# Endoscopic Optical Imaging Technologies and Devices for Medical Purposes: State of the Art
## 2 Medical Imaging Applications
### Confocal Microscopy  
In a conventional epi-reflectance microscope, the object (specimen) is uniformly illuminated using a light source, and the image is obtained from the reflected light. Biological tissue is a highly light-scattering media. Thus, the scattered photons generated from the out of focus refractive index differences represent noise causing blur in the image. In confocal microscopy (discovered in 1957), the sample is illuminated using a point illumination system, where out-of-focus light is rejected by a pinhole placed at a plane optically conjugate to the object. As a single point of the sample is illuminated at a time, scanning methods are required to generate 2-D or 3-D images using a confocal microscope. For beam scanning, it is possible to use either a single beam for scanning using micro mirrors or other fiber optic techniques discussed later in the paper, or multiple beam scanning using the spinning Nipkow disks or spectral encoding. The imaging of thick sections is accomplished using optical sectioning by imaging virtual slices of the sample. The depth of imaging can be changed by refocusing the light [10,17]. The originally proposed trans-illumination confocal microscope used two objective lenses for illumination and detection of the light passed through a pair of pinholes placed at the light source to create a point source and at the detector to avoid the out-of-focus noise light signal. Thus, the pinholes act as a filter that pass information carrying ballistic photons and reject the scattered out-of-focus photons. Consequently, this configuration generates higher resolution images. The contrast in reflectance-based confocal microscopy is based on the scattering properties of the tissue sample; whereas, the contrast in fluorescence-based confocal microscopy is given by the fluorescent properties of the sample. In the latter case, the resolution is increased by staining the sample with fluorophores that increase the contrast and sensitivity of the image [10,18]. The size of the pinholes affects the lateral and axial resolution of the obtained image and can be measured by placing the point object at the focal point of the objective lens. The resolution can also be described in terms of the Airy pattern, which is the light intensity distribution near the focal point. The Airy pattern in confocal microscopy has a narrower central lobe and attenuated side lobes, providing for high resolution. The transverse resolution is given by Equation (1), while the axial resolution can be described as [10]:  
\[\delta z=\frac{0.89\lambda}{n(1-cos\theta)}, \tag{5}\]  
where \(\theta\) is the angular aperture (half angle of the marginal ray). The lateral resolution in confocal microscopy is less than 1 \(\upmu\)m, and the axial resolution is about 1 \(\upmu\)m. In a fluorescence-based system, the resolution depends on the point spread functions of the illumination and detection signals, which differ from each other due to the Stokes shift. In fiber optic confocal microscopy, the fibers used for illumination and detection act as pinholes [10]. [PERSON] and [PERSON] fabricated one of the early fiber optic confocal microscopes, where the single mode fiber illuminates the beam on the target sample, and the beam is scanned along the sample  
Figure 2: OCT image of the human retina and comparison of macular thickness in a person without Alzheimer’s disease (AD), and patient with AD (taken with the permission of [14]). using torsional scanning mirrors [19]. The continuous development in the MEMS field facilitates the miniaturization of devices and achieving better imaging with higher resolution. [PERSON] et al. developed a 3-D scanning fiber optic confocal microscope, which is able to scan with a depth of field of over 400 \(\upmu\)m. An optical fiber carrying the illuminating light is excited at resonance by an electrothermally actuated MEMS mirror. The scanner is able to image the tissue surface with a lateral resolution of 1 \(\upmu\)m, and an axial resolution of 7 \(\upmu\)m [18].