Clive’s Corner #13: Light Sources for Microscopy

Corner articles focus on inexpensive hacks that microscopists can use to broaden the range of techniques that are possible with basic microscopes. Some of the topics were known by microscopists many generations ago, while others involve components that have only recently become available to the general consumer. This article falls into the latter category and explores the application of LED (light emitting diodes) and laser diodes as light sources to explore new horizons.

Over the last decade or two, most student/hobbyist microscopes have switched from a tungsten filament light source to an LED which offers several advantages. LEDs are more efficient which means the power consumption is reduced by >10-fold and less heat is generated as a side product. In addition, LEDs can be driven by a battery supply, making microscopes more portable. LEDs also have a longer lifetime than light bulbs and rarely need replacing. One of their downsides, at least for normal brightfield imaging, is that so-called white LEDs often have a blue or yellowish bias, which can be more obvious when taking pictures with a digital camera. This problem arises because an individual LED emits a limited range of wavelengths (≡ colors) and a so-called white LED is made up of blue, green and red LED components which combine to give the appearance of white light. This trick works because we have the three different types of cone cells in our eyes which are sensitive to blue, green and red light and when they are equally stimulated our brain interprets this as white light. Likewise digital color cameras have three different color sensors but the balance to produce a white image may differ slightly from that of the LED emitter. Consequently, the camera image may have an exaggerated blue or yellow tinge. This can be minimized by fine control of the color balance function. As a side note, white LEDs would not fool some mantis shrimps which have 12 types of color receptors!

The LEDs supplied with my Amscope 120 and Swift 350 microscope are rated as 1 W which is sufficient for standard brightfield imaging and even darkfield illumination. Why do LEDs require so much less power than tungsten filament lamps? Partly this is due to the limited wavelength output of LEDs which means there is little wasted energy in the ultraviolet and infrared region (unless of course UV or IR LEDs are deliberately designed for specialized purposes). However, the main reason concerns the size of the light source (Figure 1). A typical LED chip may be around 1 mm square (Figure 1b) and is combined with a focusing lens that ensures most of the light is emitted in one direction. A filament bulb is larger and much of the light produced is wasted because it cannot be efficiently focused to a small spot at the front focal plane of the condenser to illuminate the sample.

Apart from the built-in LED of a microscope, additional LEDs may be added to a basic microscope such as Angle Eye ring lights for darkfield or phase contrast illumination. Older microscopes are often modernized by adding an LED light source.

An LED flashlight can be used as excitation source for fluorescence microscopy. Here the limited wavelength range (typically ± 10 to 25 nm of the peak value) is an advantage as there is less light that bleeds through the emission filter. Using an LED flashlight as an excitation source by directly illuminating the sample (Figure 2a), rather than via a dichroic mirror in the body of the microscope as is done in most research-grade microscopes, allows the most basic of microscopes to be used in a fluorescence mode. However, this set-up is limited to 10x objectives or less because of the necessary working distance between the focusing lens of the flashlight and the sample slide. The problem is due to the size of the LED light source which, although maybe of the order of 1 mm, gives a minimum focused spot size of several millimeters. With higher power objectives this limitation may be overcome by using a laser pointer as a light source. Lasers emit a near parallel light beam that can be brought to focus to a very small bright spot. This property does raise potential safety issues which I will return to below, but first let me describe the arrangement.

The laser pointer is mounted on a mini-tripod and a 50 mm focal length lens is attached to the pointer to bring the beam to a focus at this distance (Figure 2b). The minimum spot size for a blue laser is about 50 µm. By increasing the distance from the sample, the spot can be defocused and enlarged to match the field-of-view of the microscope objective. However, once the spot reaches about 1 mm in diameter, the intensity of a 5 mW laser pointer is about the same as that from a focused LED flashlight, so for low power objective lenses with a larger field-of-view, an LED source is better. One problem with laser illumination results from its coherence (i.e. all the photons oscillate in phase). Any reflected beam may combine with the original beam to give bright and dark fringes, resulting in uneven illumination of the sample.

Another essential component for fluorescence microscopy is an emission filter that can be located in the body of the microscope as described previously. This filter (yellow in the case of a blue laser) blocks the scattered excitation light which generally is more intense than the longer wavelength emitted fluorescent light. An excitation filter is not usually necessary as a laser pointer emits a single wavelength with a width of <1 nm. An example of the kind of image that can be obtained is shown in Figure 3, using the fluorescent dye 4′,6-diamidino-2-phenylindole (DAPI) to stain DNA in a cell nucleus.

Regarding laser safety, laser pointers fall into the 3R laser category and should have an output of < 5 mW in the visible light region. At this power level it is deemed that the normal blink response is sufficient to prevent eye damage. However, caution is raised when 3R lasers are used in conjunction with optical instruments, although specific details are rarely given in regulatory documents. Optical instruments which magnify an object do not necessarily pose a problem because the image on the retina becomes larger and hence the laser intensity per unit area is less than in the absence of the instrument. In any event, the arrangement shown in Figure 2 is safe because most of the laser beam is directed away from the objective lens and the directly reflected light from the slide surface (typically less than10% of the initial power) also fails to enter the objective. Furthermore, when used in fluorescence mode, the emission filter will block >95% of the blue light that enters the microscope by scattering from the sample. As a further precaution, it is useful to hold a white card above the eyepiece to check for any stray light before taking a look by eye. The inclusion of a 50 mm focal length lens in front of the laser pointer to focus the beam close to the sample plane also ensures that the beam diverges beyond the microscope and becomes greater than 7 mm (the maximum pupil diameter) at a distance of ~ 35 cm (14 inches). Consequently, the laser pointer possesses less of a hazard to distant bystanders than from an inconsiderate lecturer who waves the pointer in all directions while talking. One point to be aware of is that imported laser pointers marked with a 5 mW power rating sometimes give 2 to10 times more power than stated. However, with the precautions given above they should not pose a hazard.

Apart from fluorescence, a laser pointer is useful for observing small scattering objects – indeed it is possible to detect objects smaller than the resolution limit of light microscopy with the basic microscope set-up as Figure 2b– a Nobel Prize winning topic for a future Corner.