-
Optical Simulation and Design. As LEDs have a large radiation angle (α in Fig. 1a), optical collimation towards the objective lens is essential. However, the limited space within the fluorescence filter cube makes installing spherical or aspheric lenses difficult. Our design used a 1.9 mm Fresnel lens to collimate the LED light. Because LEDs of different emission wavelengths can emit light at different radiation angles, the Fresnel lens’s position directly affects the excitation power’s delivery efficiency. In a typical epifluorescence microscope, the excitation light is reflected by a dichroic mirror into the objective lens (Fig. 1a). We performed an optical simulation in Zemax by changing the LED radiation angle α and the distance D between the LED and Fresnel lens in two microscope models, Nexcope NE900 and Olympus IX73, as illustrated in Fig. 1b.
Fig. 1 Zemax simulation to optimize the excitation power density. a The LEC is mounted inside a microscope filter cube with the excitation light collimated by a Fresnel lens and reflected by a dichroic mirror into the objective lens. b Changing the LED radiation angle α and the distance D between the LED and the Fresnel lens in Nexcope NE900 (Labc = 110 mm) and Olympus IX73 (Labc = 80 mm) microscope models.
As microscopy manufacturers adopt different designs, two typical distances from the Fresnel lens to the entrance pupil of the objective lens, for example, LNexcope NE900 = 110 mm and LOlympus IX73 = 80 mm, were investigated here. With the LED power set to 1 W (Note: Owing to the limited space of the fluorescence filter cubes and LED heat dissipation, the optical power of a single LED can hardly exceed 2 W) and using the three typical focal lengths, f = 12, 10, and 8 mm, Fig. 2 summarises the simulation results of the power delivery efficiencies as a function of the LED radiation angle α and the distance D between the LED and Fresnel lens for the Nexcope (upper panel) and Olympus (lower panel) designs, respectively. The OSRAM official website provided the LED simulation files with different radiation angles (20°–150°). By altering the parameters D (Dmin – Dmax = 5–15 mm) for the two types of microscopes (LOlympus IX73 = 80 mm and LNexcope NE900 = 110 mm), we obtained the power integral at the entrance pupil area (here we used a diameter of 6 mm). We found that a shorter focal length of the Fresnel lens favoured the design within the limited mounting space inside the filter cubes. However, the shorter focal length also resulted in a lower excitation power.
Fig. 2 Simulation analysis to optimize the LED excitation power delivery efficiency. The distances (L) between the Fresnel lens and the entrance pupil of the objective lens for Nexcope NE900 (upper panel) and Olympus IX73 (lower panel) microscopes are 110 and 80 mm, respectively. Using three typical Fresnel lenses, f = 12, 10, 8 mm, the best mounting distance D can be obtained when D is close to f.
Moreover, the highest power integral can be obtained when D is close to f. Therefore, we chose f = 11.5 mm as the Fresnel lens while D is 11.87 mm (Supporting Information Figure S1). Currently, the main manufacturers of fluorescent microscope excitation filter cubes are Zeiss, Leica, Nikon, and Olympus. Other manufacturers’ fluorescent microscope excitation filter cubes are generally the same size as those of the above four companies. Here, we demonstrate that high-efficiency illumination can be achieved within a 15 mm mounting distance (Figs. 1, 2); therefore, the LEC design should meet the above microscope installation requirements.
Selection of LEDs and Measured Optical Power. Fig. 3a shows the four typical LEDs used here, commercially available from NICHIA (360–370 nm 120°) and Yingfeng Opto-Electronic Co. (480–485 nm 60°, 540–560 nm 20°, 620–640 nm 20°). The wavelengths of the LEDs matched well with the four fluorescence channels filtered by a mercury lamp (OSRAM HBO 100W/2) through an excitation filter (Fig. 4). The LECs were mounted on a Nexcope NE900 microscope to measure the excitation powers after the three typical objective lenses (Nexcope PlanF S-Apo 20× NA = 0.5, Nikon Plan Fluor ELWD 60× NA = 0.6, Nexcope NIS 60× NA = 0.8). Fig. 3c shows the results of the final spectral measurement. To clearly show the advantage of the LEC design, we compared the power intensities of the four channels from the mercury lamp and summarised them in Table 1. Through the 20× objective lens, the optical power efficiencies of the four channels of the LECs were 97, 52, 66, and 341 times those of the mercury lamp, respectively. Similarly, through the 40× objective lens, the optical power efficiencies are 53, 60, 85 and 313 folds, respectively, and through the 60× objective lens, the optical power efficiencies are 44, 57, 84 and 361 folds, respectively, compared with those of the four filtered channels using a mercury lamp. Although objective lenses have different transmittances according to their spectral bands, LECs were at least one order of magnitude more efficient than mercury lamps (Table 1). In the red channel, particularly when it is difficult to achieve high-power excitation using a mercury lamp, the required power can be reached using the LEC.
Fig. 3 LEC integrated components and maximum power test. a 365, 485, 545, and 625 nm LED (NICHIA U365 120°, Yinfeng Opto-Electronic Co. Ltd., 480–485 nm 60°, 540–560 nm 20°, 620–640 nm 20°). b LECs were mounted in Nexcope NE900 fluorescence microscope (L = 110 mm, Objective lens: Nexcope PlanF S-Apo 20× NA = 0.5, Nikon Plan Fluor ELWD 60× NA = 0.6, Nexcope NIS 60× NA = 0.8). c The experiment result of the four test LEDs. More detailed information is in Table 1.
Fig. 4 The excitation spectrum and emission transmission curve of fluorescence filter cubes and LECs. a The purple solid line is the mercury lamp spectrum, the purple dashed line is the 365 nm LED spectrum, and the solid black line is the emission transmission curve. b The blue solid line is the mercury lamp spectrum, the blue dashed line is the 485 nm LED spectrum, and the solid black line is the emission transmission curve. c The green solid line is the mercury lamp spectrum, the green dashed line is the 545 nm LED spectrum, and the solid black line is the emission transmission curve. d The red solid line is the mercury lamp spectrum, the red dashed line is the 625 nm LED spectrum, and the solid black line is the emission transmission curve.
Light Source Conditions Rated electrical
Power(W)Optical power (mW)/
Efficiency/20×Optical power (mW)/
Efficiency/40×Optical power (mW)/
Efficiency/60×Mercury Lamp 375 nm 100 4.24 0.00424% 1.84 0.00184% 0.48 0.00048% 477 nm 2.32 0.0023% 0.67 0.00067% 0.41 0.00041% 545 nm 7.58 0.00758% 2.48 0.00248% 1.55 0.00155% 625 nm 1.58 0.00158% 0.64 0.00064% 0.36 0.00036% 365 nm LED 1.824 7.4 0.41% 1.78 0.098% 0.38 0.021% 485 nm LED 5.590 6.76 0.12% 2.21 0.040% 1.61 0.029% 545 nm LED 1.232 6.11 0.50% 2.55 0.21% 1.65 0.13% 625 nm LED 1.232 6.62 0.54% 2.41 0.20% 1.59 0.13% Table 1. The measured optical powers of mercury lamps and LECs and their optical delivery efficiencies after three typical objectives.
Selection of Emission Bands. We measured the spectra of four fluorescence excitation channels in a Nexcope NE900 fluorescence microscope, as shown in Fig. 4, and the excitation spectrum peaks were at 375, 477, 547, and 625 nm. To compare the imaging performance of the mercury lamp and LECs, we selected four high-power LEDs (365, 485, 545, and 625 nm). As the spectra of LEDs may often cover more than 80 nm (Fig. 4a−c) and the emission peaks may be shifted, a spectrometer was first used to characterise the spectra of the LEDs. Our compact LEC design also allows an excitation filter to be added in front of the Fresnel lens to purify emissions. As LEDs emit in a narrow spectrum with a well-defined wavelength range, a simplified design of either short-pass excitation filters or long-pass dichroic mirrors can be used to reduce the complexity and cost of the microscopy system. Moreover, emission filters can be saved by using time-gated luminescence microscopy.
Mechanical Design. Next, we tested the LEC design using commercial fluorescence microscopy. Fig. 5a and Supporting Information Figure S1 show the LEC designs based on the U-MF2 and U-FF modules mounted on an Olympus IX73 fluorescence microscope and a Nexcope NE900 microscope, respectively. The optimal mounting distance, D, can be adjusted by adjusting the thickness of the gasket in front of the Fresnel lens (see Fig. 5a, b). In addition, depending on the LEDs’ spectral purity and the fluorescent dyes’ Stokes shift, an emission filter and an excitation filter can be added. The distance between the LED and Fresnel lens was 13.2 mm, slightly different (~0.3 mm) from the simulation result in Fig. 1a.
Fig. 5 LEC assembly details and electrical controller. a the assembly of LEC components designed for Olympus IX73 fluorescence microscope. b the schematic layout of the assembled LEC. c The electronics circuit and control module to power LEC that allows either continuous or pulsed modes. The driver can automatically recognize the type of LEDs and adopt the appropriate voltage of Vadj and current Iadj. d The schematic layout shows that multiple LECs can be installed inside the fluorescence microscope.
Electrical Controller. A LEC driver is designed for LEDs’ identification and control. When the turntable for the fluorescent filter cubes selected one of the LEC modules, the pair of golden electrodes (shown in Fig. 5d) touched each other to power the LEC in standby mode. Fig. 5c shows the circuit control principle. With Vid as the voltage across the Zener diode, each LED type can be identified by a Zener diode with a specific voltage setting. An advanced RISC machine (ARM) STM32H743 was used as the microcontroller of the LEC, which recognised the voltage across the Zener diode and generated a corresponding PWM signal to the LM358 module with adjustable voltage Vadj that controls the SN3351 module to generate a corresponding current Iadj to power a specific type of LED. This design provides a great degree of switching ability between multiple LECs used in a fluorescence microscope via the golden touch positions, as shown in Fig. 5d. It also produces a suitable operating current and modulation frequency for the excitation pulses that can be synchronised with the optical chopper for time-gated imaging.
Contrast-enhanced fluorescence microscope by LED integrated excitation cubes
- Light: Advanced Manufacturing 4, Article number: (2023)
- Received: 28 October 2022
- Revised: 15 February 2023
- Accepted: 15 February 2023 Published online: 31 March 2023
doi: https://doi.org/10.37188/lam.2023.008
Abstract: Fluorescence microscopy is a powerful tool for scientists to observe the microscopic world, and the fluorescence excitation light source is one of the most critical components. To compensate for the short operation lifetime, integrated light sources, and low excitation efficiency of conventional light sources such as mercury, halogen, and xenon lamps, we designed an LED-integrated excitation cube (LEC) with a decentralized structure and high optical power density. Using a Fresnel lens, the light from the light-emitting diode (LED) was effectively focused within a 15 mm mounting distance to achieve high-efficiency illumination. LEC can be easily designed in the shape of fluorescence filter cubes for installation in commercial fluorescence microscopes. LECs’ optical efficiency is 1–2 orders of magnitude higher than that of mercury lamps; therefore, high-quality fluorescence imaging with spectral coverage from UV to red can be achieved. By replacing conventional fluorescence filter cubes, LEC can be easily installed on any commercial fluorescence microscope. A built-in LEC driver can identify the types of LEDs in different spectral bands to adopt the optimal operating current and frequency of pulses. Moreover, high-contrast images can be achieved in pulse mode by time-gated imaging of long-lifetime luminescence.
Research Summary
A useful illumination device for fluorescence microscopy
A compact LED fluorescence light source that can be arbitrarily used in any fluorescent microscope. Fluorescence microscopy is a powerful tool for scientists to observe the microscopic world, and the fluorescence excitation light source is one of the most critical components. Recently, Dayong Jin from China’s Southern University of Science and Technology and colleagues present a LED fluorescence light source, called LED integrated excitation cube (LEC). By replacing the conventional fluorescence filter cubes, LEC can be easily installed on any commercial fluorescence microscope, and a built-in LEC driver can identify the types of LEDs in different spectral bands to adopt the optimal operating current and the frequency of pulses. The team applied LEC to Nexcope and Olympus fluorescence microscopes and achieved high-contrast imaging of time-gated technology.
Rights and permissions
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article′s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article′s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.