LED arrays as cost effective and efficient light sources for widefield microscopy

Abstract

New developments in fluorophores as well as in detection methods have fueled the rapid growth of optical imaging in the life sciences. Commercial widefield microscopes generally use arc lamps, excitation/emission filters and shutters for fluorescence imaging. These components can be expensive, difficult to maintain and preclude stable illumination. Here, we describe methods to construct inexpensive and easy-to-use light sources for optical microscopy using light-emitting diodes (LEDs). We also provide examples of its applicability to biological fluorescence imaging.

Figure 1. A high intensity Luxeon V LED and driving circuitry.

(A) A high intensity 5 W Luxeon V blue LED is shown mounted on a standard fin type heatsink with thermal grease and an electrical insulator in between. There are no moving parts used, reducing susceptibility to mechanical damage. (B) A simple electronic circuit that can be used to drive a single blue LED is shown. Its output intensity is adjusted by a potentiometer; the light can be turned on and off by a low current TTL input. (C) An LED driven by a high square wave input follows the signal closely. The speed of a typical LED array is clearly illustrated by its response to a voltage step. The driver circuit was that shown in B.

Figure 2. Two methods for coupling an LED based illuminator to a microscope.

(A) An optical is fiber placed on the surface of an LED to direct light into a small box consisting of a focusing lens and mirror that is inserted into a side port on the microscope. (B) A homemade “lamp” consisting of an LED, heatsink, tube, and lens is coupled to the lamp housing port in the back of the microscope.

Figure 3. A standalone two wavelength illuminator.

Two different LEDs, with independent electronic driving circuitry similar to that shown in Figure 1 are used. A dichroic mirror passes light from one LED and reflects light from the other onto the end of the fiber light guide. Any necessary excitation filters can be placed on the LEDs.

Figure 4. Comparison of an LED based illuminator and Xenon arc lamp.

(A) Images of hippocampal astrocytes in cell culture labeled with antibodies against the astrocytic marker GFAP are shown for illumination with Xenon lamp and with LED. (B) Time traces of fluorescence intensity of the same region of the field (shown by white square) in the two conditions. (C) Distribution of pixel intensities of the images.

Figure 5. A custom built widefield microscope.

(A) As an alternative to a standard commercial microscope, we used a custom built setup as shown above for our in vivo widefield experiments. A CCD camera is coupled to two lenses along with optional extension tubes to adjust the magnification. The ordering of parts is as follows: CCD camera, an F to C mount adapter with an emission filter placed inside or on top of the coupler, a 62 mm, 105 mm FL lens, a 62 mm to 52 mm step down ring, a 52 mm to 52 mm coupling ring, and a 52 mm, 50 mm FL lens. (B) Shown here are sample individual frames of the dorsal surface of the olfactory bulb, under white light, revealing the blood vessel architecture, as well as resting synaptopHluorin and intrinsic optical signals from the same animal respectively under blue and far red illumination. (C) The changes in spH and intrinsic signals upon presentation of fresh air or two odorants. Images show fractional changes (ΔF/F) with increases shown by brighter pixels and decreases by darker pixels.