19 July 2002
LED fluorescence microscopy in theory and practice
by Ely Silk
Presented at the First Annual Citizen Science Conference, Philadelphia, June 28-29, 2002.
This paper deals with a novel technique of employing low-cost ultrabright LEDs for fluorescence microscopy using a standard transmission optical microscope. The author independently created and developed this technique as a research tool. First, a background look at fluorescence microscopy in general will be given. Next, a comparison of the pros and cons of one of the most widely used current techniques versus the LED approach will be tabulated. The theory and practical application of LED fluorescence, along with charts, graphs, and photomicrographs, will be presented throughout. The technique will be explained in sufficient detail to enable the experimenter to quickly incorporate it into daily laboratory use. Experiments in a number of different scientific fields will be discussed. A very brief look at additional applications of LEDs for photomicrography in science is included. Suppliers’ and manufacturers’ part numbers for the LEDs and required filters, lenses, biological stains, etc., are listed.
Introduction
Starting back in the early 20th century, the technique of using ultraviolet light to excite fluorescence in order to enhance and differentiate details within living cells was developed. This required special quartz lenses and aluminized mirrors for working with the UV light sources. Typically, fluorescence microscopy employs intense UV light sources, such as a high-pressure mercury lamp or xenon lamp, along with narrow-bandpass excitation filters to excite fluorescent compounds to luminesce. The reflected light from the UV source must be blocked to prevent it from entering the microscopist’s eyes or recording camera and severely reducing the image contrast. Barrier filters designed to pass the fluorescence radiation but cut off the excitation wavelengths are used. (The use of tungsten light with appropriate filters is limited due to weak emission in the blue, and this method will not be elaborated on in this paper.) More recently, a great improvement in overall brightness of the fluorescing image was invented by Ploem and incorporated by Leitz in its Ploemopak fluorescence microscopy system.1 Ploem developed the exciter filter + barrier filter + beam splitter in-a-cube concept ideal for incident light microscopes. The cubes are designed for different excitation wavelengths and barrier wavelengths (therefore, for different dyes) and are easy to insert or switch within an incident light microscope setup. Basically, the Ploemopak allows shorter wavelengths to irradiate the specimen (from above, through the objective) while permitting the Stokes’ shifted fluorescence to pass through the microscope’s optical train to the eyepieces or camera. The cube is properly referred to as a dichromatic beam splitter. The overall setup is called epifluorescence, which is now the commonly preferred lighting method. Now a look at fluorescence to see why it is such a useful phenomenon for the microscopist.
Fluorescence microscopy
For our purposes here, fluorescence may be defined as the absorption of light at a particular wavelength by an organic substance with the very rapid (under 10-8 seconds) emission of light at a longer wavelength. A fluorescent dye, such as Acridine Orange that in the presence of DNA absorbs light at 500 nanometers (nm) and emits it at the longer wavelength of 526 nm, is referred to as a fluorochrome and the longer wavelength emission is called a Stokes’ shift. There are instances when the emission is at a shorter wavelength than the exciting radiation, and this is referred to as an anti-Stokes’ shift. This phenomenon is not usually encountered and is mentioned for completeness. The reason for the shift in wavelength to a lower-energy emission is related to the lower-level singlet state achieved by the fluorochrome molecule during irradiation and absorption by a process called internal conversion. An important property of a fluorochrome is the quantum yield and is defined as:
Q = number of photons emitted/number of photons absorbed
Quantum yield ranges from 0 to 1. Unfortunately, there is a limit to how many times a fluorochrome molecule can absorb and emit light due, in part, to covalent bond splitting that disrupts the molecule. This can be many thousands of times for Acridine Orange, one of the hardier fluorochromes. But if there is a large number of photons in the irradiating flux, it can be expected that the fluorochrome’s useful lifetime (meaning how long before the dye’s fluorescence is so weak as to be rendered useless) will be severely reduced. Now, high-pressure mercury lamps and xenons in the 50-100 watt range produce a large flux of photons that soon fades the dye. This is referred to as photobleaching, and the rate of fading can be reduced by decreasing the illuminating flux or light intensity. Reducing light flux also reduces cellular heating and cells last longer. However, then it becomes necessary to employ ever more sensitive recording cameras to detect the weaker fluorescence radiation.
Just as staining with absorbing dyes (e.g., Methylene Blue, Janus Green, etc.) helps differentiate cellular components for the biological microscopist, fluorescence staining can greatly accentuate these components and with much lower numbers of dye molecules. Fewer dye molecules usually mean lower toxicity and vital staining, i.e., staining without killing the organism, can be achieved more easily. Observed cells and microorganisms will live longer, will probably be happier, and can be studied for longer periods. Moreover, single molecules can be detected if a sufficient quantity of fluorochrome is bound. Resolution can appear to exceed the normal diffraction-limited resolution of the optical microscope in that ultra-small components can be detected. The effect is similar to darkfield illumination in this respect. The literature is replete with the advantages of fluorescence microscopy.
Generally, fluorochromes are used to stain the specimen. However, many cells contain components that autofluoresce. For example, in observing plant tissue, chloroplasts under blue illumination (~ 436 nm) will often fluoresce a deep red with no additional staining. Fungi and actinomycetes will glow a telltale bluish-white under UV. In humans, cytoplasm, collagen, elastin, eye lens tissue, hemoglobin, and NADH in muscle tissue and elsewhere all autofluoresce under the right conditions. There are numerous instances of autofluorescence throughout the plant and animal kingdoms, and the experimenter will soon encounter this phenomenon. Often, cell preparations will combine autofluorescence and fluorochrome excitation for truly remarkable slides. Many dyes used in normal biological staining techniques (e.g., Eosin, Basic Fuchsin) also may fluoresce.2 Check existing microscope slides with the methods to be dealt with in this paper. (See Plate I for a mounted section of Elderberry stained with standard histological dyes that happen to fluoresce. The tissue section also includes autofluorescing cellular components.)
LEDs and fluorescence microscopy
In the past few years, LEDs with light outputs rated in the thousands of millicandelas have become readily available at very low prices. These ultrabrights definitely are not your grandmother’s LEDs. They are bright enough to dazzle the eyes, and caution should be exercised in their use.
Standard LEDs are made with GaP, GaAsP, and similar materials. The new ultrabrights are fabricated from newer compositions, such as InGaAlP, InGaN, and GaN. The ultrabrights can be 30-50 times brighter as measured in millicandelas (mcd), a photometric rating, which is listed alongside various LEDs in a table accompanying this article. As an example, one of the blue diodes that are ideal for fluorescence studies is the Hosfelt #25-365 (a light-emitting diode probably manufactured by Toshiba). The luminous intensity is given as 6000 mcd (6 candelas). Standard indicator LEDs may have a rating of 90-130 mcd. Whether standard or ultrabright, these are solid state devices that emit light when their PN junctions are properly biased. Most of the LEDs useful for fluorescence work operate at 10-20 milliamps. If operated at current levels low enough not to overheat the diodes, they will operate approximately 100,000 hours. The light output will drop at least in half over that period of time. A 10,000-hour lifetime is a conservative assessment for useful fluorescence.
Table 1 lists a number of LEDs along with their suppliers and part numbers, the manufacturers (when known or surmised), colors, peak wavelengths, and luminous intensities. Most of the LEDs are supplied in 5mm diameter packages, but several are 10mm. The peak wavelength can vary by several nanometers from batch to batch. Also, note the reference to IR that indicates infrared emission along with the visible light. This emission can be troublesome, especially for CCD camera work, and a suitable IR filter may be necessary to block it. Figure 1 illustrates the light output of most of the visible spectrum diodes referenced.
Fig. 1. Spectral outputs of various ultrabright diodes.
Click image to enlarge
Along with the LEDs emitting visible light, two diodes manufactured and supplied by Nichia are listed in the table. They emit long-wave ultraviolet light (~378 nm) and are useful for stimulating several important dyes not excited by the visible LEDs. Unlike the visible light diodes, the UV diodes can be particularly hazardous. The radiation emitted is nearly invisible, which could lead to mishandling by an inexperienced experimenter. Note that the UV diodes’ invisible "light" outputs are rated in m Watts, a radiometric measurement.
Dyes and fluorescence microscopy
As with standard histological practice, stains are often needed to accentuate cellular details. Typically, fluorescent dyes are aromatic, ring-bearing organic molecules. Fluorescence work requires dyes that have a high quantum yield (Q), possess a long lifetime, exhibit fluorescence emission in convenient regions of the visible spectrum (so as not to be confused with autofluorescence), are nontoxic, and have a high affinity for those parts of the cell under study. Sometimes fluorochromes are attached to biological molecules that, in turn, are attracted to cell components. Dyes can be used that do not fluoresce by themselves but may enzymatically split when combined with cellular components—and then fluoresce (e.g., fluorescein diacetate). Some other dyes may fluoresce when exposed to certain pH levels.
Dyes can act as bullets directed at specific targets. To study calcium ions intracellularly, Fura-2 or Indo-1 can be used. For aldehyde groups, the fluorescent Schiff reagents employing dyes like Basic Fuchsin are ideal. Bisbenzimide is useful for neuronal tracing. Calcofluor White M2R can distinguish between living and dead cells in plants, as well as stain cell walls. DiOC6 highlights the endoplasmic reticulum, while Hoechst stain illuminates the nuclear DNA. Mitochondria yield to Rhodamine 123 and MitoTracker Red. Bodipy Ceramide outlines the Golgi apparatus. The subject of dye selection is very rich, complex, and challenging.
Two of the most important factors in selecting an appropriate fluorochrome are the excitation and emission wavelengths characteristic of the dye. Table 2 lists fluorescent dyes alongside the LEDs most likely to excite them. (The emission characteristics of different dyes can be obtained from numerous reference works dealing with fluorescence microscopy.3) Most of the dyes listed have not been tested for fluorescence with LEDs. The list was derived in part from various sources in the literature citing fluorochrome properties. These properties were then extrapolated to correspond with LED output wavelengths. The table should be considered only as a starting point for work with those dyes.
Filters and fluorescence microscopy
To block the excitation light coming from the LED and yet allow the Stokes’ shifted fluorescence to pass through to the eyes and camera, inexpensive longpass filters are used. Figure 2 illustrates the filter blocking and longpass characteristics of a number of Schott filters.4
Fig. 2. Longpass transmittance curves of various Schott filters
Click image to enlarge.
The filter cutoff, l C, is at the 50% point on the rising part of each curve and is a useful characteristic in selecting the required blocking filter. (It is the light to the left of l C that is blocked in these longpass graphs.)
Occasionally, a narrow-bandpass filter may be needed to "sharpen" the LED output, and Figure 3 shows the output of a blue-green LED with and without an interference-type, narrow-bandpass filter.
Fig. 3. Wide and narrow bandpass transmission
Despite the rather considerable loss of intensity with use of the narrow-bandpass filter, it may be necessary if the fluorescence lies within or too near the broader, unfiltered emission curve of the LED and the blocking filter cannot adequately isolate the excitation light. Since the cutoff position is part of the Schott reference number, it is convenient to select a longpass filter for the application. For example, the OG 515 is an orange glass filter with its cutoff at 515 nm.
Figure 4 combines the output of an ultrabright blue LED with the longpass behavior of an OG 515 filter. The success of the LED technique for a specific application will depend on the proper selection of a longpass filter with the bandpass as close to the dye’s fluorescence peak as possible without allowing too much leakage from the LED.
Fig.
4. An LED spectral output combined with longpass curve of a barrier filter
Click image to enlarge.
Mercury lamps versus LEDs
High-pressure mercury lamps and xenon lamps are powerful, bright sources of light that have traditionally been used in fluorescence work.5 The light source is combined with a suitable narrow-bandpass filter that permits the desired mercury spectral line or wavelength region to pass and excite the fluorochrome. Much of the power residing in the continuum between spectral lines is wasted and goes into heating the excitation filter and laboratory. LEDs are considerably more efficient than mercury lamps, but they are considerably dimmer as well. On the other hand, dyes do not fade as rapidly using the LEDs, and antifading agents (see Summary) may not be required. The examined specimens remain cooler. Mercury lamps typically last between 100-200 hours if left on most of the workday. They will not last nearly that long if turned on and off for short periods of time. When the lamp finally does die, it may explode noisily, spreading mercury vapor in the air and glass within the microscope’s lamp housing. LEDs can operate usefully for at least 10,000 hours and then slowly fade away without a whimper. Replacement costs for the mercury lamp are between $100-$300 while the visible-light LEDs are under $5.00. There are many such comparisons and tradeoffs that can be made. The chart on the next page lists some features and drawbacks for each system.
Comparison of high-pressure mercury lamps and ultrabright LEDs
|
High-pressure mercury lamps |
Ultrabright LEDs |
|
Very high brightness |
Low brightness compared to a mercury or xenon lamp but newer and brighter diodes constantly being developed; not all dyes excited |
|
Excitation filters required for each wavelength needed |
Most have a small output bandwidth and do not usually require narrow-bandpass filters |
|
Generate short-wave UV good for many dyes |
Long-wave UV generated by UV diodes (~378 nm), so some important UV dyes not excited efficiently |
|
Work well with standard film cameras and sensitive CCDs |
Require sensitive films, sensitive CCDs, CCDs with integrating capabilities, or cooled cameras |
|
Bulbs last 100-200 hours and need to be left on for hours at a time to insure long life |
Last >>10,000 hours and can be turned on and off as needed |
|
Replacement is hazardous |
Replaced in an instant with no danger |
|
Replacement costs can exceed $300, more if lamp housing is damaged by exploding bulb |
Cost $5.00 or less except the Nichia UV diodes that cost $35.00 or less |
|
Require expensive and heavy power supply |
Can operate off a battery or low-cost DC supply |
|
Are designed to work with expensive dichromatic filter cubes that cost >$500.00 each |
Work well with inexpensive barrier filters that cost under $20.00; if narrow-bandpass filters are needed, additional costs can approach $100 |
|
Many wavelengths available across the spectrum dependent on lamp type and filter cubes |
Limited to approximately 8-12 wavelength regions |
|
Epifluorescent illumination incident on specimen |
Total Internal Reflection (TIR) and obliquely incident light are used in the methods discussed in this paper |
|
Work with all objectives, including oil-immersion, and function best with high NA (numerical aperture) objectives |
Neither TIR nor oblique illumination work with oil-immersion objectives. However, TIR will work with water-immersion lenses. Both will work well with low, medium, and high-dry objectives with high NA. |
|
Work in combination with brightfield, phase contrast, and DIC systems |
Plate II-6,8 show examples using brightfield illumination; also will work with phase contrast and DIC systems |
|
Cannot be turned on and off rapidly or repeatedly |
Are easily pulsed for high-speed stroboscopic photomicroscopy |
|
Specimens fade rapidly and can be cooked |
Rate of fluorochrome fading is much lower, and protozoans 10:1 favor LEDs |
The technique
Bill of materials
Assuming that a standard binocular or monocular transmission light microscope will be used for the fluorescence work, the following types of basic materials and components should be made available to start with initially:
|
Component |
Suggested source & part number |
|
25mm double convex lens with focal length = 25mm |
Edmund #32-490 |
|
Adjustable focus element support |
ThorLabs #SM1V10 |
|
1" stackable lens tube |
ThorLabs #SM1L10 |
|
End cap for lens tube (need 2) |
ThorLabs #SM1CP2 |
|
Retaining rings for lens (need 2) |
ThorLabs #SM1RR |
|
Spanner wrench for retaining rings |
ThorLabs #SPW602 |
|
Mounting ring for optical posts and base assemblies |
ThorLabs #SMR1 |
|
Short mounting post with 8-32 threaded stud on one end and _"-20 tapped hole on other |
ThorLabs #TR1 |
|
Articulating arm mounting system1 |
Edmund #54-799 |
|
Schott filter OG 515 (12.5mm or 1" as required) 2 See text for additional details. |
Edmund #54-653 (12.5mm diameter) or #45-069 (1" diameter) |
|
Schott filter GG 420 (as above) |
Edmund #46-425 (12.5mm) or #46-426 (1") |
|
UV LED (l peak = 378 nm, 750 m W) |
Nichia #NSHU590 |
|
Blue ultrabright LED (l peak = 465 nm) |
Hosfelt #25-365 |
|
Prism (small, right-angle unit preferred)3 |
Edmund #32-330, #45-108, or similar |
|
Glycerine or immersion oil |
Fisher, Polysciences, Sigma-Aldrich |
|
150 ohm and 270 ohm _ watt resistors |
Radio Shack |
|
LED holders |
Radio Shack |
|
12-volt DC power source (battery, variable supply, milliammeter) |
Radio Shack |
|
Acridine Orange, Calcofluor White M2R, Primulin |
Spectrum Chemical, Sigma-Aldrich, Polysciences |
|
Microscope slides, glassware, wire, sockets |
Polysciences, Spectrum Chemical, Radio Shack |
The ideal setups will include individual LED lamps (tube assembly, collecting and focusing lens, etc.) with one or two of each type spanning the LED spectrum as described in Table 1. A broad collection of dyes for the particular experimental areas of interest is vital. Note the storage conditions listed for each dye when purchasing. Some require refrigeration or deep-freezing for long-term storage. The manufacturers’ catalogs should be checked to see if the components selected above are exactly what may be required for individual setups.
1The articulating arm mounting system listed needs to be attached in some convenient manner to a supporting base that, in turn, can be clamped to the microscope table if required.
2The setup may require two filters for binocular eyepieces if a single filter cannot be inserted below the binocular head, plus one additional filter in the camera’s light path.
3Resting the prism on its longest side works best.
Some assembly required
The LED holder can be inserted into a central hole drilled into the tube end cap listed in the table. Solder a 150 ohm resistor in series with the selected visible-light LED, 270 ohms for a Nichia UV LED, or make up a cable terminated with the required resistor and a transistor socket. This will make a convenient, removable connector between the DC power source and the LED lamp. Initially, a milliammeter should be included in the circuit to monitor the LED current until a maximum voltage for each lamp is established. Insure that the manufacturer’s recommended current is not exceeded (typically 10-20 ma). Be sure to observe proper polarity when hooking up. Insert the lens in the focusing tube and assemble as shown in Figure 5.
Fig. 5. LED assembly with mounting post and focusing tube
Affix the LED lamp on an articulating arm mount attached to a weighted or clamped base. Position the lamp to obliquely illuminate the specimen from above the slide and the microscope stage. Focus the lens as needed to produce an intensely bright spot on the specimen. This method is adequate with objectives that have sufficient clearance between lens and slide (i.e., working distance) and do not obscure the spotlight. Next, insert the selected filter between the objective and binocular (or monocular) head. Some microscopes have filter slides that allow such positioning. If this is not possible, insert the filter(s) directly into the eyepiece(s). For photography, this barrier filter must be in the camera’s light path.
Note: If the focused spot appears too uneven under low power, one option is to produce a diffused surface on the LED by gently rubbing the dome with 400-grit emery. However, diffusing does reduce light intensity. A much better choice would be to have two individual LED lamps lighting the specimen from both ends with wider spots of light.
The oblique incident illumination method will encounter problems when using higher power objectives (designed to operate in air) with very little working distance. For these objectives, use can be made of total internal reflection (TIR). In effect, the glass slide acts as a light pipe, channeling light to the specimen. TIR works best with very thin specimens or smears. Oblique illumination works best with thicker specimens or sections. The TIR setup can produce much brighter illumination when properly adjusted and may be used for low-power objectives as well.
To couple the LED lamp light to the specimen more efficiently and without the annoyance of having objectives block the illumination, a prism is used. Figure 6 shows a convenient setup.
Fig. 6. Coupling prism to microslide
A small prism, resting on its longest side, is placed on the glass microslide with a drop of immersion oil (nD = 1.515-1.518) or glycerine (nD = 1.473). This allows an easy passage of light through the prism and down into the microscope slide. Bearing in mind that since the index of refraction of the glass usually used in prisms and microscope slides is 1.517, employing immersion oil is usually a better choice than using glycerine.
The next figure demonstrates the nature of TIR in the coupled-prism technique.
Fig. 7. Total Internal Reflection with prism coupling
Some lost light can be traced to inefficient coupling due to an index mismatch at the base of the prism. That is shown by the arrow reflecting at the interface in Figure 7. Also, some light does couple back out while it bounces back and forth along the axis of the glass if it encounters an easy path (illustrated by the very small arrow near base). Of course, some light (~6%) is lost at the first surface of the prism and could be circumvented by antireflection coating that prism face.
The specimen resting on the glass surface immersed in air, mounting media, or just plain water will act as a channel for the LED light. Attempting to use oil immersion objectives will prevent the LED light from illuminating the specimen as the oil immersion liquid pipes the light away. However, water immersion objectives do work. This is due to the lower index of refraction of the water in relation to the glass slide versus the higher index of oil. As such, the water does not refract away the LED light before the specimen is illuminated.
Additional brightness can be obtained by using two LED lamps to illuminate prisms on both ends of the glass microslide. The LEDs need not generate the same wavelength or operate at the same time. The results in those cases could be simultaneous or alternating fluorescence in two or more regions of the viewable passband!
For higher magnification work, high-dry with high numerical apertures will work but require high-sensitivity recording equipment or long exposures with low-noise cooled cameras. (Water-immersion lenses with high magnification and high numerical aperture should work, too, but these have not been thoroughly tested as yet, though early efforts appear promising.) Nevertheless, cooled cameras6 are used quite commonly by amateur astronomers and with color filter wheel attachments make satisfactory fluorescence recorders. Plate I was photographed with an SBIG ST-7 camera coupled to a CFW-8 filter wheel equipped with RGB filters. The experiments that follow, categorized into specific fields of interest, make good use of the capabilities possible with the new, low-cost technique described in this paper. Of course, the experiments represent only a tiny cross-section of potential applications.
Notes for experiments
Note 1: A word of caution before attempting any of these experiments—always assume that dyes used for microscopy are potentially carcinogenic. Be sure to read and observe the manufacturers’ warnings.
Note 2: A Mintron integrating color CCD camera (model #MTV-6268ND) was used to record most of the images in the following experiments. However, the camera does more than just record images. It can reveal subtle colors entirely invisible even to dark-adapted eyes and can greatly enhance the appearance of specimens. It can uncover invisible fluorescence in the far red and near infrared. Coupled with a good color monitor, an integrating camera can make a highly satisfactory demonstration unit for classroom, meeting room, lab, etc., since there is almost immediate feedback. For low light levels, the camera integrates up to a second or so. However, with motile organisms, images could smear. A low-cost, highly sensitive b&w CCD camera is available from Supercircuits (model PC-164C) that will work in real time. It is rated at 0.0003 lux sensitivity and sells for under $130. The images are black & white, of course, and somewhat grainy at very low light levels. Also required is a frame grabber for computer image capture. An inexpensive Snappy unit was used in these experiments. (For capturing fluorescent images in motion, a video input card is required. This would be of value for recording cyclosis, etc.) Plate III was taken with the Supercircuits camera. To the eye peering through the microscope, very little of the image was visible. Only the barest outline of the cell walls and a faint orange glow permeating throughout could be spotted. None of the chloroplasts were seen. This clearly indicates that a specimen should not be prejudged for suitability until viewed by a sensitive camera. The color version of the Supercircuits camera is rated at 0.05 lux and will not work for these experiments due to insufficient gain and the absence of integration.
Cooled cameras are much better for recording the very faintest of images. These are long-period integrating devices, so real-time or near real-time capture and display are ruled out. If available, image intensifiers can be quite useful. For really faint fluorescence, using a monocular rather than a binocular microscope can help since the "light-eating" prisms in the binocular head are gone. In the absence of electronic digital or analog cameras, standard film cameras (or film backs) loaded with highly sensitive film should work well, assuming that the specimen is bright enough to be brought into focus. One trick with faint specimens, or when working in the red end of the spectrum, is to place the barrier filter only in the camera’s light path. This will allow rough focusing with the full LED light, but any fluorescence is channeled to the recording camera and won’t be seen directly by eye. For eye safety, always have barrier filters (GG 420) in the eyepiece path when working with UV diodes.
Note 3: Work in a darkened room, with dark-adapted vision, and with eye cups on the microscope. Use low-power eyepieces. This is desirable even with a mercury arc lamp setup. First, locate the area of interest on the slide using low-intensity, red-filtered brightfield illumination to preserve dark adaptation. Then, switch over to LED illumination by blocking the transmitted light. Be sure to look for LED light leakage or spillover transmitted by the filter. This may be evident in specularly reflecting surfaces (e.g., crystal faces, air bubbles) and could produce artifacts that appear to be fluorescent. Careful positioning and adjustment of the LED lamp and the prism while viewing a slide through the microscope will be necessary to produce the maximum illumination. Unless the specimen is sufficiently bright, a CCD camera may be necessary. This is especially true if the image is being viewed in the red end of the spectrum. (Refer to Note 2.)
Note 4: All the photomicrographs accompanying this paper were taken with the light from single, ultrabright LEDs. However, it is not difficult to place at least four additional LEDs in a tight ring around a central unit within the LED housing described above. To do that, the LEDs will have to be inserted into the end cap without the fancy LED holders. The LEDs can be connected in parallel with individual series resistors for each LED. Allow 10-20 ma per LED for current draw. Alternatively, using two LED lamps illuminating the specimen from both ends works remarkably well. The extra light will allow higher quality imaging with sensitive CCD cameras, such as the unit sold by Supercircuits. The much brighter images produced by multiple LEDs should also be more comfortable for viewing by eye, but photobleaching will become more of a problem.
Experiments with LED fluorescence
Experiment 1: Forensic Science
A 1" x 1" square of bright red fabric "suspected" of containing minute traces of blood was examined. First, it was sprayed with a buffered solution of fluorescin (a very weakly fluorescing dye) and allowed to dry. Then the sample was sprayed with an ethanolic peroxide solution and examined under the microscope using a Hosfelt #25-365 Blue LED (l peak = 465 nm) and an OG 515 barrier filter. In the presence of hemoglobin and a source of oxygen, fluorescin is oxidized to fluorescein, a highly fluorescent yellowish-green fluorochrome.7 Sure enough, Plate II-1 clearly reveals the ghastly truth as telltale bright fluorescence bathes the fabric’s fibers.
Experiment 2: Materials Science
Various short-wave and long-wave UV phosphors were produced at high temperature in quartz crucibles in the laboratory, and it was decided to photograph a mixture to show crystal appearance. The mix was mounted in a non-fluorescent mounting medium on a microscope slide with a cover glass. Using the Nichia NSHU590 UV LED lamp and a GG 420 barrier filter, a photo was taken using the integrating CCD camera. Although the phosphors were formulated ideally for 254 nm and 365 nm excitation, many of the crystals manage to glow under the 378 nm UV from the Nichia diode. (See Plate II-2.)
Experiment 3: Educational
A commercially prepared slide of a marine polyp was selected. Though not deliberately stained for fluorescence studies, the dyes used did fluoresce under a Hosfelt #25-365 Blue LED lamp illumination. An OG 515 was used as a barrier filter. The picture is a photomosaic of 11 individual photographs taken along the length of the Sertularia polyp. (See Plate II-17.) Many slides currently on the laboratory and classroom shelf should be reexamined under LED fluorescence for possible new views.
Experiment 4: Microbiology
A petri dish containing Sabouraud Dextrose Agar,8 a medium suitable for growing fungi, was taken outdoors and the cover removed. After five minutes, the dish was covered and incubated for several days to allow luxuriant fungal growth. Selected samples of fungal hyphae from different species were placed on a microscope slide in distilled water with a drop of 0.1% Calcofluor White M2R, a fluorochrome with a strong affinity for chitin and cellulose. The hyphae were examined with a Nichia NSHU590 UV LED lamp (378 nm) and a GG 420 longpass filter. The images were recorded with the integrating camera. (See Plate II-8, 9, 10, 11, 13, 14.)
Experiment 5: Botany
A leaf from fresh Cilantro was slightly peeled and placed on a slide with a drop of 0.1% Calcofluor White M2R in distilled water. One photo was taken using a Nichia UV LED and a GG 420 barrier filter. (See Plate II-3.) Here the absorbed dye fluoresced under UV producing a bluish-white luminescence, revealing epithelium and stomata. The second photo was taken of a different area on the leaf using a Hosfelt #25-365 Blue LED and an OG 550 filter. (See Plate II-4.) As the Calcofluor does not produce significant fluorescence under these conditions, the colors seen in the second photo are due to autofluorescence. Other examples of botanical views are seen in Plate II-5 and 6.
Experiment 6: Palynology
Various plants (e.g., Ixora, Begonia, Impatiens, Kalanchoe, several weeds) were sampled for pollen. The samples were stained with Primulin, which is an excellent fluorochrome for pollen particularly at certain stages of pollen development. A UV LED and a GG 420 barrier filter were used for examining and photographing the pollen. (See Plate II-16.)
Experiment 7: Epidemiology/Pathology
This last experiment is, at least for the author, a hypothetical one and is included here because of its possible ramifications. To perform the experiment, access to a source of Mycobacterium tuberculosis is required. The tubercle bacillus is the cause of millions of deaths each year, especially in Third World nations. Screening sputum samples taken from suspected tuberculosis patients for the acid-fast bacilli often employed the relatively insensitive Ziehl-Neelson staining technique, which requires careful microscopic examination under an oil-immersion objective. The screening technique has been greatly improved upon by employing the fluorescent dye Auramine O to stain the bacteria due to the dye’s affinity for mycolic acid in the bacterial walls. Examination of the fluorescently stained bacilli using a standard (mercury arc lamp) fluorescence microscope is readily performed under a 25x objective. This magnification is well within the ideal working range for LED fluorescence as described in this paper. A low-cost, portable microscope outfitted with an inexpensive blue (Hosfelt #25-365, l peak = 465 nm) LED lamp, an OG 530 or an OG 550 barrier filter, plus a 12-volt battery and control should perform well in the field. Those researchers who routinely work with tuberculosis patients (or patients suffering from other mycobacterium-caused diseases, such as leprosy) are strongly encouraged to try the experiment using the TIR prism approach and to evaluate the technique for possible diagnostic use.
Other applications of LED fluorescence and illumination
In addition to fluorescence studies along the lines of standard mercury arc lamp techniques, the use of LEDs opens avenues previously difficult or impossible to explore. LEDs, being solid state devices, thrive on being pulsed to produce very brief flashes of light. Just applying square waveforms to an LED will not work well. The idea is to pump as much power into the LED in as brief a period of time as is safe for the component, thus yielding a very short duty cycle. There are circuits already available9 that will perform well, or circuits can be especially designed. Circuits of this type will allow stroboscopic photomicroscopy to be performed. Using pulsed, ultrabright white-light LEDs will enable the experimenter to freeze and view rapidly beating cilia or flagella, as an example, once the proper flashing rate is dialed in. Using color-producing LEDs will allow the combining of stroboscopy and fluorescence microscopy. Also, pulsing the light source could help reduce photobleaching.
Using microchemical spot-testing reagents (e.g., morin) coupled with UV LED illumination, minute traces of metals, such as calcium, aluminum, zirconium, and beryllium, can be detected in plant and animal cells.
Because fluorescent dyes can be detected in extremely small and relatively nontoxic amounts, they make ideal biological tracers in studies of living organisms. Microscopic examination of plant xylem employing time-lapse recording of fluorochrome-bearing nutrient flow is one such example. Fluorescent-microsphere ingestion by protozoa is another example.
Summary
The LED fluorescence technique detailed in this paper is intended to describe a new, inexpensive lighting approach that can serve as an adjunct to, but certainly not replace, high-pressure mercury arc or xenon arc fluorescence microscope methodologies. The fluorescence revealed using LEDs may be all that is required by the experimenter. If not, the technique should at least demonstrate the usefulness and feasibility of fluorescence microscopy for particular laboratory requirements.
Due to the fairly narrow bandwidth of ultrabright LEDs, the technique does away with the need for exciter filters for many applications. Only inexpensive barrier filters are usually required. TIR, an important part of the technique, makes it appear as though transmitted light employing darkfield optics is being used. Just the fluorescence excited in the specimen travels to the objective, so minute particles, organelles, and other cellular components can appear illuminated against a jet-black background, as they do in darkfield illumination. The low light levels make prolonged studies of living specimens feasible. Since autofluorescence is practically omnipresent in the plant world, initial studies can be made without recourse to expensive fluorochromes.
Due to its ancillary nature, the subject of antifading agents (e.g., n-propyl gallate, ascorbic acid, Vectorshield) could only be touched upon in this paper. Antifading agents are chemicals and proprietary formulations used to scavenge oxygen and reduce fading. One of the benefits of the lower luminous intensity of LEDs is a reduced need to use such additional chemicals, which may not be compatible with normal cellular growth. Nevertheless, the subject will need to be investigated when a problem with photobleaching occurs, as it will sooner or later. Also, mounting media used should be non-fluorescent and free of quenchers (e.g., certain heavy metal ions, such as iron, nickel, and cobalt) that will hinder fluorescence. Check the references for additional details.10-12
Recently, a division of Uniroyal announced that large LEDs with very impressive light outputs would be made available to its customers. Look to these LEDs, or similar introductions, as possible replacements for mercury and xenon lamps in traditional epifluorescence microscopes, if and when the proper wavelengths and luminous intensities become available.
Hopefully, the novel technique detailed in this paper will prove useful for school, forensic, epidemiology, and research labs. Unquestionably, the future of LED fluorescence microscopy is very bright.
Table 1. LED PEAK WAVELENGTH CHART
|
Supplier |
Mfg. # |
Part # |
Color |
Wavelength nm |
Intensity mcd |
|
Hosfelt |
Toshiba TLOA180AP |
25-277 |
Orange |
621 + IR |
3000 |
|
TLYH180P |
25-335 |
Yellow |
596 + IR |
8000 |
|
|
TLRH190P |
25-339 |
Red |
646 + IR |
15000 |
|
|
TLSH180P |
25-359 |
Red |
623 + IR * |
8000 |
|
|
25-365 |
Blue |
465 |
6000 |
||
|
TLGE185EP |
25-366 |
Green |
575 + IR |
3500 |
|
|
TLOH190P |
25-276 |
Orange |
620 |
18000-36000 |
|
|
B.G. Micro |
LED1051 |
Blue |
465 |
3000 |
|
|
LED1050 |
Green |
519 |
10000 |
||
|
LED1052 |
Bluish-Green |
498 |
20800 |
||
|
LED1044 |
White |
||||
|
Nichia |
NSHU550 |
UV |
377-378 |
1000 m W |
|
|
NSHU590 |
UV |
377-378 |
750 m W |
* One diode tested showed a clear double peak (@624 and 637)
Table 2. FLUORESCENT DYES FOR LED EXCITATION
|
Supplier |
Part # |
Wavelength |
Dyes |
|
Hosfelt |
25-339 |
646 |
Allophycocyanin |
|
25-359 |
623 |
SYTO 17 |
|
|
25-277 |
621 |
" |
|
|
25-276 |
620 |
" |
|
|
25-335 |
596 |
BODIPY
TR |
|
|
25-366 |
574 |
SNARF-1
(high pH) |
|
|
B.G. Micro |
LED1050 |
519 |
Astrazon
Red 6B |
|
LED1052
LED1052 (cont’d) |
498
498 |
Acridine
Orange |
|
|
Hosfelt |
25-365 |
465 |
Acridine
Orange (RNA) |
|
B.G. Micro |
LED1051 |
465 |
(Same as Hosfelt 25-365) |
|
Nichia |
NSHU550 NSHU590 |
378 |
Aniline
Blue |
REFERENCES
- E. Becker, Fluorescence microscopy, booklet published by E. Leitz, W. Germany
- Steven E. Ruzin, Plant Microtechnique and Microscopy, Oxford University Press, 1999
- F.W.D. Rost, Fluorescence microscopy Vol. II, Cambridge University Press, 1995
- Optical Glass Filters (Catalog), Schott Glass Technologies, Inc., Duryea, PA
- F.W.D. Rost, Fluorescence microscopy Vol. I, Cambridge University Press, 1992
- R. Berry and J. Burnell, Astronomical Image Processing, Willmann-Bell, Richmond, 2001
- Boan, DiBenedetto and Marie, Fluorescein as a Reagent, IAFS Seminar, California, 1999
- J.G. Cappuccino and N. Sherman, Microbiology: A Laboratory Manual, Addison, 1999
- R. Penfold, L.E.D Stroboscope, EPE Online (www.epemag.com), July, 1999
- B. Herman, Fluorescence Microscopy, second edition, BIOS Scientific, UK, 1998
- T.P. O’Brien and M.E. McCully, The Study of Plant Structure, Termarcarphi, 1981
- G. Sluder and D.E. Wolf, Video Microscopy, Academic Press, 1998
MANUFACTURERS AND SUPPLIERS
|
B.G. Micro High-power LEDs (their term for ultra brights), miscellaneous electronic components and kits www.bgmicro.com |
Polysciences Dyes, histology and microscopy supplies, organic reagents, fluorescent microspheres www.polysciences.com |
|
Edmund Optics Schott filters, lenses, mounts, lasers www.edmundoptics.com |
Radio Shack Resistors, cable, DC power supplies, connectors www.radioshack.com |
|
Edmund Scientific Mounted slide sets, microscopes, etc. www.scientificsonline.com |
Resources Un-Ltd Mintron cameras, lasers, optomechanics www.resunltd4u.com |
|
Fisher Scientific Chemicals, lab apparatus www.fishersci.com |
Sigma-Aldrich Dyes, biochemical reagents www.sigma-aldrich.com |
|
Hosfelt Electronics, Inc. Ultrabright LEDs, miscellaneous electronic components and kits www.hosfelt.com |
Spectrum Chemical Acids, bases, dyes, organic chemicals, lab equipment, glassware www.spectrumchemical.com |
|
Molecular Probes, Inc. Fluorescent dyes, microspheres, slides, probes (They issue a must-have catalog!) www.probes.com |
Supercircuits Ultra-sensitive video cameras, miniature cameras, time-lapse VCRs, C-Mount lenses, etc. www.supercircuits.com |
|
Nichia Corporation High power UV LEDs (Japanese company has U.S. office in California) www.nichia.com |
ThorLabs Optical mounts, lenses, electro-optical equipment, optical breadboards, laser diodes www.thorlabs.com |
Copyright 2002 by Ely Silk.
