Microscopy refocusing and dark-field imaging by using a simple LED array

Abstract

The condenser is one of the main components in most transmitted light compound microscopes. In this Letter, we show that such a condenser can be replaced by a programmable LED array to achieve greater imaging flexibility and functionality. Without mechanically scanning the sample or changing the microscope setup, the proposed approach can be used for dark-field imaging, bright-field imaging, microscopy sectioning, and digital refocusing. Images of a starfish embryo were acquired by using such an approach for demonstration. © 2011 Optical Society of America OCIS codes: 110.0180, 110.2945, 110.3010, 180.6900. Appropriate illumination of the specimen is an important factor in achieving high-resolution and high-quality images in microscopy and critical photomicrography. With the maturation of LED technology, the use of LEDs as the light source for optical microscopy can bring certain cost-and usage-advantages [3,7,8]. In this Letter, we demonstrate a simple and cost-effective microscopy illumination scheme by replacing the optical condenser with a programmable LED array. The proposed illumination scheme has several advantages. 1) A conventional bright-field image can be acquired by digitally matching the illumination numerical aperture (NA) to the collection NA (i.e. NA of objective lens). 2) A dark-field image of the specimen can be acquired by simply turning on the LEDs at the edge of the array, where the illumination NA is beyond the collection NA. 3) We can sequentially turn on each individual LED and capture a sequence of specimen images. These images contain the information for different view angles and therefore we can postprocess them to digitally refocus the specimen into different depths. 4) The aforementioned imaging schemes can be accomplished simultaneously by a single LED scan process. We can select the images corresponding to LEDs within the collection NA to form a bright-field image. On the other hand, the images corresponding to LEDs beyond the collection NA can be used for dark-field imaging. Furthermore, we can realign all the images (with different view angles) to digitally refocus the specimen into different depths. 5) No mechanical moving parts are involved in the proposed scheme. The scanning rate of the LEDs can easily operate in the kHz domain. The limiting factor in our prototype is the capturing frame rate/data transfer rate of the camera. Because of the rapid development of the semiconductor industry, we believe that such a data transfer rate will not be a significant bottleneck for the proposed illumination scheme in the near future. 6) The proposed scheme is cost-effective and compatible with most modern laboratory microscopes.This Letter is structured as follows. We will first describe the experimental setup of our proposed illumination scheme. Next, we will report on our results of bright-field and dark-field imaging capabilities. We will then report on our demonstration of digital refocusing of the specimen into different depths. Finally, we will draw our conclusion at the end of...

Quantitative differential phase contrast imaging in an LED array microscope

“…darkfield [15,16], phase contrast [12,16], Fourier Ptychography [17,18], digital aberration removal [19], light field refocusing [12,15], and 3D phase imaging [20]). The hardware involves simply replacing a microscope's illumination unit with an LED array (Fig.…”

3D intensity and phase imaging from light field measurements in an LED array microscope

“…Light field refocusing predicts the intensity image I Δz at Δz from the actual focal plane to be [35,38] I Δz x; y X…”

“…Our system is built on a commercial microscope in which the illumination unit has been replaced by a programmable LED array. This simple, inexpensive hardware modification enables not only 4D light field capture [35,38], but also dark field [38,39], phase contrast [35,39], Fourier ptychography [23,24], and digital aberration removal [40].…”

Angular light modulator using optical blinds

“…In the photographic setup shown in Fig. 3(a), we captured 10 images corresponding to different slat angles and performed digital refocusing using tomographic back-projection [8,9]. Figures 3(b1)-3(b4) show the refocused images at the different z planes (Visualization 3) and we highlighted the in-focus regions using red arrows.…”

“…In this case, we can sweep the slat angle of the blind structure and capture multiple images corresponding to different perspectives. These images can then be back-projected for 3D tomographic refocusing [8,9]. By using a fixed slat angle in the device, we can convert the incident-angle information into intensity variations for wavefront sensing; we can also introduce a translational shift to the defocused object for high-speed autofocusing [10].…”