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. Appropriate illumination of the specimen is an important factor in achieving high-resolution and high-quality images in microscopy and critical photomicrography. Most modern laboratory microscopes are equipped with the Köhler illumination setup, which was first introduced in 1893 by August Köhler and is now recommended by most microscope manufacturers. Such a Köhler illumination setup is composed of collector lens, field diaphragm, condenser diaphragm, and condenser lens. It can provide specimen illumination that is uniformly bright and free from glare [1]. More advanced illumination schemes have also been reported in recent years, including structured illumination [2,3], light sheet illumination [4], focus-grid illumination [5], and nondiffracted Bessel beam illumination [6]. 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 this Letter. The proposed illumination scheme is shown in Fig. 1(a), where the optical condenser is replaced by an LED array. To understand the principle, we first consider only one LED lit in the LED array. The location of this LED can be denoted as ðx i ; y i Þ, as shown in Fig. 1(b). Assuming the distance (at the z direction) between the LED array and specimen is 'H', the illumination NA of this LED can be defined as r= ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi ffi r 2 þ H 2 p ; ð1Þ Fig. 1. (Color online) Proposed illumination scheme. (a) The optical condenser is replaced by a programmable LED array. No lens is placed between the LED array and the sample stage. (b) One LED is turned on for illumination. (c) The LEDs in central part are turned on for the bright-field imaging. (d) The LEDs at the edge are turned on for dark-field imaging. (e) The actual LED array prototype used in our experiment. (f) A simple ray-trace diagram showing different z-planes result in different image shifts (also see Eq. (2)).