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Abstract
Saturated excitation microscopy, which collects nonlinear fluorescence signals generated by saturation, has been proposed to improve three-dimensional spatial resolution. Differential saturated excitation (dSAX) microscopy can further improve the detection efficiency of a nonlinear fluorescence signal. By comparing signals obtained at different saturation levels, high spatial resolution can be achieved in a simple and efficient manner. High-resolution multiplane microscopy is perquisite for volumetric imaging of thick samples. To the best of our knowledge, no reports of multiplane dSAX have been made. Our aim is to obtain multiplane high-resolution optically sectioned images by adapting differential saturated excitation in confocal laser scanning fluorescence microscopy. To perform multiplane dSAX microscopy, a variable focus lens is employed in a telecentric design to achieve focus tunability with constant magnification and contrast throughout the axial scanning range. Multiplane fluorescence imaging of two different types of pollen grains shows improved resolution and contrast. Our system's imaging performance is evaluated using standard targets, and the results are compared with standard confocal microscopy. Using a simple and efficient method, we demonstrate multiplane high-resolution fluorescence imaging. We anticipate that high-spatial resolution combined with high-speed focus tunability with invariant contrast and magnification will be useful in performing 3D imaging of thick biological samples.
© 2024 Optica Publishing Group under the terms of the Optica Open Access Publishing Agreement
1. Introduction
Fluorescence microscopy allows us to analyze and visualize complex biological events by collecting spatial and temporal information in the form of images [1,2]. Because of their great contrast, selectivity, and spatial and temporal resolution in visualizing the molecules of interest, these techniques have become essential tools for advancing life science research [3–5]. In the past, several techniques of fluorescence microscopy have been experimentally demonstrated. Each of these techniques is associated with some advantages and limitations. Optical imaging of biological samples at the nano-scale requires techniques that can cross the fundamental diffraction limit [6–11]. In the last two decades, by utilizing the characteristic properties of light-matter interaction, various super-resolution techniques have been proposed and super-resolution microscopy has become a separate topic of research in optical sciences [6–12]. Sub-diffraction limited imaging can be accomplished by stimulating nonlinear fluorescence in the saturated regime [13]. Based on absorption, emission, or depletion of fluorescence under saturation conditions, different schemes to improve spatial resolution of the microscope have been proposed [14,15]. Obtaining high-resolution three-dimensional images is a challenging task, and usually, it is performed by acquiring multiple 2D transverse plane images. In this process, optical sectioning thickness plays a crucial role in removing the out-of-focus background. Typically, due to its optical sectioning ability, confocal fluorescence microscopy is most suitable for 3D optical sectioning imaging of volumetric objects. To achieve sub-diffraction limited spatial resolution in confocal imaging, additional methods are required [16,17].
Saturated excitation (SAX) is one of the prominent sub-diffraction limited imaging techniques in which saturation of excitation is obtained by tight focusing of the excitation beam. Saturation of fluorescence can help in achieving high spatial resolution [13–18]. It was demonstrated that higher spatial frequency content generated by the nonlinear relationship between the excitation and emission processes of saturated fluorescence can improve spatial resolution beyond the diffraction limit. The nonlinear components of the fluorescence signal can be extracted by temporal modulating the excitation signal and demodulating the corresponding fluorescence signal [13,14,18].
For any super-resolution imaging technique, the requirements for special optical setup, sample preparation, and post-processing algorithms are important issues that limit their applications [17]. Differential super-resolution techniques have fewer requirements in comparison to their counterparts and can be directly implemented with simple optical setups [19,20]. The detection efficiency of the non-linear fluorescence signal was improved in differential saturated excitation (dSAX) [20]. High spatial resolution can be accomplished efficiently by comparing signals obtained at different saturation levels. This method is simple and straightforward and can be applied to confocal or multi-photon microscopy [21,14]. Despite the success of differential techniques in super-resolution microscopy, to the best of our knowledge, so far there are no reports on multiplane dSAX imaging using variable focal lenses. Here, we demonstrate the possibility of achieving 3D imaging more efficiently and straightforwardly without the need for special requirements in terms of hardware, complex post-processing algorithms, and mechanical scanning along the axial direction.
Multiplane dSAX images were acquired in a mechanical scan-free manner by utilizing a variable focus system. An electrically controlled variable focal lens is ideally suited for microscopic imaging, in particular 3D imaging. Due to its consistent performance and ability to operate at high speed, we chose an electrically controlled variable focal lens. However, focus tunability can also be incorporated with other optical elements. The direct insertion of a variable focal lens into an imaging system has some drawbacks in terms of reduced magnification and contrast across the axial scanning range [22]. In our approach, focus tunability with constant magnification and extended axial scanning range is achieved by implementing an electrically tunable lens in a telecentric design [22,23]. A relay system is used to achieve this condition where VFL is placed on the Fourier plane such that constant magnification can be obtained while scanning the object in the focal region of the objective lens. The performance characteristics of our designed system are experimentally demonstrated. To show the practical applicability of our system for 3D super-resolution imaging of biological samples, two different kinds of fluorescently labeled pollen grains were used. In contrast to the other super-resolution techniques, the present method offers many advantages, such as being easy to implement, no extra requirements for sample preparation, simple post-processing, and the ability to incorporate large sample sizes, and low cost.
2. Differential saturated excitation microscopy
The concept of saturated excitation was used to obtain super-resolution in fluorescence microscopy [8,13,14]. The nonlinearly of the saturation was utilized in the excitation and emission to collect high spatial frequency content in the signal. In typical fluorescence microscopy, the intensity of fluorescence has a linear relationship with the excitation power. However, the situation will be different when the excitation intensity levels are high and saturated of the fluoresce can occur. Enhancing the spatial resolution in SAX microscopy relies on the saturation effect, which allows for the extraction of fluorescence signals from a volume smaller than the laser focus by utilizing nonlinear fluorescence response [13,14]. By extracting this saturated component of the fluorescence signal resolution can be improved. Extraction of the signal can be done with the modulation SAX or dSAX microscopy [18–21]. Here, we are utilizing dSAX microscopy in the confocal system.
The dSAX technique extracts the fluorescence signal that exhibits nonlinear dependence on excitation intensity by comparing fluorescence signals under different degrees of excitation saturation. The measured fluorescence ${I_{FL}}$ is derived from the linear fluorescence response ${I_L}$, which would be observed in the absence of saturation. The nonlinear component ${I_{NL}}$ can then be extracted by subtracting the measured fluorescence signal from the estimated linear signal. In this condition, the fluorescence has linear and non-linear components given by
The detection efficiency of the non-linear fluorescence signal was improved in differential saturated excitation dSAX by using a differential technique. The saturated component of the signal is extracted by directly comparing the signal produced under different degrees of saturation. When the saturation is relatively low, we can ignore the higher-order nonlinear terms in Eq. (1) and obtain an equation with only the linear and second-order nonlinear term, $I_{NL}^{(2 )}({{I_{exc2}}} )$. In this case, the nonlinear term can be easily calculated using fluorescence signals measured at two different excitation intensities, as shown in Eq. (2)
3. Experimental setup
Our multiplane dSAX microscopy is based on a laser scanning confocal fluorescence microscope with an additional variable focal lens in telecentric design to achieve constant magnification and contrast throughout the axial scanning range. Axial scanning is performed digitally by controlling the focal spot of the variable focal lens and multiplane images are obtained. Figure 1 shows the experimental setup of the multiplane dSAX microscope. The light from CW laser (Cobolt SambaTM) with a wavelength of 532 nm is guided using a dichroic mirror to the scanning system. Due to high speed and stable scanning we employed a galvanometer (GVS001 Thorlabs) for point-by-point lateral scanning of the laser beam over the sample. The scanning system consists of a set of biaxial galvanometer mirrors, scanning lens (SL), and tube lens(TL) to scan excitation and emission light. The light after passing through the scanning system enters in the telecentric arrangement of the variable focal lens (VFL), and relay lens system (RL1, RL2). As mentioned before we used an electronically tunable lens as a variable focal lens in our system (Optotune-EL-10-30-TC). The VFL is placed at the Fourier plane between the TL and RL1. In this situation, while longitudinal focal shift at the object plane magnification and contrast remain constant. The parallel beam of the excitation light will enter the pupil plane of the objective lens and finally focused on the sample plane by microscopic objective (OL, NA = 0.8) for excitation and collection of the fluorescence light. We followed our previous works on the telecentric system and expressions and detailed mechanism of design can be found in [22,23]. The emitted fluorescence from the sample is captured by the objective lens (OL) and followed the reverse path of the excitation light. A low-pass dichroic mirror (DM), condensing lens (CL), and green band stop filter (GNF) is used in the detection path. A pinhole (0.64 AU) with a diameter of 15 μm is used in our experiment. The focused illumination is collected by the photomultiplier tube PMT (Hamamatsu, H7827-001). A homemade Labview software is developed to control laser power, scanning system, and photodetector to form the fluorescent images. A DAQ card is used for this purpose.
Fig. 1. Schematic diagram of differential saturated excitation microscope. A conventional confocal microscope with a variable focal lens in telecentric design. (BS: beam splitter, CL: condensing lens, LPF: long-pass filter, M: mirror, OL: objective lens, PC: computer RL: relay lens, VFL: variable focal lens SL: scan lens, TL: tube lens, GNF: Green notch filter, DAQ: Data acquisition system to interface signals to PC.)
4. Experimental results
4.1 Verification of magnification invariance
To characterize the telecentric design for magnification invariance, we used USAF high-resolution targets to image them throughout the axial scanning range. Figure 2. (a) shows the images at different z-positions. An image stack is obtained by scanning the focal length of the VFL. From Fig. 2(a), it can be observed that the magnification of our system remains constant throughout the scanning range with no change in the focal position of the VFL. Theoretically, the axial scanning range can be calculated using our previous work [23]. According to the design parameters of our system, the magnification M is ∼57. We further evaluated the performance of the VFL in the system by changing the driving current and measuring the displacement focal position. To acquire a different z-position at each step, the USAF resolution chart was defocused by the translation stage, and the focus adjustment of the lens was done by a change in driving current. In all eight images, the minimum resolvable element in group 10 element 1, shows that the image quality of our system remains constant during axial scanning. The minimum resolvable element in group 10 is element 1. These results show that the magnification of the system remains constant throughout the entire scanning range. We calibrate the system for focal tunability range. Figure 2(b) shows the axial displacement of ∼35 μm that can be obtained by changing the current in the range of (0- 300 mA). The performance of VFL is consistent and it shows the linear relationship between the driving current and the axial displacement of the focus. The focal plane moves around ∼35 μm, which is slightly less than the theoretically estimated value of ∼37.9 μm (zd = 0.146×IETL), where IETL is the current of an electrically tunable lens, and the constant factor is constant. The small difference between the theoretical and experimental values may be due to focus adjustment error or to the alignment error between the linear stage and the optical axis. To measure the resolution of the system at different depths, USAF chart images were acquired at three different depths. Figure 3(a) depicts the three images at three different focal depths with almost constant lateral resolution. Figure 3(b) shows the normalized line intensity profile along the vertical section at Group 10 Element 1 of the USAF chart at three positions, which have equal contrast with the same shape and size for three images.
Fig. 2. Test of magnification invariance for telecentric design and axial scanning performance of VFL. (a) Sequential images of standard USAF charts at different focal planes were obtained by tuning the focal length of VFL. (b) Variation of the axial position of focal spot with change in driving current of VFL.
Fig. 3. (a) Lateral resolution measurement of the confocal microscope at different focal depths using a standard USAF chart. (a) Images are acquired at depth zd = 3 μm, 17.5 μm, 32 μm. (b) Normalized line profile along the vertical section at Group 10 Element 1 of the USAF chart.
The above results demonstrate the advantage of the telecentric design in our system for getting uniform magnification and contrast for high-resolution imaging. With our simple and effective implementation of VFL, fine-tuning of axial position can be achieved in a mechanical scan-free manner with added advantages of invariant magnification and contrast that are essential for high-resolution microscopy.
4.2 Optical sectioning capability
The ability to differentiate between in-focus and out-of-focus structures can be attributed to unique point-by-point illumination and detection mechanism of confocal microscope. By precisely illuminating and detecting signals only from the focal plane, the microscope effectively minimizes out-of-focus blur, resulting in high-resolution images. This optical sectioning capability is particularly valuable in biological research, as it allows for the visualization of intricate cellular structures and processes within thick specimens.
To evaluate the optical sectioning capability of our confocal microscope, fluorescence beads with a diameter of 470 nm (Polyscience, Warrington, PA, USA) were embedded within a 2.5% agarose (Invitrogen) solution. For precise examination of the sample, a systematically controlled linear stage was employed to scan the sample. In Fig. 4, the intensity profile is obtained through axial scanning of five different fluorescent microspheres of the same size (470 nm), and an average value has been calculated for each point, serving to measure the optical sectioning capability of the proposed system. The full width at half maximum (FWHM) of the intensity profile, is approximately 3.2 ± 0.27 μm. To effectively eliminate excitation light and ensure that only fluorescence signals originating from the focal plane were captured, a band-pass filter was utilized. These results show our confocal microscope is effective in removing the out-of-focus background and can be used for the volumetric imaging.
Fig. 4. Measurement of optical sectioning capability of the confocal microscope at single focal depth by scanning florescence beads of size 470 nm. The measured FWHM of the line profile is around 3.21 ± 0.27 μm.
4.3 Multiplane dSAX microscopy measurement
The excitation light intensity of 35 W/cm2 and 52 W/cm2 was used to derive the quadratic nonlinear curve of two fluorescent agents, Rhodamine B and Rhodamine 6 G. The fluorescent agent Rhodamine B begins to have fluorescence saturation when the excitation light intensity is 350 W/cm2. When the excitation light intensity increases to 1000 W/cm2, the quadratic nonlinear curve also begins to produce saturation. The fluorescent agent Rhodamine 6 G must have fluorescence saturation occurs only when the excitation light intensity is 950 W/cm2, and the saturation phenomenon of the quadratic nonlinear curve begins at 3000 W/cm2. Measurement of fluorescence intensity curves of two fluorescent agents are shown in Fig. 5.
Next we measured the lateral resolution of confocal and the dSAX microscopy at different depths and results are compared as shown in Fig. 6. In Fig. 6, the average intensity value is determined by measuring the intensity values of fluorescently labeled microspheres in the field of view for each point, illustrating the lateral resolution of the proposed system and comparing it with traditional confocal microscopy at three different depths. Average fluorescence intensity images for 200 nm fluorescence beads (Polyscience, Warrington, PA, USA) were measured in non-saturated and saturated excitation regime. First, fluorescent beads 470 nm (Polyscience, Warrington, PA, USA) diameter was used as sample and the excitation light intensity varied from 300 to 1750 W/cm2 and fluorescent intensities were observed. It was observed that intensities more than 1650 W/cm2 is sufficient to achieve saturation level. In order to reduce noise levels, each measurement is performed five times and final image is obtained by averaging. Oversaturated fluorescent microscopy does not require image averaging process. The images retrieved from dSAX clearly demonstrate the high spatial resolution. Corresponding, line profile plot is shown in the bottom of the Fig. 6. For comparison, a confocal image and corresponding dSAX image is depicted in Fig. 6. As expected, the FWHM show dSAX provided better resolution compared to the corresponding confocal images more than 10% improvement in lateral resolution can be observed for each case. The performance of dSAX with depth variation is consistent and it demonstrate the capability of our system to perform multiplane dSAX microscopy. Imaging speed of the proposed system to scan 300 × 300 points, is 135.9 seconds (since time taken for signal to stay in single point is 1.5 ms) for corresponding pixel dwell time of 10μs.
Fig. 6. Measurement of lateral resolution of confocal and dSAX microscope at different focal depth using 200 nm fluorescent beads. (a) zd = 2.92 μm. (b) zd = 17.5 μm. (c) zd = 32.12 μm.
4.4 Multiplane dSAX imaging of biological samples
To show the practical applicability of our system for 3D super-resolution imaging, two different kinds of fluorescently labeled pollen grains (mixed pollen grains (Carolina, USA)) for red wavelength emission (Rhodamine B (max. Absorption wavelength: 551 nm, max. emission wavelength: 577 nm)) were used for imaging. Excitation intensities of 826 W/cm2 for linear and 3542 W/cm2 for saturated regions were used for imaging.
The size of pollen grains is around 50 nm and it emits fluorescence light in the red region of the spectrum. Multiplane optically sectioned images were obtained by varying the focal length of the lens. Figure 7 shows high-resolution optically sectioned fluorescent images of Ericaceae and Helianthus pollen grains obtained with dSAX microscopy. Focus tunability is achieved by changing the current of an electrically tunable lens. Due to their better spatial frequency mapping ability, dSAX images show better resolution and contrast than corresponding confocal images.
Fig. 7. Multiplane dSAX images of Fluorescently labeled pollen grains. (a) Ericaceae pollen. (b)Helianthus pollen. Images are obtained by the varying current of the varifocal lens.
To further analyze imaging results, contrast in the section of the image is measured. Figure 8 shows the confocal and dSAX images with corresponding line profiles. As shown in Fig. 8, (e) and (j), we can see an improvement in contrast values corresponding to line profiles across the sample region. With the application of the dSAX a significant improvement in the resolution is clearly evident. The image contrast is measured as C=(Imax−Imin)/(Imax+Imin). Where Imax and Imin are maximum and minimum intensity points on the line plot. Due to its simplicity and less stringent requirements for hardware, the proposed multiplane technique can also be applied to the multiphoton microscopy spatial resolution improvement for 3D image reconstruction. Compared to other multiplane imaging techniques, our mechanical scan-free approach can easily be implemented.
Fig. 8. High-resolution imaging of fluorescently labeled pollen grains. (a) Confocal image, (b) dSAX image. Images are obtained at focal depth z = 7.3 µm. (c), (d) Zoomed-in images corresponding to yellow box in (a) and (b). (e) Line profile corresponding to the blue, green arrow in (c) and (d). High-resolution fluorescent imaging of Helianthus pollen grain. Images are obtained at focal depth z = 3.65 µm. (f) Confocal image, (g) dSAX image. (h), (i) Zoomed-in images corresponding to yellow box in (f) and (g). (j) Line profile corresponding to the blue and green arrow in (h) and (i).
5. Discussions
The resolution degradation observed in our experiment is attributed to the presence of the aberrations of VFL and other hardware issues including the alignments and other procedures. The degradation in resolution and image quality and resolution becomes noticeable when the control current is set to high values. This drop in resolution can be mainly attributed to inherent aberrations induced by the lens when operating at high currents and low positive focal lengths. Nevertheless, following the calibration process outlined above and considering the measured tunable range, the image resolution and quality may be useful and acceptable for many applications as we can see from Fig. 3, By varying the axial position, the image quality is reasonably acceptable, and the resolution and contrast are almost uniform. These results show that the VFL is effective and can be utilized for axial scanning in a confocal microscope system. In addition, VFL has also been used in a variety of imaging systems with good performances, which can be a sufficient reason to implement it for focus tunability in the confocal system. In our experiment, the focus tunability is around 35 μm. The image quality shown in Fig. 3 shows that the intensity remains uniform for the axial range, which suggests that the height of the focus and the intensity to get the saturation effects will be sufficient to perform dSAX microscopy. A small deviation may be minutely small enough to be considered reasonable for practical purposes.
One of the limitations with the electrically tunable lenses is hysteresis. Hysteresis refers to a phenomenon where the response of a system depends not only on its current input but also on its history. Prolonged exposer and usage can lead to accumulation of charges in electrically controllable parts that can lead to hysteresis. Hysteresis can be controlled using control algorithm, material and design, and feedback system. However, in our system we used commercially available (Optotune-EL-10-30-TC) and we did not perform hysteresis measurements. Additional information regarding the hysteresis of electric tunable lens used in our system can be found in [24]. While we haven't characterized our system's aberrations from the VFL and other components, their presence might explain the enlarged laser focus. These aberrations can be quantified and potentially corrected using established methods. Given the limited resolution and image quality observed, a thorough aberration characterization and correction study is necessary which can be performed following earlier methods [25].
The dSAX microscopy principle is based on the Taylor expansion of the saturation function is used to combine information from several expansion orders. Although this technique enhances the signal-to-noise ratio, it eliminates a substantial portion of the signal's energy by sorting the higher resolution images based on the varying orders of the extracted nonlinear signal. Alternatively, such as novel computational saturated absorption (CSA) that utilizes a joint deconvolution fusion algorithm called super-deconvolution imaging (SDI) can be used to [26,27]. The CSA technique's strength lies in its ability to harness all nonlinear components collectively rather than individually, circumventing the reliance on information solely contained within a specific harmonic to acquire super-resolution data.
6. Conclusions
In conclusion, we developed a multiplane differential saturated excitation microscope for biomedical applications. A variable focal lens is integrated with the telecentric design to provide focal tunability and magnification invariance to the system. Imaging performance is evaluated by fluorescence imaging of two different kinds of pollen grains, which indicates that the present method has the potential to perform high-resolution optical sectioning of thick biological samples. Without any stringent requirement for hardware and optical measurements, spatial resolution can be significantly improved with our method. Another key advantage of our method is that it combines all the advantages of conventional confocal microscopy with those of differential saturated excitation microscopy, such as optical sectioning and improved spatial resolution, without any computational complexity. We anticipate our method will find important applications for high-resolution imaging of biological samples.
Funding
National Science and Technology Council (NSTC 112-2221-E-002 -055 -MY3, NSTC 112-2221-E-002 -212 -MY3, NTU-113L8507, NTU-CC-113L891102).
Disclosures
The authors declare no conflicts of interest.
Data availability
Data underlying the results presented in this paper are not publicly available at this time but may be obtained from the authors upon reasonable request.
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