use of one or more optical techniques to improve the system's overall performance and maximum utility. In this article, we demonstrate a multimodal system with oblique illumination that combines two different techniques; fluorescence micro-endoscopy and spectroscopy simultaneously and can be utilized to obtain diverse information from the same location of biological sample. In present system, use of graded index (GRIN) rod-lens makes it highly compact and oblique incidence decouples illumination geometry with collection geometry, preventing CCD cameras from saturation and reduces number of optical elements, thereby making system further miniaturized and field-portable. It also overcomes the disadvantages of undesired reflections from different optical elements. The experimental results of simultaneous imaging and spectroscopy of the biological samples are presented along with quantitative spectroscopic parameters; peak wavelength shift, area under the curve and full width half maximum (FWHM). The spatial resolution, spectral resolution and field of view of the system are found to be 4.38 µm, 0.5 nm and 2.071×1.548mm2 , respectively. Furthermore, we have obtained the red shift for cancerous oral tissue with respect to normal oral tissue 5.79 ± 1.071 nm. This could be important indicator for oral cancer screening.

An apparatus for qualitative assessment of the shading ratio of oblique illumination and real-time high-contrast imaging.

Oblique illumination imaging can significantly improve the contrast of transparent thin samples. However, in traditional oblique illumination methods, either the condenser is offset or a block is added to the condenser, which makes it complicated and challenged to build a stable oblique illumination imaging. Herein, we present a method to measure the optimal shading ratio of oblique illumination in an inverted microscope, and develop an apparatus for stable high-speed high-contrast imaging with uniform brightness. At optimal shading ratio, the oblique illumination imaging has better imaging quality than differential interference contrast, which characteristic is independent on sample. In oblique illumination with low magnification objective, the images have uneven brightness. According to target brightness, we have developed a brightness unevenness correction algorithm to form uniform background brightness for oblique illumination. Integrating the algorithm with imaging acquisition, corrected oblique illumination microscopy is appropriate to observe living cells with high contrast.

Quantitative phase microscopy with enhanced contrast and improved resolution through ultra-oblique illumination (UO-QPM).

Recent developments in phase contrast microscopy have enabled the label-free visualization of certain organelles due to their distinct morphological features, making this method an attractive alternative in the study of cellular dynamics. However tubular structures such as endoplasmic reticulum (ER) networks and complex dynamics such as the fusion and fission of mitochondria, due to their low phase contrast, still need fluorescent labeling to be adequately imaged. In this article, we report a quantitative phase microscope with ultra-oblique illumination that enables us to see those structures and their dynamics with high contrast for the first time without labeling. The imaging capability was validated through comparison to the fluorescence images with the same field-of-view. The high image resolution (~270 nm) was validated using both beads and cellular structures. Furthermore, we were able to record the vibration of ER networks at a frame rate of 250 Hz. We additionally show complex cellular processes such as remodeling of the mitochondria networks through fusion and fission and vesicle transportation along the ER without labels. Our high spatial and temporal resolution allowed us to observe mitochondria "spinning", which has not been reported before, further demonstrating the advantages of the proposed method.

Oblique illumination in microscopy: A quantitative evaluation.

Many biological objects are barely distinguished with the brightfield microscope because they appear transparent, translucent and colourless. One simple way to make such specimens visible without compromising contrast and resolution is by controlling the amount and the directionality of the illumination light. Oblique illumination is an old technique described by many scientists and microscopists that however has been largely neglected in favour of other alternative methods. Oblique lighting (OL) is created by illuminating the sample by only a portion of the light coming from the condenser. If properly used it can improve the resolution and contrast of transparent specimens such as diatoms. In this paper a quantitative evaluation of OL in brigthfield microscopy is presented. Several feature descriptors were selected for characterising contrast and sharpness showing that in general OL provides better performance for distinguishing minute details compared to other lighting modalities. Oblique lighting is capable to produce directionally shadowed differential contrast images allowing to observe phase details in a similar way to differential contrast images (DIC) but at lower cost. The main advantage of OL is that the resolution of the light microscope can be increased by effectively doubling the angular aperture. OL appears as a cost-effective technique both for the amateur and professional scientist that can be used as a replacement of DIC or phase contrast when resources are scarce.

Endoilluminator-assisted pediatric cataract surgery with hazy cornea.

The purpose of this study was to demonstrate the usefulness of endoilluminator in pediatric cataract with hazy corneas. We describe a series of three cases of pediatric cataract where visualization of intraocular structures was inadequate under the operating microscope. The endoilluminator was held at the limbus with light directed obliquely to visualize the details of intraocular structures against the hazy cornea using oblique illumination. It allowed structures behind the hazy cornea to be seen with ease. A simple modification in surgical procedure of pediatric cataract using an endoilluminator helps in better visualization of intraocular structures in difficult situations.

Spatially-incoherent annular illumination microscopy for bright-field optical sectioning.

Numerous advanced microscopic imaging techniques have been proposed for optical sectioning, but they generally employ a complex and costly optical system. Here we report a microscopy termed spatially-incoherent annular illumination microscopy (SAIM). It allows for simple, effective, non-fluorescence, and bright-field optical sectioning. The proposed technique is implemented by installing an annular array of light emitting diodes (LEDs) on a standard bright-field microscope for illumination. The LED array produces distinctive illumination, that is, each LED provides coherent, large-angle oblique illumination while all LEDs generate spatially-incoherent annular illumination. Such a distinctive illumination can improve both lateral resolution and axial resolution. The improvement of lateral resolution is due to the coherent and large-angle oblique illumination. The spatially-incoherent annular illumination can improve the axial resolution. It is because, for defocused structures, each LED results in a blurred image with a different lateral shift and all LEDs result in an incoherent stagger superposition of the defocused images. The superposition looks much more blurred, which improves the contrast of the in-focus image remarkably. We experimentally demonstrate that SAIM is able to provide bright-field optical sections with 600-nm axial resolution and 150-nm lateral half-pitch resolution by using a 525-nm wavelength LED array and an objective with 100X, numerical aperture (NA) 1.25.

Dual-axis illumination for virtually augmenting the detection view of optical-resolution photoacoustic microscopy.

Optical-resolution photoacoustic microscopy (OR-PAM) has demonstrated fast, label-free volumetric imaging of optical-absorption contrast within the quasiballistic regime of photon scattering. However, the limited numerical aperture of the ultrasonic transducer restricts the detectability of the photoacoustic waves, thus resulting in incomplete reconstructed features. To tackle the limited-view problem, we added an oblique illumination beam to the original coaxial optical-acoustic scheme to provide a complementary detection view. The virtual augmentation of the detection view was validated through numerical simulations and tissue-phantom experiments. More importantly, the combination of top and oblique illumination successfully imaged a mouse brain in vivo down to 1 mm in depth, showing detailed brain vasculature. Of special note, it clearly revealed the diving vessels that were long missing in images from original OR-PAM.

Low-cost oblique illumination: an image quality assessment.

We study the effectiveness of several low-cost oblique illumination filters to improve overall image quality, in comparison with standard bright field imaging. For this purpose, a dataset composed of 3360 diatom images belonging to 21 taxa was acquired. Subjective and objective image quality assessments were done. The subjective evaluation was performed by a group of diatom experts by psychophysical test where resolution, focus, and contrast were assessed. Moreover, some objective nonreference image quality metrics were applied to the same image dataset to complete the study, together with the calculation of several texture features to analyze the effect of these filters in terms of textural properties. Both image quality evaluation methods, subjective and objective, showed better results for images acquired using these illumination filters in comparison with the no filtered image. These promising results confirm that this kind of illumination filters can be a practical way to improve the image quality, thanks to the simple and low cost of the design and manufacturing process.