High-Speed Neural Imaging with Synaptic Resolution: Bessel Focus Scanning Two-Photon Microscopy and Optical-Sectioning Widefield Microscopy

Abstract

Brain is composed of complex networks of neurons that work in concert to underlie the animal’s cognition and behavior. Neurons communicate via structures called synapses, which typically require submicron spatial resolution to visualize. To understand the computation of individual neurons as well as neural networks, methods that can monitor neuronal morphology and function in vivo at synaptic spatial resolution and sub-second temporal resolution are required. In this chapter, we discuss the principles and applications of two enabling optical microscopy methods: two-photon fluorescence microscopy equipped with Bessel focus scanning technology and widefield fluorescence microscopy with optical sectioning ability, both of which could be combined with optogenetic stimulation for all optical interrogation of neural circuits. Details on their design and implementation, as well as example applications, are presented.

1 Introduction

With the development of genetically encoded fluorescence reporters that convert neural activities into fluorescence fluctuations [1,2,3,4,5,6,7], optical microscopy has been widely applied to in vivo studies of brain function due to its non-invasiveness and subcellular spatial resolution. Over the past decades, major efforts have been made to improve the performance of optical microscopes in terms of their resolution, speed, and imaging depth [8,9,10,11,12,13]. Depending on the specific application, a distinct set of criteria should be applied in order to select the most appropriate imaging modality. In the case where synaptic responses need to be characterized (e.g., map** all the synaptic inputs of a neuron), the ideal method should have high spatial resolution to resolve individual synapses and sufficient temporal resolution to capture all the ongoing activities in the observation area/volume. For studies in opaque samples (e.g., adult mammalian brains), multiphoton fluorescence microscopy is often the method of choice, because its nonlinear excitation leads to optical sectioning and its near-infrared illumination light provides optical access to structures at depth in highly scattering tissue [10,11,12]. Despite its ability to image scattering tissues, the main limitation of multiphoton microscopy is its speed, which is limited by the point-scanning scheme of its standard implementation [12], especially when applied to three-dimensional (3D) volumetric imaging. Bessel focus scanning technology, where a Bessel-like beam is utilized for fluorescence excitation [14,15,16,17], has been used to improve the volumetric imaging speed of multiphoton microscopy via an extended depth of field (DOF), and is discussed in Subheading 2 of this chapter. In contrast, for studies in transparent samples with minimal tissue scattering, widefield fluorescence microscopy can be applied, with the advantages of high imaging speed and simple hardware implementation (see also Chap. 2). However, widefield microscopy does not have optical sectioning ability [18,19,20,21]. As a result, significant out-of-focus signal contributes to a blurry and sometimes overwhelming background, which obscures the in-focus objects, especially small structures like synapses. One approach to impart optical sectioning ability to widefield fluorescence microscopy is via structured illumination (SI) [9, 22,23,24,25], where the sample is illuminated with a structured rather than uniform pattern of light to distinguish the in-focus information from the background signal. The details of this technology are discussed in Subheading 3 of this chapter [9].

2 Bessel Focus Scanning Two-Photon Fluorescence Microscopy

2.1 Background

2.1.1 Multiphoton Excitation

Widefield fluorescence microscopes with optical sectioning ability enable high spatiotemporal resolution imaging in neurobiological model systems that are transparent (e.g., zebrafish larvae and Drosophila larvae), but cannot visualize deep structures in opaque samples (e.g., adult mammalian brains) due to tissue scattering. In multiphoton excitation fluorescence microscopy, the longer wavelength of excitation light renders the system more resistant to scattering and grants optical access to structures at depths up to 1–2 mm in the mouse brain [10,11,12]. Furthermore, the nonlinearity of multi-photon excitation can significantly suppress out-of-focus fluorescence generation, thus requiring no additional device or computation to realize optical sectioning.

2.1.2 Challenges of Volumetric Imaging with Multiphoton Microscopy

In a conventional multiphoton fluorescence microscope, fluorescence excitation is three-dimensionally confined. Thus 2D or 3D scanning of the focus is required to obtain a planar image or a volume stack, respectively. Such a point-scanning scheme ensures optical sectioning ability and high spatial resolution even in opaque tissue, but limits imaging speed. Therefore, to record neural dynamics with sufficient frame rates, scanners that can move the excitation focus at high speed are desired. Lateral beam steering (translation of the focus in a plane perpendicular to the optical axis) in multiphoton microscopy is typically achieved by a pair of galvanometer-based optical scanners (or “galvos”). Conventional raster-scanning galvos can achieve line scan rates of a few kHz [12]. By replacing one of the raster-scanning galvos with a faster scanner (e.g., resonant galvos [26]), line scan rates of tens of kHz and 2D frame rate of tens of Hz can be realized [12]. However, information observed from a single optical section can be incomplete. Since both neural circuits and individual neurons therein are 3D structures, volumetric imaging with subsecond temporal resolution is required to capture their dynamics completely. To perform volumetric imaging in multiphoton microscopy, in addition to scanning the focus in the lateral xy plane, axial movement of the excitation focus along z direction is required. In contrast to lateral focus scanning, axial scanning of the focus is more challenging. A common method to scan the excitation focus axially is to translate the microscope objective along z direction, the speed of which, however, is limited by mechanical inertia associated with the objective. Wavefront sha** devices such as electrically tunable lenses (ETLs) [27], spatial light modulators (SLMs) [28], or acousto-optic deflectors (AODs) [29,30,31] allow the control of wavefront divergence at the objective back focal plane, which translates the focus axially. These methods avoid the problems posed by objective inertia, but introduce additional aberrations to the system and degrade image quality at large focal shifts, since most microscope objectives are designed to produce optimal performance for light of a specific divergence (e.g., multiphoton excitation objectives are designed for collimated beams) [32]. A dual-objective remote focusing method, where the excitation light travels through two microscope objectives with conjugated pupil planes [33,34,35], has aberrations caused by the two objectives cancelled out and can achieve high-speed volumetric imaging at diffraction-limited resolution over a large axial range. The requirement of two objectives nevertheless increases the system cost and complexity, requiring custom optical design [36]. Moreover, all the above volumetric imaging methods are sensitive to axial motions of the sample, which cause the objects of interest to move out of focus and the loss of dynamic information. Bessel focus scanning multiphoton microscopy provides an alternative to the volumetric imaging methods described above [15, 16, 37, 38] and will be discussed in detail below.

2.1.3 Bessel Focus Scanning Technology

During most brain imaging experiments in vivo, neurons exhibit variations in their fluorescence signal associated with their functional activity, but their positions remain unchanged. Therefore, when the temporal dynamics of neurons is the subject of interest, one does not need to constantly track their 3D locations, but can instead obtain axially projected views of 3D volumes via extended depth-of-field imaging by scanning an axially elongated focus, e.g., a Bessel-like beam [14,15,16, 39,40,41] (Fig. 1). Since all structures along the elongated focus are probed simultaneously within a single 2D scan, the 2D frame rate becomes a projected 3D volume rate. Although axial resolution is reduced substantially during Bessel focus scanning, the 3D positions of neurons and neuronal structures can be obtained from a conventional 3D stack by scanning a Gaussian focus. Because at the same numerical aperture (NA), a Bessel focus has a higher lateral resolution than a Gaussian focus [41], synapse-resolving lateral resolution is maintained even for a 0.3-NA Bessel focus [42]. With Bessel focus scanning, imaging throughput can improve by tens to a hundred times, with the image data size reduced by the same amount [15]. Bessel focus scanning technology is compatible with other fast scanning methods mentioned in Subheading 2.1.2 and, when combined together, can further boost the volumetric imaging speed of a multiphoton microscope [15, 38]. The extended depth of field of a Bessel focus also makes the imaging process insensitive to axial motion, which eliminates axial motion artifacts. Together with the much reduced data size, it substantially simplifies image processing [15, 42]. Although Bessel focus scanning technology can be incorporated into both two-photon and three-photon fluorescence microscope systems [37], the sections below focuses on Bessel focus scanning two-photon fluorescence microscopy for volumetric imaging.

Fig. 1

The concept of Bessel focus scanning multiphoton microscopy. (a) 2D scanning of a conventional Gaussian focus obtains information from a single plane (the plane in yellow). (b) 2D scanning of a Bessel focus covers a volume (the volume in orange)

2.2 Materials and Equipment

A typical two-photon fluorescence microscope includes the following optical components (Fig. 2): a femtosecond laser, an excitation power control unit such as an electro-optical modulator (EOM) (i.e., a Pockels cell), a pair of scanners/galvos, a scan lens and a tube lens, a dichroic mirror, an objective (often mounted on a piezoelectric stage to perform axial scanning), detection filters, one or two photomultipliers (PMTs).

Fig. 2

The Diagram of two Bessel focus modules. (a) A two-photon microscope with an SLM-based Bessel module (gray box). (b) An axicon-based Bessel module. L2 can be translated along optical axis to change the axial length and numerical aperture of Bessel beam. D is defined as 0 mm when the mask is at the front focal plane of L2 and positive when L2 moves away from the mask. Ti:Sa Ti:Sapphire laser, EOM electro-optical modulator, BE beam expander, M mirror, L lens, Obj objective

A Bessel focus module includes the following optical components (Fig. 2): two mirrors to switch the light path between Gaussian and Bessel modes, an SLM or an axicon, a lens, an annular mask, and a pair of conjugation lenses.

2.3 Methods

2.3.1 Two-Photon Fluorescence Microscope

The essential components in the excitation light path of a two-photon fluorescence microscope are shown in Fig. 2a. The output of a Ti:Sapphire laser with a Gaussian intensity profile first passes through a Pockels cell, and then a beam expander (see note 1 in Subheading 2.4). The Gaussian beam is then directed either straight to the galvos or into a Bessel module, by moving two mirrors M1 and M2 out of and into the light path, respectively. The galvo pair could be either placed close together or conjugated with a pair of scan lenses. The latter can eliminate beam wandering on the second galvo as well as the objective back focal plane during scanning, but leads to more power loss due to adding additional optical elements. In some systems, a resonant scanner is incorporated in addition to the two raster-scanning galvos, with the three scanners together enabling high-speed imaging in a small subfield positioned anywhere inside a large field of view [36, 38]. After the galvos, a scan lens (L4) and a tube lens (L5) conjugate the scanners to the back focal plane of the objective. The emission light path (e.g., the dichroic mirror and the detectors) is not shown in the diagram.

2.3.2 Design and Setup of a Bessel Focus Module

An annular illumination pattern at the objective back focal plane generates a Bessel-like focus. This annular illumination can be created with a phase mask [14], an SLM [15, 42], or an axicon [16, 39, 40].

In an SLM-based Bessel focus module (Fig. 2a, rectangle box), a reflective phase-only SLM is placed at the front focal plane of a lens (L1). A circular binary phase pattern (alternating 0 and π) on the SLM diffracts the incident Gaussian beam preferentially into the ±1 diffraction orders, which form an annular ring at the back focal plane of L1. An annular aperture mask is placed at the back focal plane of L1 to selectively transmit the desired annular electric field, which is conjugated to the galvos by a pair of lenses L2 and L3, and then to the objective back focal plane.

An axicon-based Bessel focus module (Fig. 2b) has a similar configuration to an SLM-based module, except that an axicon is placed at the front focal plane of L1. The conical surface of the axicon refracts the light according to Snell’s law, which forms a ring at the back focal plane of L1. The annular aperture mask at the back focal plane of L1 is not necessary for an ideal axicon (with infinitely small conical tip), but is necessary in practice to block the unwanted light refracted through the tip of an imperfect axicon.

Fig. 3

Electrical field distribution at the mask plane in an SLM-based Bessel module. (a) Amplitude and (b) phase of the (c) electric field at different radial positions. 0 is the location of the optical axis. The red and green dashed lines in (a) and (c) represent the inner and outer diameters of the annular masks for Case I and Case II discussed in Subheading 2.3.3.4. (This figure is adapted from Ref. [15])

When it comes to choosing between an SLM and an axicon-based Bessel module, several factors need to be considered. An SLM-based module offers more flexibility in terms of point spread function (PSF) engineering [15] and allows the NA and axial length of the Bessel focus to be adjusted independently [15]. Details on designing Bessel foci with different PSF profiles are discussed in Subheading 2.3.3, but a brief overview is presented here. In both methods, the axial length of the Bessel focus can be altered by varying the size of the Gaussian beam im**ing on the SLM or the axicon, for example, by adding a beam expander or reducer at the entrance of the Bessel module. With the beam size fixed, a user can adjust the NA and axial length of the Bessel focus independently by changing the phase pattern on the SLM and the dimensions of the annular mask. Therefore, an SLM-based module is ideal for systems utilizing multiple objectives with different NAs (e.g., a 1.05-NA objective for neocortical imaging or a 0.5-NA microendoscopic lens for deep brain imaging). In contrast, an axicon-based module does not allow users to adjust NA and axial focal length independently, without varying the beam size or introducing a different axicon [39, 40]. Translating one of the conjugation lenses (L2 or L3) along the optical axis concurrently changes the NA and axial length of the Bessel focus [16]. However, despite being less flexible in PSF engineering, the axicon-based Bessel module is nevertheless an attractive alternative for the following reasons: first, its transmissive layout occupies less space and makes it easier to incorporate into an existing system [38]; second, an axicon module costs much less (~$5000, rather than $30,000 for an SLM-based module) to set up; third, an axicon works with a larger wavelength range compared with an SLM (e.g., compatible with three-photon microscopy [37]).

2.3.3 Design of a Bessel Module

We use MATLAB^® to calculate the PSF profiles of Bessel foci and to guide the design of Bessel module. The underlying physics is described below, and the MATLAB codes can be found in Refs. [15, 16].

2.3.3.1 Generation of Annular Illumination with an SLM (Adapted from Ref. [15])

Concentric binary grating patterns with phase values alternating between 0 and π are applied to a phase-only SLM to diffract most of the incident electromagnetic field into the ±1 orders (see note 2 in Subheading 2.4), which after lens L1 forms a ring at the mask plane. The radius of the ring (ρ), determined by the period of the grating (d), the focal length of L1 (f₁), and the wavelength of the light (λ), is calculated from the grating equation as:

$$ \rho =\frac{f_1\lambda }{d}. $$

For an annular mask to transmit the ring and block the other diffraction orders, its inner and outer diameters D_i and D_o should satisfy the relation:

$$ {D}_{\textrm{o}}+{D}_{\textrm{i}}=4\rho . $$

Combining the two equations above, to generate an annular illumination pattern that centers on an annular mask with inner and outer diameters D_o and D_i, the period of the circular binary grating on the SLM is:

$$ d=\frac{4{f}_1\lambda }{D_{\textrm{o}}+{D}_{\textrm{i}}}. $$

With the size of the SLM pixel defined as p, the period of the circular binary grating in units of pixels S is:

$$ S=\frac{d}{p}=\frac{4{f}_1\lambda }{p\left({D}_{\textrm{o}}+{D}_{\textrm{i}}\right)}. $$

The thickness of the annulus at the mask plane generated by the above circular binary grating is ~2f₁λ/beamD, with beamD being the diameter of the excitation laser on the SLM [43].

2.3.3.2 Generation of Annular Illumination with an Axicon

For an axicon with an apex angle A, the angle of incidence α on the conical surface is: $\alpha =\frac{\pi -A}{2}$. The refraction angle is then derived using Snell’s law: α_r = nα, given that α is small and n is the refractive index of the axicon. Therefore, the angle between the refracted light and the optical axis is:

$$ \theta ={\alpha}_{\textrm{r}}-\alpha =\left(n-1\right)\alpha . $$

The radius of the ring at mask plane is:

$$ \rho =\theta {f}_1=\left(n-1\right)\alpha {f}_1. $$

The annular mask to transmit the ring should again satisfy:

$$ {D}_{\textrm{o}}+{D}_{\textrm{i}}=4\rho . $$

Therefore, the apex angle and refractive index of the axicon should meet:

$$ \left(n-1\right)\alpha =\frac{D_{\textrm{o}}+{D}_{\textrm{i}}}{4{f}_1}. $$

2.3.3.3 Calculation of Two-Photon Excitation PSF

Two-photon excitation PSF can be calculated using Richards and Wolf integrals [44, 45], from which both the lateral and axial full width at half maximum (FWHM) of the PSF can be determined. The information required for PSF calculation includes: the wavelength of excitation light, the objective information (i.e., NA, magnification, immersion media), and the electric field distribution at the objective back focal plane.

The following equations hold for all infinity-corrected objectives:

$$ F{L}_{\textrm{obj}}=\frac{FL_{\textrm{tube}}}{M_{\textrm{obj}}}; $$

$$ BP{D}_{\textrm{obj}}=2N{A}_{\textrm{obj}}\ F{L}_{\textrm{obj}} $$

where the FL_obj is the focal length of the objective, FL_tube is the focal length of the tube lens, M_obj is the magnification of the objective, and BPD_obj is the back pupil diameter of the objective. The Bessel annulus at the back focal plane with an outer diameter D_o and inner diameter D_i gives rise to an excitation NA as:

$$ N{A}_{\textrm{Bessel}}=\frac{D_{\textrm{o}}}{BP{D}_{\textrm{o}\textrm{bj}}}N{A}_{\textrm{o}\textrm{bj}}. $$

Or:

$$ N{A}_{\textrm{Bessel}}=\frac{D_{\textrm{o}}}{2F{L}_{\textrm{o}\textrm{bj}}}. $$

The PSF profile is determined by D_o, D_i, and the electrical field distribution within the annulus, which is discussed below.

2.3.3.4 Design of the Annular Aperture Mask in an SLM-Based Bessel Module (Adapted from Ref. [15])

The annular mask is designed to be conjugated to the objective back focal plane, with a magnification factor M, which is determined jointly by focal lengths of lenses L2–L5. In most cases, the objective, L4, and L5 (scan lens and tube lens) are already selected and built prior to the design of the Bessel module as an add-on. Therefore, one only needs to choose L2 and L3 to fit into the available space and conjugate the mask plane to the galvos. It has been demonstrated previously that a 0.4-NA Bessel focus works well for in vivo two-photon fluorescence imaging in the brain [15], a 0.3-NA Bessel focus has better performance than 0.4-NA one when combined with two-photon fluorescence microendoscopy [42] (due to the substantial off-axis aberrations of gradient refractive index lenses), and a larger NA Bessel beam is more suitable for three-photon microscopy [37]. With the objective, L2–L5, and the desired NA selected, the outer diameter of the annular mask is determined as:

$$ {D}_{\textrm{o}}=\frac{2N{A}_{\textrm{Bessel}}F{L}_{\textrm{o}\textrm{bj}}}{M}. $$

With NA (i.e. D_o) fixed, the axial length of the Bessel beam is dictated by the thickness of the ring, which is determined by the focal length of L1 and the beam diameter on SLM, with smaller f₁ and larger beamD generating thinner rings and longer Bessel foci. The axial length of the Bessel focus can be further adjusted by fine-tuning D_i and S.

The electrical field distribution at the mask, including its amplitude and phase, can be visualized via simulation (Fig. 3). The annular mask selectively passes the electric field within D_i < r < D_o, where r is the radial coordinate at the mask plane, and is usually centered around the largest electric field amplitude (r_max, blue line, Fig. 3). Two cases are of particular interest. In Case I, D_i and D_o are selected to have the annulus fall on the two amplitude peaks closest to and on either side of r_max (Fig. 4b, red lines in Fig. 3a, c). In Case II, the edges of the annulus are located at the two zero-amplitude crossings that are closest to and on either side of r_max (Fig. 4e, green lines in Fig. 3a, c). Even though Case I has a thicker annulus than Case II, the resulting Bessel focus has a longer axial FWHM than that of Case II (Fig. 4c, f). This is because the negative parts of the electric field (between the green and red lines, Fig. 3a, c) destructively interfere with the positive parts (between the green lines) and broaden the axial FWHM. Reducing the thickness further from Case II (e.g., between the purple lines) increases the axial FWHM again. As shown in Fig. 4, to ensure that r_max is located at the center of the annulus, without changing D_o (i.e., the NA of the Bessel beam), the period of the SLM pattern, S, can be concurrently altered with D_i.

Fig. 4

Profiles of Bessel foci using masks with different ring thickness but the same outer diameter. SLM phase pattern, the dimension of an electric field at annular mask, and a measured profile of a 2 μm diameter fluorescent bead, corresponding to (a–c) Case I and (d–f) Case II in Fig. 3. (g, h) Simulated axial profiles of Bessel foci generated by annular masks of different widths. (This figure is adapted from Ref. [15])

In summary, the two cases discussed above represent typical examples of long and short Bessel focus and can be used as a starting point for the mask design. If space permits, inserting a beam expander or a beam reducer before the SLM can further vary the axial length of the Bessel focus (see note 3 in Subheading 2.4). The MATALB code to facilitate this module design can be found in Ref. [15].

2.3.3.5 Design of an Axicon-Based Bessel Module

When using an axicon for Bessel beam generation, one should start with selecting a high-quality axicon (e.g., 1-APX-2-H254-P, ALTECHNA Inc.; XFL25–010-U-B, ASPHERICON). Once the axicon is selected, the apex angle α is determined (see Subheading 2.3.3.2); thus, the radius of the ring at the mask plane is only determined by the focal length of L1 (Fig. 2b). From here, one can simulate the electric field at the mask plane and the 3D PSF (code can be found in Ref. [16]) to guide the selection of L1, L2, and L3. Example simulation results are presented in Fig. 5. In this simulation, lens L2 is translated along the optical axis. The location where the front focal plane of L2 coincides with the back focal plane of L1 (i.e., the mask plane) is indicated as D = 0 mm (Fig. 5, second column). When L2 is moving toward the mask plane (negative D), more power is allocated to the central region of the objective back focal plane (Fig. 5, first column), which yields a smaller effective NA and, together with the phase distribution of the pupil function, a longer Bessel focus. In contrast, moving the lens away from the mask plane distributes more power to the edge of the objective pupil function, leading to a bigger effective NA and a shorter focus (Fig. 5, third column).

Fig. 5

Engineering Bessel foci by displacing Lens L2 in an axicon-based Bessel module. (a, e, i) 2D and (b, f, j) 1D (along x direction) representations of the amplitude and phase of the electric fields at the objective back focal plane, (c, g, k) axial, and (d, h, l) lateral two-photon excitation point spread functions for L2 displacements of D = −20 mm, 0 mm, and 20 mm, respectively. (This figure is adapted from Ref. [16])

The mean radius of the annular mask becomes well defined once L1 and axicon are both selected (see Subheading 2.3.3.2), although one can jointly vary the outer and inner radii of the ring and obtain different ratios of transmittance and axial profiles. The mask is intended to eliminate the unwanted light passing through the imperfect, typically round, tip of the axicon, which if not blocked can interfere with the rest of the refracted electric field and cause the measured PSF to deviate from simulation. The thinner the annular mask is, the more effectively it can block the unwanted light, but more power loss will be introduced to the system. For the case when limited excitation power is available, one can use a thicker mask, or even no mask, and obtain non-theoretical yet still usable PSF profiles [16].

2.3.4 Alignment of Bessel Module (Adapted from Ref. [15])

2.3.4.1 Installation of the Optical Components

The Bessel focus module is located between the laser and the two-photon fluorescence microscope. First, obtain locations of the front and back focal planes of lenses L1–L3 (e.g., from their Zemax models or vendor specifications). Second, place lenses so that the back focal plane of L3 is at the first scanner (or between the two scanners, if they are not conjugated), the front focal plane of L3 is superimposed with the back focal plane of L2, and the front focal plane of L2 is superimposed with the back focal plane of L1 (i.e., the mask). At last, place the SLM or axicon at the front focal plane of L1 (see note 4 in Subheading 2.4). It is helpful to have two mirrors with tip/tilt mounts between L3 and the first scanner to co-align Gaussian and Bessel paths. Alternatively, if there is not enough space, set up one mirror with both tip/tilt and translational controls.

2.3.4.2 Gross Alignment of the Bessel and Gaussian Beam Paths

Set up two irises in the optical path shared by the Gaussian and Bessel focus modalities between the alignment mirror(s) mentioned in Subheading 2.3.4.1 and the first scanner. They should be sufficiently separated and not conjugated with each other. Ideally, the first alignment iris in the Bessel module should be placed right after the alignment mirror(s) and the second iris as close to the first scanner as possible to increase the accuracy of the alignment. Adjust the positions of the irises to center on the (well aligned) Gaussian beam. For SLM-based Bessel module, apply a concentric binary grating on the SLM, and the reflected beam appears as concentric rings. Then adjust the mirror(s) between L3 and the first scanner iteratively so that the Bessel concentric rings pass through and center on the two irises (see note 5 in Subheading 2.4).

2.3.4.3 Fine Alignment

For fine alignment, dismount the objective and place a camera (e.g., DCC1545M, Thorlabs) at the back focal plane of the objective. Under the Bessel mode, one should see a ring on the camera. (For a well conjugated system, where the scanners are all conjugated to each other, swee** one or more scanners should not move the position of the ring on the camera. But if the microscope does not have its scanners conjugated, place the camera at the plane with minimal movement.) Adjust the axial positions of L2 and L3 so that the ring is sharpest on the camera, which indicates that the back focal plane of the objective is conjugated to the annular mask (see notes 6–8 in Subheading 2.4).

Placement of annular filter mask: Place the annular filter mask at the back focal plane of L1. Apply the corresponding concentric binary grating as calculated in Subheading 2.3.3 and adjust first the lateral position of the mask until the post-mask ring is symmetric and then the axial position so that the power passing through the annular filter is maximized (see note 9 in Subheading 2.4).

2.3.5 Results and Data Analysis

In this section, representative in vivo demonstrations of Bessel focus scanning 2PFM are presented. In an SLM-based Bessel module, using the method described in Subheading 2.3.4, the Bessel beam of different NAs and lengths was employed to image the cortical neurites of an awake Thy1-YFP line H mouse (Fig. 6). With the increase of NA or axial length of the Bessel focus, more energy is distributed to the side rings, which reduces image contrast. 0.4-NA Bessel foci provide high signal-to-background ratio while maintaining the ability to resolve synapses laterally in two-photon microscopy (Fig. 7). A single 2D scan of a Bessel focus probes all structures from the entire volume (e.g., a depth range of 60 μm in Fig. 7), and the calcium activities in individual dendritic spines can be characterized (Fig. 7c). In contrast, a minimum 36 scans of Gaussian beam (calculated by the ratio of axial range of structures and Gaussian focus axial FWHM) are required to cover the same volume (60 2D scans were used to generate Fig. 7a). By using longer Bessel foci, imaging throughput can be improved by more than 100 folds, with data size reduced by the same factor.

Fig. 6

In vivo images of cortical neurites taken with Bessel foci of different NA and axial lengths, in a head-fixed awake Thy1-YFP mouse. An SLM-based Bessel module was used. (a) Images obtained by 2D scans of Bessel foci and their axial point spread functions. (b) A Gaussian image stack with structures color-coded by depth obtained with a 1.05-NA Gaussian focus. (From Ref. [15])

Fig. 7

Bessel focus scanning technology improves imaging throughput while maintaining synaptic lateral resolution in calcium imaging of GCaMP6s⁺ neurites in an awake mouse brain in vivo. (a) Average intensity projection of an image stack acquired by 60 2D scans of a Gaussian focus (1.05 NA), with structures color-coded by depth. (b) A single 2D scan of a Bessel focus (0.4 NA, 53 μm FWHM) probes the same volume. Insets: zoomed-in views of dendritic spines. (c) Calcium activity traces from axonal varicosities (putative boutons) and dendritic spines (arrowheads in b). (From Ref. [15])

With an axicon-based Bessel module, translating L2 allows Bessel foci of different lengths and NAs to be generated. Functional recording from neurons labeled with calcium indicator dye (Cal-520 dextran) in zebrafish larvae is presented in Fig. 8. With the axial length of the Bessel focus gradually increasing (Fig. 8a–h), more and more structures are brought into focus using a single 2D scan.

Fig. 8

An axicon-based Bessel module enables 50 Hz volumetric calcium imaging of spinal projection neurons in zebrafish larvae. (a) Image acquired by Gaussian focus scanning at 127 μm from the dorsal surface of the head (relative depth z = 17 μm). (b) Averaged calcium transients of neurons evoked by the acoustomechanical tap** stimuli. (c, e, g) Volumetric images obtained by scanning a short (14 μm axial FWHM), medium (24 μm axial FWHM), and long (39 μm axial FWHM) Bessel foci, respectively. (d, f, h) Averaged calcium transients of responsive neurons. (i, j) Average intensity projections of a 66-μm-thick image stack acquired by Gaussian focus scanning. Color in (i) encodes relative depth. Eleven trials were averaged in (b), (d), (f), and (h). Shadow represents standard deviations. (From Ref. [16])

As mentioned in Subheading 2.1.3, Bessel focus greatly reduces axial motion artifacts due to its longer axial extent. As a result, only lateral 2D registration is required for correcting sample motion, instead of the much more computationally intensive 3D registration/interpolation. As a demonstration, a Thy1-YFP mouse was imaged in vivo and the fluorescence traces of the same dendritic ROI measured with Gaussian and Bessel focus scanning, respectively, were plotted (Fig. 9). Since the neurons were labeled with yellow fluorescent protein (YFP) rather than activity sensors (e.g., GCaMP6), fluctuations in the traces were motion artifacts, which only showed up in Gaussian but not Bessel traces.

Fig. 9

Bessel focus scanning is resistant to axial motion artifacts. (a, b) Images obtained with 2D scans of a Gaussian focus and a Bessel focus, respectively, from an awake Thy1-YFP line H mouse. (c, d) Brain motion (upper panel, quantified as the lateral image displacement with time) causes (c) large changes of fluorescence signal from two YFP+ dendrites (ROI1 and ROI2) in Gaussian focus scanning mode, (d) but not Bessel focus scanning mode. (From Ref. [15])

2.4 Notes

• 1. The beam expander after the EOM (Fig. 2a and Subheading 2.3.1) is optional, but included in all our custom-built 2PFM systems. The output from typical laser usually has a small (e.g., ~1 mm) diameter and short Rayleigh length, thus diverging quickly during propagation. Expanding the beam increases Rayleigh length and reduces divergence during beam propagation.

• 2. Whenever a grayscale liquid-crystal SLM is utilized in the system, calibration, i.e., the process of generating a lookup table between the command values (gray level, e.g., 0–255 for an 8-bit SLM) and the phase exerted onto the wavefront of the light, is necessary to ensure accurate wavefront control. For Bessel beam generation (Subheading 2.3.3.1), since only 0 and π phase shifts are required, a simple method can be used: First carefully align the SLM, L1, and the mask following the steps in Subheading 2.3.4. Then sequentially apply a series of concentric binary grating images with the correct spatial period, but different gray-scale values for high phase value rings onto the SLM. Measure the power right after the mask. The image giving the maximum post-mask power (i.e., yielding highest diffraction efficiency) corresponds to the optimal 0-π binary grating used for generating Bessel beam.

• 3. Inserting beam reducers or expanders into the beam path may cause beam shifts, so one needs to double-check the optical alignment after additional beam reducers/expanders are used to change the Bessel beam size (Subheading 2.3.3.4). Refer to Subheading 2.3.4 for detailed alignment instructions. Alternatively, beam reducers/expanders should be mounted with tip, tilt, and translational control.

• 4. When installing the SLM for Bessel beam generation (Subheading 2.3.4.1), the incidence angle of the laser beam on the SLM should be kept small to minimize pixel crosstalk that would lead to decreased diffraction-efficiency (for our systems, the incidence angle is ≤10°).

• 5. In the Bessel beam path, and especially close to the planes conjugate to the objective focal plane, iris(es) used for alignment (Subheading 2.3.4.2) should be carefully chosen so that when fully opened, Bessel beam can pass through without being clipped. (Closing down an iris near a plane conjugated to objective focal plane to intentionally clip the Bessel rings can shorten the Bessel beam, providing a convenient, if not easily controlled, method of PSF engineering.)

• 6. Tilted Bessel focus: Even with perfect conjugation along the optical pathway, one may still encounter an issue that the Bessel focus is tilted away from the optical axis (i.e., when moving a sample, say, fluorescent beads, up and down, bead images shift laterally). It is caused by the annular illumination not being centered on the objective back focal plane, often due to minor alignment errors along the Bessel beam optical path. Often, a small amount of tilting does not impact experimental interpretation and does not need to be corrected. If not, adjust iteratively the mirror(s) between L3 and the first scanner (Subheading 2.3.4) so that the image of the sample does not move laterally as it moves up and down. To position the annular ring more easily on the objective focal plane, a laser positioner consisting of two prism mirrors mounted on two translation stages can be included in the system. The motion axes of the two translation stages are orthogonal to each other, which enables both x and y position adjustment.

• 7. Sometimes tilting the Bessel beam intentionally can help with the following issue: in Bessel modality, bright structures right above or below the objects of interest may appear as bright rings in the acquired image (e.g., when observing the superficial apical dendritic tree of an L2/3 cortical pyramidal neuron, the soma in L2/3 can contribute to background). By tilting the Bessel focus, the unwanted ring-like fluorescence is shifted away from the structures of interest.

• 8. Asymmetric side rings in Bessel focus: Make sure that the laser beam is incident at the center of the circular grating pattern on the SLM or the conical tip of the axicon. When the beam size on the SLM or axicon is small (e.g., an additional beam reducer is used), this effect is particularly severe; thus, careful alignment is needed (Subheading 2.3.4).

• 9. In both modules, a single mask with arrays of annular filters can be mounted on a 3D translation stage with large motion ranges in x and y, which allows the user to easily swap annular filters for generating distinct Bessel foci. Even if only one annular mask is needed, the mask should still be mounted on a 3D translation stage, although the lateral motion range can be small, to allow fine adjustment of mask position (Subheading 2.3.4.3).

3 Widefield Fluorescence Microscopy with Optical Sectioning

3.1 Background

In standard widefield fluorescence microscopy, excitation light simultaneously illuminates the sample area, from which the emitted fluorescence is collected by a microscope objective and forms an image of fluorescent structures on a camera. The parallel nature of illumination and fluorescence detection enables widefield microscopy to reach high frame rates. However, in standard (i.e., non-light-sheet illumination) widefield microscopy, emitted fluorescence comes from both structures in the focal plane and structures above and below the focal plane. Its incapability to eliminate the out-of-focus background fluorescence limits its application to thin samples such as cultured cells or ultrathin tissue sections. A powerful approach that imparts optical sectioning capability to widefield fluorescence microscopy utilizes structured illumination (SI) [9, 23,24,25]. Because a structured illumination has its highest contrast in the focal plane, the in-focus fluorescence image is modulated while the out-of-focus background fluorescence is unmodulated. Optically sectioned images can be reconstructed by taking advantage of the difference in signal modulation [23, 24]. SI can also be used to down-modulate sample spatial frequencies and leads to super-resolution imaging [18, 20, 21, 46]. In this section, we describe how to build, align, and operate a widefield structured illumination microscope (SIM). Because super-resolution SIM (SR-SIM) has been reviewed extensively previously [46], we focus on an optical sectioning SIM (OS-SIM) implemented with a refined image reconstruction method for accurate structural and functional imaging of neurons and synapses in vivo [9].

Fig. 10

Detailed optical layout of a structured illumination microscope. (a) An amplitude mask in a rotational mount selectively transmitting first-order diffraction beams. (b) Structured illumination generated by two-beam interference at the sample plane. See Subheading 3.1 for detailed information of the optical components

3.2 Material and Equipment

3.2.1 Optical Components

Figure 10 shows an example schematic diagram of a SIM setup. After passing through an acousto-optic tunable filter (AOTF; AA Opto-Electronic, AOTFnC-400.650-TN) (Fig. 11), an excitation laser beam is expanded to match the active area of a spatial light modulator. To produce sinusoidal illumination patterns at the focal plane, the laser light reflects off the spatial light modulator (SLM; Forth Dimension Displays Ltd., QXGA-3DM) displaying binary gratings. Two achromatic half-wave plates (HWP1 and HWP2; Bolder Vision Optik, BVO AHWP3) and a polarizing beam splitter (PBS; Thorlabs, PBS251) direct the laser to the SLM and maximize its diffraction efficiency off the SLM, by ensuring the right polarization direction. The diffracted light then transmits through the PBS and has its polarization further controlled by another HWP (HWP3; Bolder Vision Optik, BVO AHWP3) and a quarter-wave plate (QWP; Bolder Vision Optik, BVO AQWP3), both of which are mounted in a fast rotator (FR; Finger Lakes Instrumentation, A24201). A dichroic mirror (D1; Semrock, Di-R405/488/561/635-t3–25 × 36) reflects the illumination laser and transmits the emitted fluorescence, and an identical compensating dichroic mirror (D2, shown in Fig. 12) is used to minimize polarization scrambling (more details below). An objective lens (Nikon, CFI Apo LWD 25X, 1.1 NA and 2 mm WD) is used for both illumination and fluorescence collection. The emitted fluorescence is focused and imaged on a camera (Hamamatsu, Orca Flash 4.0). Focal lengths of all lenses (L1-L8) in the microscope: 150 mm, 125 mm, 400 mm, 400 mm, 175 mm, 300 mm, 85 mm, and 75 mm.

Fig. 11

Laser beam multiplexing with single-edge laser dichroic beam splitters

Fig. 12

The polarization scrambling by a dichroic mirror (D1) can be compensated by another identical dichroic mirror (D2) properly oriented

3.2.2 Fixed Mouse Brain Slices Preparation

To demonstrate structural imaging with OS-SIM, we prepared brain slices from a Thy1-GFP line M transgenic mouse (The Jackson Laboratory, stock 007788). After being completely sedated with isoflurane (Piramal), the mouse was transcardially perfused first with phosphate buffered saline (PBS, Invitrogen) followed by 4% paraformaldehyde (PFA, Electron Microscopy Sciences). The mouse brain was dissected and immersed in 2% PFA and 15% sucrose in PBS solution overnight at 4 °C. Then the immersion solution was replaced with 30% sucrose in PBS. After 24 h, the mouse brain was sectioned on a microtome (Thermo Scientific™, Microm HM430) to 100-μm-thick slices, and then mounted on microscope slides to dry. After ~45 min, we placed cover glasses with mounting medium (Vectashield^® Hardset™ Antifade mounting medium, H-1400) on top of the microscope slides with brain slices.

3.2.3 Drosophila Larvae Preparation

To demonstrate functional imaging with OS-SIM, we used transgenic Drosophila third instar larvae. A GCaMP6f-based postsynaptically targeted genetically encoded calcium indicator was expressed in Drosophila larval muscle throughout development (genotype: w¹¹¹⁸; OK6-Gal4/UAS-Cpx^RNAi (BDSC Line #42017); MHC-CD8-GCaMP6f-Sh/+). Larvae were dissected using a traditional semi-intact fillet preparation in HL3 solution (concentration in mM: 70 NaCl, 5 KCl, 0.45 CaCl₂·₂H2O, 20 MgCl₂·6H₂O, 10 NaHCO₃, 5 trehalose, 115 sucrose, 5 HEPES, and with pH adjusted to 7.2) before imaging. During imaging, to maintain viability, the larval fillet was immersed in HL3 containing 1.5 mM CaCl₂·2H₂O and 25 mM MgCl₂·6H₂O.

3.3 Methods

In this section, we provide instructions on how to successfully build a structured illumination microscope. Most instructions apply to both OS-SIM and SR-SIM. Throughout this section, we provide useful notes where SR-SIM differs from OS-SIM in implementation.

3.3.1 SIM Setup

The illumination and emission light paths for a structured illumination microscope follow that of a standard widefield microscope, with added components/modules to generate and optimize the structured (e.g., sinusoidal fringe) illumination at the sample plane (Fig. 10). In the following subsections we discuss critical steps in generating SIM illumination.

3.3.1.1 Laser Beam Multiplexing, Shuttering, and Expansion

Widefield fluorescence microscopes usually use continuous-wave (CW) lasers as excitation sources. In this section, we demonstrate the imaging of GFP (green fluorescence protein)-expressing samples; therefore, a single 488-nm CW laser is shown in Fig. 10. For multi-color imaging, multiple CW lasers with different wavelengths can be multiplexed using dichroic beam splitters at 45° angles of incidence. The output power of multiple CW lasers can be controlled with a single AOTF placed right after the combined laser beams. When combining multiple laser beams, it is important to ensure that they are all perfectly directed into the entrance of AOTF and follow the same light path to the sample plane. We recommend starting with a single CW laser (with the shortest wavelength, i.e., the laser closest to the AOTF) to build the microscope. Once its alignment is optimized, other CW lasers can be added to the light path. Each laser beam must have independent tip and tilt adjustments, which can be done by using two successive mirrors between the laser and the dichroic beam splitter. Beam expansion can be done by using either pairs of achromatic lenses or integrated beam expanders. Either way, we need to ensure that each laser beam is collimated after expansion, which could be qualitatively tested with a commercially available shearing interferometer (e.g., SI035 from Thorlabs) that consists of a wedged optical flat (mounted at 45° to the light path) and a diffuser on top. If the beam is collimated, the interference fringes produced by the reflections from the front and back surfaces appear parallel to the reference line on the diffuser. Some beam expanders have the sliding lens design, facilitating easy divergence adjustment with a collimation adjustment ring (e.g., GBE02-A from Thorlabs). One needs to rotate the adjustment ring and meanwhile observe the interference fringes on the diffuser until they are parallel to the reference line.

3.3.1.2 Beam Modulation Module

• 1. Structured Illumination Generated by Two Beam Interferences

The structured illumination at the sample plane can be produced by two-beam interference. In our implementation, a spatial light modulator (SLM) is placed in conjugation to the sample plane (Fig. 10). To generate a sinusoidal illumination pattern, the SLM displays a binary grating pattern. The two first-order diffraction beams are selected to transmit through an amplitude mask (Fig. 10a) positioned at the focal plane of L1, and then imaged onto the objective back focal plane by a pair of lenses (L2–L3). The two beams exiting the objective interfere at the sample plane, producing sinusoidal patterns (Fig. 10b). It is recommended to use a flip mount for the amplitude mask, allowing an easy switch between uniform and structured illumination.

• 2. Producing the Desired Illumination Patterns

There are a few important parameters to consider when choosing the sequence of gratings displayed on the SLM: the pattern period, pattern phase, pattern orientation, and the number of SI images, which are optimized for specific samples and applications.

For a binary grating displayed on the SLM with the period d, the diffraction angles θ_m are:

$$ \sin \left({\theta}_m\right)=\frac{m\lambda}{d}, $$

where m is the diffraction order, and λ is the illumination wavelength.

At the focal plane of L1 (focal length FL₁), the distance between the ±1-order diffraction beam focal spots, h_±1 is:

$$ {h}_{\pm 1}=2{FL}_1\tan {\theta}_1\approx 2{FL}_1\times \frac{\lambda }{d}. $$

The focal spots on the mask are then imaged onto the objective back focal plane by L2 and L3 (focal lengths FL₂ and FL₃). H_±1, the distance between the two dots at the back focal plane, becomes:

$$ {H}_{\pm 1}=2{F}_1\times \frac{\lambda }{d}\times \frac{FL_3}{F{L}_2}. $$

The SI frequency/period at the sample plane is determined by the ratio of H_±1 to the diameter of objective back pupil. A ratio equals 1 means the two beams are focused to the edge of the objective’s back pupil (i.e., occupying the full numerical aperture of the objective), and then exit the objective at the largest-possible angles, generating a sinusoidal interference pattern of the highest-possible spatial frequency at the focal plane (i.e., diffraction-limited spatial frequency).

To change the phase or the orientation of the illumination pattern, we laterally shift or rotate the binary grating on the SLM, respectively. This results in the sinusoidal interference pattern at the focal plane shifting by a specific phase or rotating to a corresponding orientation.

For OS-SIM, the SI period should be selected based on the desired optical section thickness and the sample. In theory, the patterns that provide the maximal optical-sectioning strength (i.e., generate the thinnest optical sections) have spatial frequency that is half of the diffraction limit [23, 47]. In practice, when imaging a thick or densely labeled sample, the strong fluorescence background sometimes makes it difficult to detect the modulated signal in the focal plane if the optical section is too thin. In such cases, the SI spatial frequency should be empirically determined to optimize OS-SIM image quality. For SR-SIM, SI patterns with higher spatial frequency lead to higher lateral resolution. SI with spatial frequency at the diffraction limit, however, loses optical sectioning capability (because the resulting standing wave illumination maintains contrast even out of the focal plane). In thick samples, a compromise between optical sectioning strength and spatial resolution is required [8] for SR-SIM.

The number of SI images varies with different reconstruction methods. For example, the OS-SIM implementation proposed by Neil. et al. [23] requires three SI images with the same orientation but equally spaced phases. For 2D SR-SIM [20], to achieve the most resolution improvement, SI images with multiple (≥ 3) orientations and phases (≥ 3) are typically used.

3.3.1.3 Maximizing Diffraction Efficiency and Pattern Contrast at the Sample Plane

A polarizing beam splitter (PBS) is used to reflect the excitation beam toward the SLM and transmit the diffracted beam traveling away from the SLM (Fig. 10). As the PBS reflects the s-polarized component and transmits the p-polarized component of the incident light, an achromatic half-wave plate (HWP) is placed before the PBS to control excitation beam polarization. The HWP is rotated to minimize the transmitted power, thus maximizing the beam power delivered to the SLM. Our SLM has maximal diffraction efficiency for p-polarized light. Therefore, another HWP is placed between the SLM and the PBS. With a binary pattern displayed on the SLM, we rotate this HWP to minimize the power in the 0th-order diffraction beam.

High modulation contrast is essential for both OS-SIM and SR-SIM imaging. For OS-SIM, high contrast ensures strong modulation of in-focus signals, providing maximal optical sectioning. For SR-SIM, high contrast ensures large magnitude of high frequency components, supporting the extended spatial resolution. To maximize the illumination contrast, the two interfering beams should have s-polarization at the sample plane. In principle, one can use a HWP set at an optimized angle for the grating orientation for OS-SIM, or mount a HWP in a fast rotator and maintain s-polarization for all illumination orientations used for SR-SIM. However, in practice the s-polarization state may not be maintained when the illumination beams reach the sample plane. This is because optical components in the illumination light path may alter beam polarization. For example, a dichroic mirror used to separate excitation and emission light (Fig. 10, D1) may reflect and transmit the p- and s-polarization components differently, and therefore scramble the polarization of the illumination and make an originally linearly polarized light to become elliptically polarized.

To solve this problem, we can use the combination of a HWP and a QWP mounted on fast rotators (HWP3 and QWP on FRs, Fig. 10) to compensate for the ellipticity altering effect and provide desired polarization at the imaging plane [48]. For each pattern orientation (i.e., each desired polarization), we need to find the rotational angles of the two waveplates that maximize the contrast at the imaging plane. In practice, we take a series of SI images while rotating the two waveplates, until image contrast is maximized. For OS-SIM, the angles only need to be optimized for a single orientation. For SR-SIM, angle optimization is required for all SI orientations and the waveplates must be rotated during image acquisition to ensure maximal contrast for all SI orientations. In our system, this is achieved by mounting the waveplates in two fast rotators synchronized with SLM display and imaging acquisition.

Alternatively, we can utilize an additional, identical dichroic mirror oriented at a specific angle relative to the first dichroic mirror [49] (Fig. 12), with the reflections upon the two dichroic mirrors interchanging the s- and p-polarization components and therefore canceling out the polarization scrambling by the two dichroic mirrors.

3.3.1.4 Fine Alignment

SIM is very sensitive to misalignment, and here we provide two useful methods to ensure perfect alignment. With both methods, fine tip/tilt adjustment is made with two successive mirrors before the SLM.

The first method utilizes the back-reflection pattern from the objective lens. We first move the mask out of the light path (or flip down if a flip mount is used). We then let the SLM display a flat pattern so that it acts as a mirror and allows standard widefield illumination. To achieve perfect alignment with the illumination beam entering the objective center along its optical axis, we hold a piece of white paper with a hole between L3 (Fig. 10) and the objective, letting the illumination beam transmit through the hole and reach the objective lens. We then can observe the light reflected from the lens components inside the objective onto the white paper. In the case of perfect alignment, the pattern appears as concentric rings (Fig. 13a); when it is misaligned, the pattern appears off-centered and scrambled (Fig. 13b).

Fig. 13

Back-reflected illumination light observed before the objective. (a) Perfect alignment pattern. (b) Misalignment pattern

With the second method, we directly observe the two interfering beams below the objective lens. For this, we let the SLM display a grating pattern and put the mask back in path. Here we display on the SLM a fine grating pattern so that the two diffraction orders enter the objective lens close to its edge (typically at 80% of the NA; see Subheading 3.3.1.2 for detailed information). In the case of perfect alignment, the two beams below the objective appear symmetric in shape and with similar brightness (Fig. 14a). Choosing a fine pattern makes any clip** of the two beams easy to detect. If the light path is misaligned, the two beams appear asymmetric and with different brightness (Fig. 14b). This procedure should be repeated for different orientations to ensure perfect alignment along all directions.

Fig. 14

Transmitted illumination light observed below objective. (a) Perfect alignment. (b) Misalignment

3.3.2 SIM Detection Path

In our system, the emitted fluorescence is collected by the same objective and focused onto a camera for imaging (Fig. 10) with achromatic lenses (L4–L8). Magnification from the sample plane to the camera should ensure Nyquist sampling for the desired resolution. For SR-SIM that can double the diffraction-limited resolution, the pixel size should be smaller than a quarter of the diffraction-limited widefield resolution.

3.3.3 Optical-Sectioning Widefield Imaging and Its Application Examples

In this section, we introduce how structured illumination is utilized for optical sectioning and describe a refined OS-SIM reconstruction method optimized for in vivo imaging.

3.3.3.1 Refined OS-SIM Reconstruction Method

The basic idea of OS-SIM is that only in-focus information can be effectively modulated. Figure 15 shows how structured illumination only preserves its contrast for structures in the focal plane (blue arrows). When a structure is out-of-focus (orange arrows), its fluorescence signal is not or only weakly modulated. Taking advantage of this difference in signal modulation, we could computationally reject out-of-focus background and retrieve in-focus information.

Fig. 15

Structured illumination only modulates in-focus signals effectively. Images at two sample planes with different structures in focus. Blue arrows: in-focus structures, orange arrows: out-of-focus structures. Scale bar: 3 μm

One popular OS-SIM implementation was proposed by Neil et al. [23], which requires three SI images with equally spaced phases 0°, 120°, and 240°. This “basic OS-SIM” method reconstructs an optically sectioned image using Eq. (1):

$$ {\textrm{I}}_{\textrm{basic}\ \textrm{SIM}}=\sqrt{{\left({\textrm{I}}_0-{\textrm{I}}_1\right)}^2+{\left({\textrm{I}}_1-{\textrm{I}}_2\right)}^2+{\left({\textrm{I}}_2-{\textrm{I}}_0\right)}^2}, $$

(1)

where I₀, I₁, I₂ is the intensity of each image at 0°, 120°, and 240° phases, respectively. The pairwise subtractions discard the out-of-focus (un-modulated) signal, and the summing removes the non-uniform illumination patterns. As shown in Fig. 16, compared with the widefield image (Fig. 16a), basic SIM (Fig. 16b) effectively suppressed the background fluorescence. However, the signal-to-noise ratio (SNR) is suboptimal because of the positive bias created by the non-linear operation, making it difficult to resolve fine structures such as dendritic spines (Fig. 16b inset). We proposed a method [9] to suppress high-frequency noise while maintaining optical sectioning: we first low-pass (LP) filtered the basic SIM image, high-pass (HP) filtered the noise-averaged (free of positive bias) widefield image (I_WF = [I₀ + I₁ + I₂]/3), and then carried out a weighted summation of the two in order to reconstruct the final optical section using Eq. (2):

$$ {\textrm{I}}_{\textrm{SIM}}=\textrm{LP}\left({\textrm{I}}_{\textrm{basic}\ \textrm{SIM}}\right)+\alpha \bullet \textrm{HP}\left({\textrm{I}}_{\textrm{WF}}\right), $$

(2)

Fig. 16

Widefield, basic SIM, refined OS-SIM, and two-photon fluorescence images of Thy1-GFP line M brain slices. (a–d) Maximum intensity projections (MIPs) of 8-μm-thick widefield (WF), basic SIM, refined OS-SIM, and 2-photon image stacks (0.1 μm Z step, 21–29 μm depth, 440 × 396 pixels at 86 nm pixel size), respectively. Insets are single optical sections. Scale bar: 5 μm; insets: 1 μm

With this refined algorithm, we observed a substantial improvement in SNR, which helped to better reveal the morphology of dendrites and dendritic spines (Fig. 16c).

We imaged the same dendritic structures using a two-photon fluorescence microscope (Fig. 16d), the most popular OS imaging method for brain tissue. Comparing the images taken with OS-SIM and two-photon, we found that they provided comparable optical sectioning capability. In addition, one-photon excitation of OS-SIM led to higher resolution, resulting in crisper and sharper OS-SIM dendritic spine images.

3.3.3.2 Fast Functional Imaging Using OS-SIM

Applying OS-SIM to functional imaging, we demonstrated the capability of OS-SIM in capturing in vivo calcium events at high frame rate. We performed functional imaging at Drosophila larval neuro-muscular junctions (NMJs), where the muscle was labeled with post-synaptically targeted GCaMP6f-based genetically encoded calcium indicator [50].

Similar to the brain slice data, OS-SIM provided excellent optical sectioning, resulting in images with much higher contrast (Figs. 17a–c). We recorded the calcium activity at the NMJs at 25 Hz OS-SIM frame rate (75 Hz for raw image frames). By calculating fluorescence change ΔF/F from eight regions of interest, we compared the sensitivity of widefield and OS-SIM imaging in reporting calcium activity (Fig. 17d). The suppression of the out-of-focus fluorescence background by OS-SIM gave rise to a ~8× larger ΔF/F than widefield imaging (Fig. 17e). Furthermore, without the contribution of the often unevenly distributed out-of-focus fluorescence, OS-SIM allowed accurate measurement of the amplitudes of calcium transients and quantitative comparison of in vivo activity in different structures.

Fig. 17

In vivo functional imaging of quantal releases at the neuro-muscular junctions (NMJs) of a Drosophila larva with OS-SIM. (a–b) Averages of widefield (WF) and OS-SIM image sequences (frames without calcium activity) of NMJs (at a depth of 20 μm, 492 × 492 pixels at 86 nm pixel size). Scale bar: 5 μm; insets: 2 μm. (c) Lateral line profiles across the structure in the insets (b, along red dashed line). (d) Spontaneous calcium transients from 8 regions of interests (8 s of recording, orange circles in a). Widefield transients were increased by 8 times for better visualization. (e) Averaged calcium transients over 5 events (black asterisks in d) measured with widefield and OS-SIM

3.3.3.3 Parameter Selection in Image Reconstruction

For optimal image reconstruction, parameters should be carefully selected based on the imaged sample as well as the desired sectioning strength. Theoretically, the maximum sectioning strength is obtained when using an illumination period twice the diffraction limited resolution [47]. In practice, there exists a tradeoff between the optical sectioning strength v.s. modulation depth and signal-to-noise ratio. As a result, when imaging a sample with strong background fluorescence, a larger illumination period should be used so that the modulated signal can be more easily detected. When extracting low- and high-spatial frequency information from the basic SIM and widefield images, the crossover frequency, σ, was chosen to balance artifact suppression and sectioning strength. Larger σ means that we take more information from the basic SIM image, while small σ means more information from the widefield image, which typically has higher SNR than the basic SIM image. Thus, when imaging samples with higher SNR such as fixed brain slices, we used larger σ to better exploit the optical sectioning capability from the basic SIM image; when imaging noisy samples, we used smaller σ to sacrifice optical sectioning for better image quality. The scaling factor, α, weights the widefield image to ensure continuity in the Fourier domain. The value of α was determined by the modulation depth, which can be either precisely calculated using the correlation-based algorithm [51] or empirically estimated by final image quality.

3.3.3.4 Other Optical Sectioning Reconstruction Methods

We described and demonstrated our refined OS-SIM method for in vivo structural and functional imaging in previous sections. There are other optical sectioning reconstruction methods as well as structured illumination strategies. For example, differential illumination focal filtering (DIFF) microscopy [25], a variant of HiLo, reconstructs one optical section from two images with structured and complementary illumination patterns. HiLo microscopy [24, 52], another SIM method, reconstructs one optical section from one SI image and one uniform illumination image. HiLo has faster imaging speed and is less sensitive to illumination distortion and sample motion. For systems that cannot provide precise SI translation (e.g., by an SLM), HiLo is a better option. The OS-SIM system described here can also implement these alternative SIM methods. A method should be chosen after considering the application, implementation difficulty, and budget.

3.3.3.5 Other Considerations for In Vivo Imaging

In addition to the often low signal-to-noise ratio, two other issues of applying OS-SIM to in vivo imaging are sample-induced wavefront distortion and motion-induced reconstruction artifacts. Previously [9], we demonstrated that aberration correction by adaptive optics is essential for OS-SIM in both structural and functional imaging of in vivo structures. For example, the imaging of the Drosophila larva suffered from spherical aberrations coming from the muscle layers above the focal plane and the high sucrose concentration immersion saline, resulting in severe reconstruction artifacts and abnormal calcium dynamics. All our presented results in this section were aberration corrected. For motion artifacts, we used a phase-corrected algorithm to remove motion-induced reconstruction artifacts [9], which is especially important for in vivo imaging.

Since the demonstrated 25 Hz rate is more than sufficient for calcium imaging, we did not push for the highest imaging speed that our camera is capable of. The imaging speed of OS-SIM is theoretically limited by the frame rate of the camera, with the maximum full chip (2048 × 2048) frame rate at 100 Hz. The frame rate can be increased by simply reducing the line number of readout. The camera in our system (Hamamatsu, Orca Flash 4.0) can operate at 400 Hz at 512 lines and 800 Hz at 256 lines. By implementing an interleaving OS-SIM reconstruction [53], the OS-SIM frame rate equals that of the raw image frame rate. Thus, for an imaging area of 256 × 2048 pixels, the 800 Hz frame rate makes voltage imaging possible with OS-SIM.

4 Discussion

Both Bessel focus scanning two-photon fluorescence microscopy and optical-sectioning widefield microscopy can perform high-speed imaging of neural activities at synaptic resolution. OS-SIM can have faster frame rates; however, its application for deep imaging of optically opaque samples is challenging due to tissue scattering. Volumetric imaging with OS-SIM requires physical movement of the objective or sample, thus maybe slower than Bessel focus scanning two-photon fluorescence microscopy, which allows video-rate volumetric recording of neurons at hundreds of microns deep into the highly scattering mouse brain. One should choose from the two methods based on sample and application, for example, whether tissue scattering is a concern and whether activity information over large volume is required. For thin or transparent samples, OS-SIM enables functional imaging with synaptic resolution at hundreds of hertz; for opaque samples, Bessel focus scanning two-photon fluorescence microscopy would be the method of choice for volumetric activity imaging. Furthermore, both methods can be combined with other cutting-edge techniques and labeling strategies, e.g., adaptive optics [13, 54,55,56] and near-infrared sensors [57,58,59], to further enhance the imaging resolution and depth, respectively.

Recent advances in optogenetic actuators and microscopy techniques to activate them have allowed all-optical manipulation of neuronal activity at single-cell resolution (see also Chap. 3 and 11 of this book). They can be combined with both microscopy methods described here to realize all optical writing and reading of neuronal activity. The high volumetric imaging throughput of Bessel focusing scanning two-photon fluorescence microscopy makes it particularly suited to study the effect of selectively activating a subpopulation of neurons on the activity dynamics of extended 3D networks [38]. If large imaging depths or high volumetric rates are not required, OS-SIM designed to have a large FOV can be combined with optogenetic stimulation to monitor activity over a mesoscopic area [60].