Experiment on Deep-Ultraviolet 222nm Solid-State Laser 3
5.2.4 Output of 222nm Deep-Ultraviolet Pulsed Laser
Figure 5.16 Variation of Average Power and Pulse Width of 457nm Pulsed Laser with Injected Pump Power at a Repetition Frequency of 15kHz
The left axis represents the average power of the 457nm laser in mW (200-700), the right axis represents the pulse width in ns (40-120), and the horizontal axis represents the injected pump power in W (30-42).
One curve corresponds to the average power at f=15kHz, decreasing from approximately 650mW to around 350mW. The other curve corresponds to the pulse width, increasing from approximately 50ns to around 110ns.
Figure 5.17 Spot and Beam Quality of 457nm Pulsed Laser at a Maximum Average Power of 600mW with a Repetition Frequency of 10kHz (See Color Diagram)
The left part is a spot diagram, and the right part is a beam quality diagram.
In the beam quality diagram, the horizontal axis is the position along the propagation axis in mm (0-200), and the vertical axis is the spot diameter in mm (0-4). There are two curves labeled "x horizontal" and "y vertical", with My²=1.32 and Mx²=1.15. The curves first decrease and then increase, with the minimum value around 100mm and a spot diameter of approximately 0.5mm.
From the aforementioned 457nm pulsed laser experiment, the 457nm pulsed laser output with the highest peak power is obtained when the pump spot radius is approximately 200μm, L1 and L2 are approximately 83mm and 31mm, and the repetition frequency is 10kHz. This 457nm pulsed laser is used as a light source to generate 222nm pulsed laser through frequency doubling with a BBO crystal. For the placement positions of the focusing mirror M3 and the BBO crystal to achieve continuous 222nm output, since the beam quality of the 457nm pulsed laser output differs from that of 457nm, it is necessary to appropriately adjust the placement positions of the focusing mirror M3 and the BBO crystal. This ensures that after the 457nm pulsed laser passes through the focusing mirror M3, its Rayleigh length matches the length of the BBO crystal to improve the frequency doubling efficiency. Finally, a 222nm pulsed laser output with a maximum average power of 35mW and a pulse width of 36ns (measured by direct laser incidence on the detector) is achieved. While commercial options like uvc 222 nm led or 222nm uvc lamp are available for broader applications, this solid-state system provides precise pulsed output. Figure 5.18 shows the ultraviolet laser spectrum. The average output power of the 222nm pulsed laser increases with the increase in the injected power of the 457nm pulsed laser, and the variation relationship is shown in Figure 5.19. Figure 5.20 is the laser spot diagram of the 222nm pulsed laser at the highest average output power.
Figure 5.18 Ultraviolet Laser Spectrum
The horizontal axis is the wavelength in nm (200-260), and the vertical axis is the intensity (0-15000). The peak is around 222nm.
Figure 5.19 Variation of Average Output Power of 222nm Pulsed Laser with Injected Power of 457nm Pulsed Laser at a Repetition Frequency of 10kHz
The horizontal axis is the injected power of the 457nm laser in mW (200-600), and the vertical axis is the average power of the 222nm laser in mW (0-40). The curve rises linearly, from approximately 5mW to around 35mW.
Figure 5.20 Laser Spot of 222nm Pulsed Laser at the Highest Average Output Power (See Color Diagram)
The spot has a ring shape with a square outline.
Figure 5.21 Stability of 222nm Pulsed Laser Output Power
When the maximum average output power of the 222nm pulsed laser is 35mW, the stability of the laser output power is measured. As shown in Figure 5.21, the laser output stability within 2 hours is within 2%.
The horizontal axis is time in min (0-120), and the vertical axis is the average power of the 222nm laser in mW (0-50). The curve is basically stable, fluctuating around 35mW.
5.3 Experiment on Bacterial Inactivation by 222nm Pulsed Laser
5.3.1 Principle and Application Advantages of Far-Ultraviolet Light for Bacterial Inactivation
In 1903, Niels Finsen was awarded the Nobel Prize for discovering that ultraviolet light can kill bacteria. Over the next century, ultraviolet light has been widely used as a disinfection method in disinfecting items, wards, and other public places. During the ongoing COVID-19 pandemic, countries worldwide urgently need new methods to inactivate viruses in the air. There are two main methods of photodynamic sterilization and disinfection: photochemical action and photothermal action.
(1) Photochemical Action The genetic information nucleic acids (DNA/RNA) of bacteria absorb a large amount of ultraviolet light when irradiated. This leads to the formation of isomers of diazabenzenes and diazabenzenes in the body, as shown in Figure 5.22. This substance disrupts the metabolic functions of bacteria, preventing them from reproducing until they die. This photochemical action is the main disinfection mechanism of traditional ultraviolet light. However, for mold and spore microorganisms, ultraviolet light has difficulty penetrating their dense cell wall structures, so DNA cannot absorb ultraviolet light, resulting in relatively low disinfection efficiency for these microorganisms.
Figure 5.22 Schematic Diagram of Thymine Dimerization of Double-Stranded DNA under Ultraviolet Light Irradiation (See Color Diagram)
It shows the process of double-stranded DNA forming thymine dimers under UV radiation.
(2) Photothermal Action Pulsed light irradiation can rapidly increase the surface temperature of cells, destroying the cell walls of bacteria, evaporating cell fluid, and completely damaging cell structures, leading to death. Photothermal action is the temperature rise caused by the absorption of light energy by substances. When microorganisms are irradiated by intense pulsed light at close range, they absorb a large amount of light energy in a short time, causing their surface temperature to rise sharply and their surface structures to be completely destroyed, resulting in death. Since the entire photothermal action process is very short, there is no temperature rise inside the irradiated object, so it basically does not affect nutrients. According to the photothermal disinfection mechanism, intense pulsed light can effectively kill all microorganisms. Pulsed ultraviolet light combines photochemical and photothermal actions for sterilization and disinfection, so its theoretical disinfection efficiency is higher than that of a single method. The traditional ultraviolet light source for sterilization is a mercury vapor lamp. However, the peak wavelength of ultraviolet light emitted by mercury vapor lamps is 254nm, which is harmful to human cells and tissues, and can cause skin cancer [4] and cataracts in severe cases. Research in the past decade has shown that ultraviolet light in the 200-230nm band can inactivate bacteria, influenza viruses in the air, and pathogens such as SARS-CoV-2 without harming human cells. Internationally, deep-ultraviolet light in the 200-230nm band is called "far-ultraviolet light," often implemented via far uv light 222nm systems. In 2022, the Beijing Winter Olympics widely used far-ultraviolet light for sterilization and disinfection, referring to it as a "light vaccine." For those seeking 222 nm uv light for sale, options include uvc 222 nm led modules or 222nm uvc lamp fixtures, which offer similar far uv light 222nm benefits in compact forms. Compared with the typical 254nm ultraviolet light for sterilization, the biophysical principle that far-ultraviolet light is harmless to human cells is that proteins have an absorption peak in this band. In this experiment, a spectrophotometer is used to measure the protein absorption spectrum, as shown in Figure 5.23. It can be seen from Figure 5.23 that proteins have very low absorption of light in the typical 254nm band but relatively high absorption in the 200-230nm band. Far-ultraviolet light can pass through microorganisms much smaller than human cells (the typical diameters of bacteria and viruses are 1μm and 0.1μm) [11], while the diameter of typical human cells ranges from 10 to 25μm. Far-ultraviolet light is strongly absorbed by proteins in human cytoplasm and sharply attenuated before reaching the human cell nucleus [6]. For example, the outermost layer of human skin is the stratum corneum, composed of dead anucleated keratinocytes. The main function of the stratum corneum is to protect the underlying subcutaneous tissue. Most of the irradiated far-ultraviolet light is absorbed by proteins in the cytoplasm of the skin stratum corneum and cannot penetrate the skin stratum corneum to reach the key basal cells or melanocytes below, as shown in Figure 5.24. For human eyes, the tissue sensitive to ultraviolet light is the lens. However, the lens is located at the back of the cornea, and the cornea is about 500μm thick. Therefore, the transmittance of far-ultraviolet light through the cornea to the lens is basically zero.
Figure 5.23 Protein Absorption Spectrum
The horizontal axis is the wavelength in nm (200-300), and the vertical axis is the absorption value (0-3.0). There are three points on the curve: (222, 1.41), (230, 1.25), and (254, 0.168). The absorption value decreases as the wavelength increases.
Figure 5.24 Schematic Diagram of the Propagation of Far-Ultraviolet Light in Skin and Pathogens
The left side shows the skin structure, where far-ultraviolet light is absorbed by the stratum corneum. The right side shows the enlarged pathogen, where far-ultraviolet light penetrates and acts on bacteria and viruses.
5.3.2 Experiment on Bacterial Inactivation by 222nm Pulsed Laser
Bacterial Preparation Escherichia coli is widely present in nature and is a key pathogen in human public health, one of the most drug-resistant species in the Enterobacteriaceae, and is often used in research on ultraviolet disinfection and environmental hygiene. Bacillus is closely related to humans in causing food poisoning, and due to the heat resistance and acid resistance of its spores, it cannot be eliminated by pasteurization or normal hygiene procedures. Therefore, the bacterial strains used in this experiment are Escherichia Coli and Bacillus Cereus. Escherichia coli and Bacillus are Escherichia coli [Escherichia coli CMCC (B) 44102] provided by the Key Laboratory of Tropical Island Ecology of the Ministry of Education, Hainan Normal University, and Bacillus thuringiensis subspecies kurstaki HD-1 from the Environmental Microbiology Ecology Research Laboratory of Hainan Normal University, respectively. Escherichia coli and Bacillus are cultured in nutrient agar medium, placed in an incubator at 35°C and 5% CO₂, with a culture period of 24h. The nutrient agar medium is provided by Guangdong Huankai Microbial Technology Co., Ltd., and its components include peptone, beef extract powder, sodium chloride, and agar, with a final pH ≈ 7.3. The cultured bacteria are observed by slide preparation. Taking Escherichia coli as an example, its slide preparation steps are shown in Figure 5.25, and the observation results are shown in Figure 5.26.
Slide taking: The slides are stored in an alcohol solution, taken out and dried with a hot air blower.
Sterile water drop: Under a super-clean workbench, drop a drop of sterile water on the slide (not too large) for diluting bacteria.
Sampling: Use the flat end of a sterilized toothpick to gently dip bacteria from the colony; the toothpick cannot be reused.
Stirring: Stir the bacteria clockwise to make them fully and evenly distributed in the water drop.
Baking: Dry the treated slide to fix the bacteria on the slide.
Staining: Drop Coomassie Brilliant Blue reagent (toxic, operate with gloves) on the slide, making the drop just cover the bacterial plaque, and let it stand for more than 15s.
Rinsing: Rinse off the excess Coomassie Brilliant Blue reagent with slow water flow, being careful not to rinse the bacterial plaque directly to avoid washing it away.
Observation: Place the slide on the microscope stage, drop a drop of oil on the bacterial plaque, and then observe with an oil immersion lens.
Figure 5.25 Bacterial Slide Preparation Steps
It includes a picture process of eight steps: "slide taking and drying", "sterile water drop", "sampling", "stirring", "baking", "staining", "rinsing", and "observation".
Figure 5.26 Image of Bacillus Observed under a Microscope
It shows a large number of rod-shaped Bacillus.
Effect of Bacterial Inactivation The irradiation source is a 222nm all-solid-state pulsed far-ultraviolet laser developed in this work, with a spectral linewidth less than 0.1nm. The entire experimental operation is carried out in a clean room. Experimental tools are sterilized using a high-pressure sterilizer, and the workbench is irradiated with ultraviolet light for 1h before the experiment to avoid bacterial contamination from the environment. The cuvette is sealed after placing the bacterial suspension to avoid direct contact of the suspension with the outside air. Throughout the experiment, the bacteria are in nutrient fluid to avoid experimental errors caused by natural death. Take 1ml samples of Escherichia coli and Bacillus suspensions with a certain concentration and place them in a cuvette with high transmission in the short-wave ultraviolet band. Turn on the laser, and by adjusting the laser output power and the placement position of the cuvette, the irradiance of 222nm laser on the cuvette is 0.1mW/cm². Use 0.1mW/cm² 222nm laser to irradiate Escherichia coli and Bacillus suspension samples for different irradiation times. Figure 5.27 (a) shows 222nm laser passing vertically through the Escherichia coli suspension. The control samples and irradiated samples are cultured for another 24h, and the distribution of unirradiated and irradiated bacteria is shown in Figures 5.27 (b) and (c). The nutrient agar plate counting method is used to determine the bacterial count of Escherichia coli in the control group and after irradiation. To improve the accuracy of the experimental data, each sample is repeated 3 times under the same irradiation dose, and the average value is taken.
Figure 5.27 222nm Pulsed Laser Sterilization Experiment
(a) Escherichia coli suspension under irradiation;
(b) Distribution of Escherichia coli after 0s, 10s, and 20s of irradiation, with the number of colonies on the plate decreasing with increasing irradiation time;
(c) Distribution of Bacillus after 0s, 30s, and 60s of irradiation, with the number of colonies on the plate decreasing with increasing irradiation time.
The test results are listed in Table 5.1. When the 222nm laser irradiates the Escherichia coli suspension for 10s (1mJ/cm²), the inactivation rate is 90.7%; for 15s (1.5mJ/cm²), the inactivation rate is 96.9%; and for 20s (2mJ/cm²), the inactivation rate is as high as 100%. When the 222nm laser irradiates the Bacillus suspension for 30s (3mJ/cm²), the inactivation rate is 88.4%; for 45s (4.5mJ/cm²), the inactivation rate is as high as 98.6%; and for 60s (6mJ/cm²), the inactivation rate is as high as 100%. This experimental study shows that irradiation with far-ultraviolet 222nm pulsed laser at doses of 2mJ/cm² and 6mJ/cm² can effectively inactivate Escherichia coli and Bacillus, respectively. It can be seen from this experiment and other literature reports that the irradiation dose required for inactivating Bacillus by ultraviolet light is larger than that for Escherichia coli. The reason is that the irradiation dose required for inactivating bacteria and viruses by ultraviolet light is mainly related to factors such as the size of microorganisms, the thickness of cell membranes (walls), and the structure of nucleic acids (single-stranded or double-stranded). Generally, the larger the size of the microorganism and the thicker the cell membrane, the more irradiation dose is required; microorganisms with double-stranded structures have stronger repair capabilities than those with single-stranded structures, so more irradiation dose is also required. Both Bacillus and Escherichia coli have double-stranded structures, but the size and cell wall thickness of Bacillus are (1.0~1.2) nm × (3.0~5.0) nm and 20~80nm, respectively, while those of Escherichia coli are only (0.5~0.8) nm × (1.0~3.0) nm and 11nm. Therefore, the ultraviolet irradiation dose required to inactivate Bacillus is larger than that for Escherichia coli.
