Low-cost, high-speed near infrared reflectance confocal microscope

: We have developed a low-cost, near-infrared (NIR) reflectance confocal microscope (RCM) to overcome challenges in the imaging depth and speed found in our previously-reported smartphone confocal microscope. In the new NIR RCM device, we have used 840 nm superluminescent LED (sLED) to increase the tissue imaging depth and speed. A new confocal detection optics has been developed to maintain high lateral resolution even when a relatively large slit width was used. The material cost of the NIR RCM device was still low, ~$5,200. The lateral resolution was 1.1 µm and 1.3 µm along the vertical and horizontal directions, respectively. Axial resolution was measured as 11.2 µm. In vivo confocal images of human forearm skin obtained at the imaging speed of 203 frames/sec clearly visualized characteristic epidermal and dermal cellular features of the human skin.

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Imaging performance test
Lateral resolution of the low-cost NIR confocal microscope was measured by imaging a USAF resolution target. FWHM of the line spread function (LSF) was calculated along the spectrally-encoded and slit-length directions. Axial resolution was measured by translating a mirror along the objective lens optical axis with a motorized stage and calculating the FWHM of the axial response curve. The source power was attenuated during the resolution measurement to ensure that the pixel values were not saturated. Tissue imaging performance was evaluated by imaging human forearm in vivo at different imaging depth levels. The forearm skin surface was placed parallel to the focal plane of the objective lens. Ultrasound gel with a similar refractive index to that of water was applied between the forearm and objective lens. The exposure time was set at 4.8 msec and the resulting frame rate was 203 fps. The microscope was translated relative to the forearm using a motorized stage. The motor speed was set to 1 mm/sec and the scan range 500 µm. The maximum acceleration of the motor was 4 mm/sec 2 , which produced the acceleration time of 0.25 sec and deceleration time 0.25 sec. At the center of the axial scanning, the uniform speed of 1 mm/sec was maintained over 250 µm range. Within the uniform speed region, the axial step size between frames was 5 µm. The skin surface was located at the beginning of the uniform velocity region. A bi-directional axial scan was conducted. The resulting 3D volume acquisition rate was 1.33 volumes/sec. Images were saved as an AVI file using a custom LabVIEW code (National Instruments, Austin, TX). At the end of each axial scanning, the confocal FOV was manually moved to a new imaging location and the axial scanning was conducted at the new imaging location. After image acquisition, the AVI file was segmented into multiple image stacks with each stack representing one axial scan. The image stacks were analyzed in ImageJ [18]. The background intensity level was measured and subtracted. 3D rending of the image stacks was conducted using 3D Slicer [19]. The speckle noise contrast was calculated by analyzing dermis images and dividing the standard deviation of the intensity values by the mean value at four 100 × 100-pixel regions that exhibited grossly uniform reflectivity without observable cellular features. The speckle noise contrast was measured at three different imaging depth levels.

Results
A photograph of the low-cost confocal microscope is shown in Fig. 4. The confocal microscope had a dimension of 15 cm (W) × 16 cm (H) × 4.5 cm (D), and the weight was 0.57 kg. The material cost for the confocal microscope was $5,188. The optical power on the specimen was 2.2 mW.
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Discussio
In this paper, low-cost NIR cellular featur rate of 203 fr microscope a imaging speed 5 large-area imaging of the entire skin lesion within a short procedural time. We also expect that the low cost of the device will facilitate a wide adaption of the device in various clinical settings. There were several remaining technological challenges found during the preliminary testing. Even though a relatively wide slit was used, the speckle noise was still prominent in confocal images, which hindered the image interpretation. Use of the wide slit degraded the axial resolution. In the future development, we will address these two issues by using a highpower LED, which has a significantly reduced spatial coherence and therefore allows for use of a narrow slit width. The volumetric imaging rate was limited to 1.33 volumes/sec mainly due to the acceleration and deceleration of the axial scanning stage. A piezoelectric transducer (PZT)-based scanner can be used to achieve higher volumetric imaging rate. In the new confocal detection optics, the CMOS sensor is located on the same side as the tissue, which will make it challenging to image certain anatomical locations such as back or face. A fold mirror can be used between the grating and camera lens to move the CMOS sensor away from the tissue and allow for imaging of a wider range of skin locations. In the future study of imaging suspicious skin lesions, we will evaluate the image quality of our microscope in comparison with the commercial confocal microscope and evaluate feasibilities of large-area scanning and real-time 3D imaging.

Funding
National Institutes of Health/Fogarty International Center (R21TW010221).