The LED light source in the fluorescence microscope has the advantages of convenience and environmental protection. These LEDs maintain the effectiveness of the research components, especially for imaging and sensitive samples.
LED technology plays an increasingly important role in our lives. In the past 50 years, the application of this technology has expanded from simple electronic product indicators to alternative incandescent lamps to save a lot of energy. LEDs have the characteristics of high strength, long life, controllability and stable spectral output, so they have developed some professional lighting applications that are not well known.
Figure 1. Basic structure of a fluorescence microscope
Replacing a bulb in a fluorescent microscope with an LED has greatly reduced its operating costs. This technique is widely used in life sciences for the study of single- or multi-cellular biological specimens. The study involved the use of an optical microscope (Figure 1) to excite light of a particular wavelength. Longer wavelengths of light are re-excited by a process known as Stokes displacement, such as fluorescence and Raman spectroscopy. The Stokes shift is the difference in wavelength or frequency unit between the same electronic transition absorption and the top position of the emission spectrum.
Using different fluorophores to label the sample, ie using unused fluorescent colors in different areas, can form high-contrast multi-color photographs (Figure 2).
Figure 2. Fluorescently labeled skin image taken with CoolLED pE-300white
Meet spectral needs
Live cell imaging is required in some studies, and cells that are fixed by chemical agents for a specific life period are required. In either case, the type and design of the light source used to illuminate the sample has a significant impact on the hardware required for the microscope and the quality and effectiveness of the recorded image.
An important reason for the early use of LED systems is the convenience of the user and the person in charge of the laboratory. The most common bulbs, such as 100W high pressure mercury lamps, have a very short life span of about 300 hours. The user usually records the time when the light bulb is turned on in the notebook, because if the light bulb is used for too long, the risk of explosion will increase. However, LED products have a service life of tens of thousands of hours.
The use of the bulb requires preheating and cooling and is turned on throughout the day. The LED light source can be turned on or off electronically when needed, that is, the light source is turned on during observation of the sample or photographing, and can be turned off during use. Although the advantages of using LEDs instead of light bulbs are numerous, the application development of LEDs has stagnated due to the two main problems of high intensity and spectral range.
Due to the complexity and cost of the LED light source, the light emitted is not a broad spectrum but a Gaussian spectrum containing up to six discrete wavelengths and has a full width at half maximum (FWHM) of about 10 nm to 40 nm. Therefore, light source designers need to use multiple LEDs to meet the spectral needs of researchers, which will create new optoelectronic and mechanical design complications that are not present with conventional light bulb sources. Researchers need to capture and calibrate the Lambertian emission of the LED chip, and then use a dichroic mirror to combine multiple colors. Lambert's law shows that the intensity of the luminescence observed from the diffuse surface is proportional to the cosine of the angle θ between the incident light direction and the surface normal.
A novel and patented approach employs the concept of wavelength grouping, ie LED wavelengths with similar spectra are a user selectable channel. Four LEDs with similar spectra are combined according to the demand for high speed applications. The key is to recognize that certain wavelength-closed packets are rarely used in the same sample. Today, researchers can use LED light sources with up to 16 wavelengths, which can improve LED intensity, spectral range, and cost. 
The power of the currently available LED chips is quite different from the radiation produced by a medium ion arc of a 100 W mercury bulb. The bulb is capable of emitting energy over a very wide spectral range, but within a given spectral range of about 20 nm, the LED is more advantageous than even a large portion of the mercury bulb in the range of 360 nm to 800 nm.
Excessive use of LEDs is very common in fluorescent applications, making thermal management extremely important. Cooling techniques include Peltier cooling and placing unpackaged LED chips on large copper heat sinks.
For a long time, the green area of ​​the spectrum is the weakest compared to the bulb. This part of the area is called “green blank†in solid-state lighting and is also a very weak area in the LED spectrum. Researchers can solve this problem by using multiple forms of fluorescent materials. A method patented by Philips uses a light stick consisting of a series of bright blue LEDs. Using this method on a common fluorescence microscope can increase cost and is inconvenient to use compared to a single LED. A recent study of the power of blue LED chips offers a simpler solution of placing the phosphor directly on the LED and selecting only the phosphor that provides the largest green spectral region. The red light excited by the bright green LED through the Stokes shift is shown in FIG.
Figure 3. Fluorescently labeled bovine pulmonary artery endothelial cells. The blue region is the nucleus, the green region is tubulin, and the red region is actin. Image source: Jordi Recasens/Izasa Scientific.
Imaging enhancement
The ultimate goal of the microscope is to obtain quality images. However, sample observation is critical to the experiment due to the effects of phototoxicity. By enhancing the inherent properties of the LED, it is possible to simultaneously improve the image signal-to-noise ratio and the reaction of the image.
After the introduction of metal halide lamps, liquid-filled lamps began to be widely used, eliminating the need to calibrate the bulbs to improve illumination uniformity. This improvement in uniformity serves as a guiding function for manufacturing optical scramblers with good performance. LEDs can be directly connected to the microscope due to their solid-state properties, eliminating the need for recalibration and using Köhler illumination, a modern scientific optical microscope sample illumination technology. With this approach, the optics in the light source can image the LED onto the rear aperture of the microscope objective. This reverse working objective is capable of evenly dispersing light throughout the field of view of the sample. However, some LEDs are still used with the light guide to reduce the weight and vibration of the microscope.
Filters with good blocking and reflection areas can improve the signal-to-noise ratio of the image. In excitation and emission filters for ordinary 4',6-diamidino-2-phenylindole fluorescence (DAPI) imaging (Figure 4), the excitation filter needs to block most of the mercury (Hg) spectrum. The energy of the blue area.
Figure 4 shows the spectrum of the 385 nm LED and the Hg spectrum of DAPI absorption and emission covered using an exemplary excitation and emission filter.
In contrast, the LED used to excite DAPI produces very low energy on the corresponding excitation band, including the blue region imaged by the relatively weak sample. The result of the comparison is that using the LED as a light source results in a better image signal-to-noise ratio because the LED source can reduce the background level of the sample. Research by Sandrine Prost and colleagues at the University of Edinburgh showed that the use of wavelength-controlled, independent LED sources has improved signal-to-noise ratios beyond the bulb system and even surpassed other white wide-source LEDs.
Since the cells cannot be exposed to high intensity light, the observations will also affect the observations. The adverse effects caused by observation of the sample include photobleaching and phototoxicity, resulting in weakened signals and death of living cells or improper operation caused by illumination over time. Reducing the illumination time of the sample is critical to reducing the effects of these reactions. Conventional bulb sources control exposure through a mechanical shutter, which sometimes causes long delays that cause the sample to be unnecessarily illuminated during exposure.
This problem can be solved by using an automatically controlled LED light source. The direct TTL control of the LED complements the USB communication method by providing on/off time in microseconds. Many high-end cameras use a TTL output signal when the camera is exposed, which can be precisely matched to the camera exposure and fed directly to the LED light source control switch. Since sequential imaging of two to three fluorescent markers is common, the latest LEDs program a step sequence of wavelengths into the source, exposing with the order of each camera. For this type of circuit connection, there is no need to manipulate mechanically controlled shutters and computer control modules in real time, thereby reducing unnecessary sample exposure.
Recently, research by Claire Brown and colleagues at McGill University has shown that by controlling the amount of sample exposure, the effects of photobleaching and phototoxicity are greatly reduced. A fluorescent atom that absorbs a photon will enter the excited single-energy state, which theoretically will release most of its energy and excite a new photon of longer wavelength. However, fluorescent atoms are also likely to enter the toxic triplet state. If the fluorescent atom in the triplet absorbs more photons, the extra energy will cause the covalent bond to break. This method also helps to reduce phototoxicity and photobleaching effects.
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High-power LEDs of 340nm have been put into the market in the near future, and they use calcium fluorescent indicator (Fura-2) for calcium imaging. The application conducts research on Alzheimer's disease and other similar conditions by acquiring activity information from the neural network. According to Peter Tinning and colleagues at the University of Strathclyde, using a strong LED system at 340 nm or 380 nm allows the calcium fluorescence indicator concentration in cells to be 25% lower than standard cell preparation protocols.
This study not only saves money for experimental research, but more importantly, it can reduce the cytotoxic effects of fluorescent labeling to observe the more typical natural behavior of biological samples.
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