Two-photon light-sheet microscopy with isotropic near-diffraction-limited resolution in three dimensions

Summary

Achieving isotropic high-resolution three-dimensional imaging across a large field of view (FOV) remains a key challenge in two-photon light-sheet microscopy (TPLSM). To address this limitation, we developed a TPLSM system that stitches multiple short Bessel beams into a large, ultrathin illumination light sheet and employs a dedicated control protocol to suppress stray light. The system delivers isotropic near-diffraction-limited resolution across a 332.8 μm FOV, enabling uniform visualization of fine structural details in biological samples and avoiding morphological misinterpretation. In vivo imaging of zebrafish embryos validates the system’s performance, confirming its utility for high-fidelity biological observation. Furthermore, the proposed approach is broadly applicable to other microscope modalities using Bessel-beam illumination, extending its utility well beyond TPLSM.

Graphical abstract

Highlights

• •Isotropic near-diffraction-limited resolution in three dimensions
• •FOV > 300 μm
• •Applicable to other microscope modalities using Bessel-beam illumination

Physics; Photonics; Applied sciences

Introduction

Fluorescence microscopy has long served as a cornerstone for visualizing dynamic biological processes. Among advanced imaging modalities, two-photon light-sheet microscopy (TPLSM)¹ stands out as a powerful hybrid technique that combines the deep-tissue penetration of two-photon excitation² with the low-phototoxicity volumetric imaging capability of light-sheet illumination.³ Even though, compared with point-scanning two-photon microscopy, the distribution of laser power across an extended sheet substantially reduces the photon density at any given point, generally necessitating higher total laser power. Moreover, the orthogonal arrangement of illumination and detection objectives constrains sample geometries and complicates the imaging of larger, less transparent specimens. Nevertheless, by scanning near-infrared femtosecond laser pulses into a thin light sheet, TPLSM achieves intrinsic optical sectioning and confines excitation to the focal plane, thereby minimizing out-of-focus photobleaching and photodamage—advantages that are especially critical for long-term live imaging. These advantages make TPLSM particularly valuable in diverse applications, such as brain neuroimaging,^4,5 pathological studies,^6,7 and real-time organ activity monitoring.^8,9

TPLSM uses light-sheet illumination to selectively illuminate thin planes within a sample, with the detection objective collecting fluorescence signals exclusively from this illuminated region. This configuration means the axial resolution is determined by the light sheet thickness, while the imaging field of view (FOV) is determined by the lateral extent of the light sheet. Therefore, achieving large FOV, high-resolution imaging hinges on the generation of a large, thin illumination light sheet. Meanwhile, a thinner light sheet further confines the excited volume to a more localized axial range, reducing off-target fluorophore excitation and the associated phototoxicity and photobleaching.

Over the past decades, multiple strategies have been proposed to address this challenge. A comparison of the prior systems is given in Table 1. However, up to date, there have been no reports of TPLSM achieving isotropic, near-diffraction-limited resolution in three dimensions within an FOV of hundreds of micrometers.

Table 1.

Comparison table of the prior systems

Author	Method	Lateral resolution	Axial resolution	FOV
Chen et al.10	focus scanning with TAG	420 nm	800 nm	170 μm
Gao et al.11	tiling lattice light sheets	260 nm	600 nm	170 μm
Fiolka et al.12	convert lateral scanning into axial focusing	400 nm	400 nm	100 μm
Betzig et al.13	Bessel beam illumination	300 nm	300 nm	60 μm
Côté et al.14	Bessel beam illumination	2.4 μm	2.4 μm	1.3 mm

In this work, we develop a TPLSM that achieves isotropic, near-diffraction-limited three-dimensional imaging across a 332.8 μm FOV. Isotropic high resolution in large FOV ensures that biological samples exhibit equally fine structural details along three dimensions, preventing morphological misinterpretation caused by axial blurring, and is critical for neuronal tracing and dynamic analyses of subcellular organelles, among other 3D structural analyses.

Results

Imaging results with fluorescent microbeads

To characterize the imaging performance of our TPLSM system, 100-nm-diameter fluorescent microbeads (Thermo Fisher, F8803) embedded in agarose gel were imaged. The sample was translated along the z axis at 100 nm increments via a motorized translation stage (Thorlabs, MTS25/M-Z8). One frame of raw data was acquired after each translation step, yielding a sequence of optical sections with a z interval of 100 nm. The imaging throughput is approximately 0.16 s per optical section, corresponding to a total acquisition time of about 10 s for 50 consecutive sections. The image of the microbeads across the entire 332.8 μm FOV is shown in Figure 1A, where the projections of a single microbead onto the XY, XZ, and YZ planes are presented in Figure 1B. Given that the fluorescent microbeads are sufficiently small, Figure 1B also represents the 3D point spread function (PSF) of the system. The imaging resolution of the system was obtained by Gaussian fitting the grayscale profiles of individual microbeads in the lateral and axial directions, followed by calculating the full width at half maximum (FWHM). We statistically analyzed 100 microbeads distributed across the central two thirds of the FOV. The best lateral and axial resolution achieved are 394 nm and 388 nm, respectively, demonstrating isotropic diffraction-limited 3D resolution, as illustrated in Figures 1C and 1D. For all 100 analyzed microbeads, the lateral resolution falls in the range of 424 ± 33 nm, while the axial resolution is 419 ± 35 nm, which is approximately 1.08 times the diffraction limit. Outside the 332.8 μm diameter, circular (red circle in Figure 1A) FOV, TPLSM exhibits degraded resolution. We measured 30 microbeads located in the four corner regions outside the circular FOV, and the results show a lateral resolution of 527 ± 71 nm and an axial resolution of 513 ± 60 nm, which is approximately 1.3 times the diffraction limit. To more intuitively demonstrate the isotropic resolution capability of the proposed TPLSM system, a locally magnified 3D projection video is provided in Video S1.

Figure 1.

Results with microbeads

(A) Image of microbeads across the entire 332.8 μm FOV (Scale bars, 50 μm) and zoomed-in views at representative positions (Scale bars, 2 μm).

(B) Projections of a single microbead onto the XY, XZ, and YZ planes.

(C) Imaging intensity profile of an individual microbead in the lateral direction.

(D) Imaging intensity profile of an individual microbead in the axial direction.

Imaging results with transgenic zebrafish in vivo

To demonstrate the bioimaging applicability of TPLSM, we imaged 5 day post-fertilization (dpf) Tg(fli1:EGFP) transgenic zebrafish with green fluorescent protein (GFP) expression in blood vessels (purchased from the China Zebrafish Resource Center [CZRC]). Figure 2A shows a detailed cardiac morphology captured in vivo. Video S2 provides a video of a single-plane time series revealing cardiac dynamics and heartbeat. The imaging speed is about 0.14 s/frame. Moreover, we imaged 5 dpf Tg(elavl3:EGFP) transgenic zebrafish with GFP expression in neurons cell (purchased from CZRC). Figure 2B displays the imaging results of the spinal region. Video S3 provides a video reconstructed from 300 optical sections at 100 nm intervals along the z axis, clearly revealing the connectivity between spinal nerves. The imaging speed is about 0.36 s/section, requiring 2 min for 300 sections.

Figure 2.

Results with transgenic zebrafish in vivo. (Scale bars, 50 μm)

(A) Cardiac morphology.

(B) Spinal nerves.

Imaging results with acridine orange-stained wild-type zebrafish in vivo

In order to further demonstrate the high-resolution imaging capability of TPLSM in living biological tissues, wild-type zebrafish stained with acridine orange at 5 dpf were imaged. A z stack acquisition of 500 optical sections was conducted at 200 nm intervals across an imaging depth of 50–150 μm. The imaging speed is about 0.31 s/section, requiring 3 min for 500 sections. A representative single-plane image in the XY plane is presented in Figure 3A, and the reconstructed cross-sectional images in the XZ and YZ planes are shown in Figures 4A and 4B, respectively. Through Gaussian fitting of intensity profiles and FWHM calculations, the spatial resolutions in the XY, XZ, and YZ planes were measured to be 483 nm (Figure 3C), 462 nm (Figure 4E), and 436 nm (Figure 4F), respectively. We have also statistically analyzed 10 similar structures in each of the XY, XZ, and YZ planes, and the measured resolutions are determined to be 496 ± 78 nm, 476 ± 57 nm, and 492 ± 66 nm, respectively. These results demonstrate fine agreement with the resolution results obtained using fluorescent microbeads. As shown in Figure 4G, the signal-to-noise ratio (SNR) of the image decreases gradually as the imaging depth increases. Additionally, the image contrast in the left half of Figures 3A and 4B is lower than that in the right half. This phenomenon arises from the fact that the illumination light is incident from the right side of the image, and must pass through the zebrafish spine to reach the left imaging region. Due to the relatively low light transmittance of the spinal bone, the illumination beam becomes scattered after penetrating the spine, which ultimately reduces the imaging contrast.

Figure 3.

Results with acridine orange-stained zebrafish

(A) Image in the XY plane (Scale bars, 50 μm).

(B) Enlarged view of the boxed region in (A) (Scale bars, 2 μm).

(C) Imaging intensity profile along the line indicated by red arrow in (B).

Figure 4.

Reconstructed cross-sectional images

(A) Cross-sectional image in the XZ plane (Scale bars, 10 μm).

(B) Cross-sectional image in the YZ plane (Scale bars, 50 μm).

(C) Enlarged view of the boxed region in (A) (Scale bars, 2 μm).

(D) Enlarged view of the boxed region in (B) (Scale bars, 2 μm).

(E) Imaging intensity profile along the line indicated by red arrow in (C).

(F) Imaging intensity profile along the line indicated by red arrow in (D).

(G) SNR variation with imaging depth.

Discussion

In our system, the FOV is limited to 332.8 μm due to constraints from the objective working distance, piezoelectric actuator (PZT) travel range, and camera parameters (pixel number and size). However, this FOV can be expanded—for instance, by adding a second illumination objective and redesigning the optical path to illuminate the sample from both sides (instead of the single-side illumination shown in Figure 5A), which would double the FOV. Of course, maintaining the same spatial resolution would require an imaging camera with a higher pixel number.

Figure 5.

Working principle of our TPLSM

(A) Optical setup.

(B) Control schedule.

(C) Single Bessel illumination beam (top) and stitched five Bessel illumination beams (bottom) (Scale bars, 50 μm).

(D) Comparison between the thickness of the single Bessel beam and stitched five Bessel beams in (C).

(E) Normalized intensity of the light sheet generated by the single Bessel beam.

(F) Normalized intensity of the light sheet generated by stitched the five Bessel beams.

Currently, we achieve stitching of multiple Bessel beams by mechanically scanning the illumination objective via a PZT. This method is simple and straightforward, but limits the FOV due to PZT travel range and objective working distance. Moreover, the relatively slow speed of the PZT results in low imaging frame rates. A more efficient approach involves precisely designing the phase pattern on a spatial light modulator (SLM) to simultaneously modulate and position multiple Bessel beams. This method eliminates mechanical movement, removes range limitations, thereby enables a larger FOV and much higher imaging speeds (SLMs can currently respond in a few milliseconds). We are currently working on this method.

Although the stitching operation reduces the imaging frame rate, it significantly improves the axial resolution of the system. Meanwhile, the overlap between adjacent Bessel beams enhances the uniformity of the imaging grayscale across the FOV. Notably, although the imaging process for the entire FOV is presented as a single exposure, it is essentially equivalent to the stitching of multiple sub-region scanning images along the illumination axis, according to the control schedule shown in Figure 5B (see more in STAR Methods). This configuration allows independent modulation of the laser power (via the EOM) or scanning dwell time for each sub-region, which can further homogenize the imaging intensity along the illumination axis. Furthermore, despite the long total exposure time per full frame, the effective fluorescence excitation duration for each sub-region remains short. Consequently, phototoxicity and photobleaching are not elevated; instead, the thinner light sheet confines excitation to a more localized volume, leading to reduced photobleaching.

Limitations of the study

Our system adopts an 80 MHz femtosecond laser, which is currently a widely used illumination source in the field of two-photon imaging. However, Maioli et al.¹⁵ have reported that the 80 MHz pulse frequency can induce significant linear absorption and thermal effects, and reducing the pulse repetition frequency is a superior strategy to enhance the fluorescent signal, compared to increasing the laser power.¹⁵ Hence, a femtosecond laser, tunable in both wavelength and pulse repetition frequency, may be the ideal illumination source to optimize in vivo imaging conditions.

In addition, the current system is only a preliminary prototype developed to validate the proposed method, and there still remain several unresolved issues. For example, as can be observed from Figures 4A and 4B, discrete black line artifacts exist in the reconstructed images obtained on the XZ and YZ planes. All these issues require further refinement and improvement in future research work.

Resource availability

Lead contact

Requests for further information and resources should be directed to and will be fulfilled by the lead contact, Xingyun Zhang (xyzhang@ciomp.ac.cn).

Materials availability

This study did not generate new unique reagents.

Data and code availability

• •All data reported in this paper will be shared by the lead contact upon request.
• •This paper does not report original code.
• •Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

Acknowledgments

This work was funded by the National Natural Science Foundation of China (62475258), the Jilin Provincial Scientific and Technological Development Program (20250601067RC), and the Jilin Provincial Key Research and Development Plan Project (20250201087GX).

Author contributions

Conceptualization, X.Z. and J.L.; methodology, X.Z. and L.Y.; investigation, X.Z., L.Y., W.W., and J.Z.; writing – original draft, X.Z. and J.L.; writing – review and editing, X.Z., R.Q., S.L., H.Z., Y.Z., W.X., and Z.L.; funding acquisition X.Z., R.W., and J.L.; resources, R.W. and J.L.; supervision, J.L.

Declaration of interests

The authors declare no competing interests.

STAR★Methods

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Biological samples
Transgenic zebrafish	China Zebrafish Resource Center, CZRC	Tg(fli1:EGFP)
Transgenic zebrafish	China Zebrafish Resource Center, CZRC	Tg(elavl3:EGFP)
Wild-type zebrafish	China Zebrafish Resource Center, CZRC	–
Chemicals, peptides, and recombinant proteins
Rhodamine 6G	Beyotime	Y268252
Agarose	Beyotime	D0156A
Fluorescent microbeads	Thermo Fisher Scientific	F8803
Acridine orange	Beyotime	C0233S
MS-222	Leagene	R03050
E3 culture medium	Fish Leap Future	FLF-E3-010X-500
Software and algorithms
MATLAB R2014a	The MathWorks	https://www.mathworks.com/
ImageJ 1.53c	National Institutes of Health, NIH	https://imagej.net/ij/

Experimental model and study participant details

Zebrafish type and culture

Three types of zebrafish embryos were used for the in vivo imaging experiments in this study, including Tg(fli1:EGFP) transgenic zebrafish, Tg(elavl3:EGFP) transgenic zebrafish, and wild-type zebrafish. All zebrafish embryos were cultured in E3 medium and maintained in a biochemical incubator under strictly controlled conditions: a constant temperature of 28.5 °C, and a 14 h light / 10 h dark photoperiod. The culture medium was refreshed every 24 h to maintain a stable nutrient environment. In this work, zebrafish embryos were utilized exclusively as the imaging test objects to verify the imaging performance of the custom-built microscope, rather than being subjected to independent experimental manipulations for biological research. As a result, the sex of the embryos does not exert any influence on the final imaging results. To ensure the zebrafish reached the optimal size suitable for imaging, embryos at 5 days post-fertilization (5 dpf) were selected as the experimental subjects.

Method details

Optical setup

As shown in Figure 5A, the whole optical setup of our TPLSM is divided into two mutually perpendicular optical paths. 1) Illumination path: femtosecond laser passes through an electrooptical modulator (EOM) and a half wavelength plate (HWP), and is expanded by beam expander (BE), then reflected by a spatial light modulator (SLM) with small angle (less than 10°). SLM is modulated with an axicon phase Φ_B as shown in Equation 1, so the incident Gaussian beam will be modulated to Bessel beam. Then the Bessel beam is transmitted with two 4F systems (L1-L2 and L3-IO) to form a Bessel illumination into the sample. And finally, by scanning the galvanometric mirror (GS) in X-direction, the Bessel beam forms a thin light sheet in the sample. Note: The path from mirror M3 to GS involves a vertical climb along the X direction; consequently, the optical path before and after M3 do not lie within the same XY plane. 2) Detecting path: The two-photon fluorescence excited by illumination light is collected by the detection objective DO (perpendicular to IO), and then captured by Camera passing through Filter and lens L4. The details of the optical devices are as follows: the illumination laser is ALCOR 920–2 from Spark Lasers, with 2W average power, 920 nm wavelength, 100 fs pulse width and 80 MHz repetition rate. Through EOM adjustment, the average laser power at the sample plane during actual scanning imaging is maintained at approximately 100 mW. SLM is HSP512L from Meadowlark Optics, with 512 × 512 pixels. BE is BE02-05-B from Thorlabs. The focal lengths of lens L1∼L4 are 200 mm, 80 mm, 180 mm, 200 mm respectively. GS is GVS011 from Thorlabs, with maximum scan angle of ±20°. Both IO and DO are N40X-NIR from Nikon, with effective focal length 5 mm, working distance 3.5 mm and NA 0.8. Filter is FESH0700 from Thorlabs, with cutoff wavelength 700 nm. Camera is an Andor Zyla 4.0 sCMOS with 2048 × 2048 pixels and 6.5 μm pixel size. At the excitation wavelength of 515 nm, the diffraction-limited resolution of the system is approximately 393 nm (0.61λ/NA, according to the Rayleigh criterion).

$${\mathrm{\Phi }}_B\left(x,y\right)=\mathrm{mod}\left(2\pi \sqrt{x^2+y^2}/r_0,2\pi \right)$$ (Equation 1)

Where (x, y) are the pixel coordinates of SLM, mod(a, b) denotes the modulo operation of a by b. Φ_B has the form of concentric rings, and parameter r₀ denotes the width of the ring (where the phase changes from 0 to 2 π), and its value determines the width and length of the Bessel beam.

In this optical layout, we get rid of the annular mask which is commonly used to block the non-diffracted light when modulate Bessel beams with SLM,¹⁶ since we experimentally find that non-diffracted light has barely any effects to two-photon excitation if high efficiency (>90%) dielectric mirror coating SLM is used. We verified this with Rhodamine 6G solution, and the excited Bessel fluorescence beams (parameter r₀ = 133 μm) without and with annular mask are shown in Figures S1A and S1B, respectively. No measurable difference in beam thickness or length was observed between the two excitation modes.

Illumination with stitched multiple bessel beams

Theoretically, the thickness of the Bessel beam is proportional to r₀/M, while the diffraction-free propagation length is proportional to Rr₀/M². Where R represents the radius of the light spot incident on SLM, M represents the magnification of the illumination path. Due to the limited size of the spatial light modulator (SLM), R is inherently limited, making it difficult to obtain a Bessel beam with hundreds of micrometers length and sub-micron thickness by simply designing appropriate r₀ and M. To address this, we stitch together five relatively short and thin Bessel beams by using a PZT (piezoelectric actuator) to move the illumination objective and adjust its focus in the Y direction, thereby creating a large, thin light sheet for illumination.

To achieve Bessel beam stitching, we designed a delicate control scheme for TPLSM, as illustrated in Figure 5B. The imaging camera operates in “External Trigger Exposure” mode, maintaining continuous exposure while the external trigger signal remains at TTL High. Both EOM and GS are triggered only when the PZT reaches its target position, ensuring laser illumination and GS scanning occur precisely at the desired location. Laser illumination is disabled during PZT motion with EOM at TTL Low, preventing unwanted exposure. Although the camera remains in exposure, it receives no optical signal during PZT motion, eliminating stray light interference. This approach allows a single camera exposure to capture a stitched light sheet composed of five individual Bessel light sheet, ensuring high-fidelity imaging without motion artifacts.

We verified the effectiveness of the proposed method with Rhodamine 6G solution, and the two photon fluorescence images excited by the Bessel illumination beams are shown in Figure 5C. The parameter r₀ of the axicon phase for single Bessel beam and stitched Bessel beams is 533 μm and 133 μm, respectively, representing a 4-fold reduction in r₀. Comparison between the thickness of the single Bessel beam and stitched five Bessel beams along the propagation axis is shown in Figure 5D. Along the entire propagation path, the thickness of the single Bessel beam exceeds 3 μm, with a thickness fluctuation of over 30%. In contrast, the stitched Bessel beams exhibit an average thickness of merely 0.85 μm, with a fluctuation of less than 10%. Normalized intensity of the light sheet generated by single Bessel beam and stitched Bessel beams is presented in Figures 5E and 5F, respectively. The results confirm that multi-Bessel-beam stitching also improves the imaging intensity uniformity, which can be further enhanced by precisely tuning the overlap ratio between adjacent Bessel beams.

Sample preparation protocol

Two types of samples, namely fluorescent microbeads and zebrafish embryos, were prepared and imaged during the experiment. The preparation protocol for fluorescent microbead samples is described as follows: 1) The stock solution of fluorescent microbeads was firstly fully homogenized via vortex mixing. Then the homogenized microbead solution was aspirated by pipette, and diluted 10-fold with sterile physiological saline. 2) The diluted microbead suspension was then mixed with pre-melted 1% (wt) agarose solution at a volume ratio of 1:50, followed by additional vortex mixing to achieve a homogeneous dispersion of microbeads in the agarose matrix. 3) The microbead-agarose mixture was aspirated into a capillary glass tube with an inner diameter of 1.5 mm using a metal wire fitted with a Teflon sealing plug. Then the agarose rapidly gelled at room temperature. 4) An appropriate volume of ultrapure water was added to the sample cell to ensure complete immersion of the objective lens. The gelled agarose was then fixed together with the capillary tube onto a sample holder, suspended above the sample cell, as shown in below figure. For imaging, a portion of the agarose embedded with microbeads was pushed out of the capillary tube. The 1% (wt) agarose concentration provides sufficient stiffness to prevent the gel and embedded samples from floating or shifting in the aqueous environment during imaging.

Custom-made components

3D-printed sample cell (A) and sample holder (B).

In vivo imaging was performed on two types of zebrafish: transgenic zebrafish labeled with fluorescent protein, and wild-type zebrafish stained with acridine orange. The staining protocol for acridine orange is detailed as follows: 1) The 5 mg/mL acridine orange stock solution was diluted with buffer at a ratio of 1:1000 to prepare the working staining solution. 2) Zebrafish was carefully transferred into the acridine orange working solution using a pipette to avoid mechanical damage, and incubated in the dark at 28.5 °C for approximately 30 minutes. From this step until imaging, all samples were kept protected from light. 3) The acridine orange working solution was replaced with ultrapure water, and the container was placed on a shaker for gentle shaking for 10 minutes. This washing step was repeated three times to remove excess unbound dye. 4) After removing the ultrapure water with a pipette, zebrafish was anesthetized with MS-222 for approximately 5 minutes until no obvious spontaneous movement was observed.

All zebrafish samples were prepared using an agarose embedding method: 1) 1% (wt) agarose was heated in short, multiple intervals in a microwave oven until fully melted. 2) The melted agarose solution was transferred onto the surface of a disposable culture dish by pipette. During transfer, the pipetting speed was carefully controlled to avoid introducing air bubbles into the agarose solution, which would compromise imaging quality. 3) The temperature of the agarose was tested via thermometer, and when it cooled to approximately 40 °C, anesthetized zebrafish was gently transferred into the agarose solution to avoid thermal injury. 4) Before the agarose solution was completely solidified, the agarose embedded with zebrafish was quickly aspirated into capillary glass tube. To facilitate subsequent observation, the posture of zebrafish in the agarose column was adjusted during preparation to ensure that the body of the zebrafish was aligned parallel to the capillary tube. 5) E3 culture medium supplemented with MS-222 was added to the sample cell to maintain zebrafish in a sustained anesthetized state throughout imaging. After natural cooling of the agarose, the capillary tube containing the sample was mounted on the sample holder, and the portion of agarose embedding the zebrafish was gently pushed out using the metal wire for imaging.

Quantification and statistical analysis

Beam thickness evaluation

The image grayscale intensity of the Bessel beams along the thickness direction were first readout via MATLAB, followed by a Gaussian curve fitting procedure to extract the standard deviation σ of the corresponding Gaussian distribution. The thickness of the beam was finally defined as the full width at half maximum (FWHM) of the fitted Gaussian curve, as follows:

$$FWHM=2\sqrt{2\ln 2}\sigma$$ (Equation 2)

Resolution evaluation

The acquired raw image was imported and opened in ImageJ, followed by spatial calibration via the Analyze > Set Scale, where the pixel physical dimension was set to the pre-calibrated value corresponding to the imaging system. The straight-line tool in the ImageJ toolbar was then selected to draw a line across the target region for spatial resolution assessment. By sequentially invoking the Analyze > Plot Profile, the grayscale intensity distribution along the drawn line was extracted and visualized in the dedicated profile plot window. Subsequently, the Data > Add Fit option was selected within this Plot Profile window, ‌Gaussian‌ was chosen from the Fit Function dropdown menu in the pop-up fitting dialogue, and the operation was confirmed with OK. All parameters of the fitted Gaussian model were output in the ImageJ Log window, where the parameter d corresponds exactly to the standard deviation σ of the Gaussian distribution. The resolution was defined as the FWHM of the fitted Gaussian curve, which was calculated consistently using Equation 2.