Single Objective Light-Sheet Microscope (SOLS) Guide 

Build and Alignment Guide

The written manuscript can be found here: https://www.jove.com/pdf/65411/jove-protocol-65411-design-building-customizable-single-objective-light-sheet 

The video protocol can be found here: [insert JoVE link when published] 

Introduction to the Project

Over the summer and fall of 2022, our lab constructed a single objective light-sheet microscope from commercially available parts. We designed and built this system with two intentions: 

1. 1. 1. First, we required a better tool for the research projects that we aimed to undertake. In particular, our lab images the mechanics of cytoskeleton networks, which tend to be dense, three-dimensional samples. Acquiring better images of cytoskeleton samples necessitated a tool which could image these dense networks without producing a large amount of background signal, and do so over a long time scale without photo-bleaching the sample. A light-sheet microscope, which illuminates only the sample plane and offers low amounts of photobleaching, was an appropriate choice. Given the mechanics of our samples, we required top-down slide mounting capabilities, so a single-objective light sheet (SOLS) was an applicable tool. 

1. 1. 2. Second, we aimed to address the general absence of user-friendly build guides for SOLS-type instruments. Although there exist a number of excellent light-sheet build guides, the overwhelming majority of these are tailored towards light-sheet instruments which utilize multiple objectives at the sample plane. For biologists and other researchers who require a light-sheet with traditional top-down slide mounting abilities, our SOLS guide aims to provide a straightforward protocol to build a single-objective light-sheet, allowing samples to be mounted in a way that researchers are comfortable and familiar with. 
         
         
         

After the completion of our SOLS system, we worked with the Journal of Visualized Experiments (JoVE) to produce a video protocol of the construction and alignment procedure. This video protocol and the accompanying written manuscript can be found at the links directly above. This website page serves as a home for additional information not included in the written manuscript, the large majority of which is meant to assist readers in designing their own LSFM system should they wish to deviate from the exact specifications detailed in the JoVE manuscript and video protocol. 

Introduction to the Single Objective Light Sheet Design 

Light-sheet fluorescence microscopy (LSFM) is catch-all term, used to reference a large variety of high-resolution fluorescence imaging modalities in which samples are excited with a thin sheet of light 1, 2. This includes Oblique Plane Microscopy (OPM), Selective Plane Illumination Microscopy (SPIM) and Swept Confocally-Aligned Planar Excitation (SCAPE), among many others 3–7. Beyond the high-resolution imaging that LSFM setups provide, the primary advantage of LSFM microscopes is a very low amount of phototoxicity, especially when compared to more common fluorescence imaging techniques, such as epi-fluorescence and confocal imaging. The low amount of phototoxicity is made possible through the thin excitation sheet, which is aligned to illuminate only the focal plane being imaged (Figure 1). In turn, the low amounts of phototoxicity greatly increase the timescales over which samples can be imaged 8–10. The thin excitation sheet also provides excellent optical sectioning capabilities, making it possible to image dense, three-dimensional samples without scattering or background signal overwhelming the fluorescence signal from the focal plane. Accordingly, LSFM has become an increasingly popular imaging tool for biologists, physiologists, neuroscientists, and more 3, 4, 6, 11. Following the original development of LSFM in 2004 light-sheet systems have seen significant advances, making it possible to visualize biological structures and dynamics at increasingly smaller scales. As such, LSFM systems have since been utilized to image everything from tissue 11, 12, to cells, and all the way down to sub-cellular structures 13–17.  

To achieve optical sectioning, early LSM implementations utilized two microscope objectives, one each for excitation and for detection, in a perpendicular configuration to image samples mounted in capillaries or embedded in cylinders of gel4, 18 (Figure 2A). Further developments in LSM design extended the system capabilities to be compatible with traditional slide samples, easing the sample mounting and imaging processes. However, these LSM systems were still limited in terms of their resolution, as the multi-objective interface at the sample restricted bulky, high NA objectives from being used with each other or required special adaptive optics to correct for aberrations19, 20, 21, 22. In contrast, recent Single-Objective Light-Sheet (SOLS) implementations circumvent this limitation by utilizing a single objective for both excitation and detection at the sample plane (Figure 2C)5, 6, 8, 13, 23. However, this comes at the expense of a much more complex build, as two additional objectives must be used further along the optical path to de-tilt and re-image the imaged object onto the camera (Figure 2D).  

Design, Hardware, and Layout Considerations 

1. Designing the system: When planning one’s SOLS setup, two major design considerations will dictate the particular hardware required: 

1.1 System magnification: The ideal magnification of the system will depend on what the system will be used to image. This is specific for each use case, but as a rule of thumb, some reasonable values are 5x for organismal, 20x for cellular, and 60x for sub-cellular imaging. For reference, the SOLS system described here is used to image sub-cellular structures: this design has a theoretical magnification of 66.67x and was calibrated at 61.905x.  

1.2 Optical resolution: The optical resolution determines how close two points can be within the sample and still be distinguished in the resulting image, also known as the “minimum resolvable distance” or the “diffraction limit.” The diffraction limit (d) for the completed SOLS system is set by the system’s numerical aperture (NA), which can be calculated using:

where λ is the wavelength of the excitation light. The system NA is set by the objectives used in the system, so one must develop a sense of the desired resolution, and accordingly, an appropriate system NA, before one can select suitable objectives. Once again, the specific requirement for system resolution is determined by the types of samples that will be imaged and the types of information one needs from the images. However, since it is in imaging thick and dense samples where LSM excels, it is in general worthwhile to optimize the resolution — or, in other terms, to maximize the NA — of the instrument. One must note that there is a tradeoff between a higher system NA and the field of view (FOV)/ lateral scan range at the sample, which is further explained in Kumar et al.24 For reference, the SOLS system described here has a theoretical numerical aperture (NA) of  (NAx, NAy) = (0.88,1.0), which can be used to calculate the diffraction limit at different excitation wavelengths. The NAx is smaller than NAy due to the loss of NA at the O2-O3 interface, which occurs more in the x-plane than the y-plane. 

1. Selecting hardware:  

2.1 Objectives: The objectives are the main determinants of the overall magnification and the NA of the system. To compute both the magnification and the system NA of different objective setups, we point the reader to a helpful guide developed as part of a previous SOLS design23. For readers more interested in how the tilted relay interface and multiple objectives affect the in toto system NA, we refer to the descriptions written by Yang et al.8. Before designing a system on one’s own, a reader might also consider objective sets which have already been proven to be compatible in a SOLS style design: a number of guides have been published with lists of suitable objectives23, 25. More considerations specific to each objective are provided below.  

2.2 The primary imaging objective (O1): This objective determines the upper bound for numerical aperture (NA) for the full system. The NA of O1 also determines the maximum tilt angle of the light sheet. Just as the angular aperture of an objective determines the maximum angle of the incoming light cone that the objective can collect, the angular aperture determines the maximum angle at which the light sheet can leave the objective: a higher NA O1 allows for a more extreme tilt of the light sheet from the optical axis of O1. To ensure that a particular objective is capable of tilting a light sheet to the requisite angle, utilize the common NA equation which considers the refractive index of the immersion medium n and the half-angle of the maximum longitudinal angle of the collected light cone α: 

This can be backsolved to obtain an equation for α, given an objective with a specified NA and immersion medium: 

In terms of the light sheet, α is equivalent to the angle of the maximally tilted light sheet from the optical axis of O1. Because the tilt of O3 relative to O2 must match the angle of the light sheet from the surface of the objective, a larger tilt of the light sheet from the optical axis of O1 allows for a smaller tilt of O3 from the optical axis of O2. This decreases the loss of fluorescence signal in the O2-O3 relay interface, ultimately proving why a bigger tilt on the light sheet corresponds with a higher system NA. Additionally, because O1 is mounted independent of surrounding optical components in a SOLS build, O1 does not need to satisfy any particular working distance requirements. This allows the user to consider different immersion media, usually water, to increase NA without complicating the system build and maintenance. 

2.3 The relay objective (O2) and re-imaging objective (O3): These objectives both contribute to system NA, but less so than O1. The primary consideration for these objectives is that their working distances be high enough, and bodies tapered enough, to achieve confocal (sharing a focal point) alignment, as depicted in Fig 1D. For selecting O2 and O3, at least one of the two should be a high working distance objective (WD > 5 mm), then consult the manufacturers drawings to confirm that the pair can be mounted at the correct distances from each other without colliding. For reference, the system described here has WD = 2.00 mm for O2 and WD = 5.3 mm for O3. Once again, for readers who are interested in following an objective setup that has already been proven to be mechanically compatible, we point readers to the systems designed and cataloged in the papers written by Kumar et al. and Millett-Sikking and York23, 25. Last, one might consider immersion objectives for O2 and O38, 23, but this requires custom horizontal immersion chambers at the relay interface, complicating the alignment process.  

2.4 Camera: Beyond the three objectives, another very influential component of the SOLS system is the camera used for imaging. Although a large number of cameras within a range of prices and performance capabilities are compatible with this type of SOLS design, there are basic requirements that any camera used for this type of system must meet. Most importantly, the pixel size of the camera must pass the Nyquist criterion given the diffraction limit and magnification of the system, which describes the minimum sampling density required to impart all optical information from the microscope onto the image. The theoretical diffraction limit can be calculated using the Rayleigh formula for minimum resolvable distance, shown in Section 1.2 (Optical Resolution). To meet the Nyquist criterion, the pixel size of the camera at the systems magnification must be smaller than ½ of the theoretical diffraction limit of the system: 

To maximize the efficiency of the camera and image acquisition process, it is recommended that the magnified pixel size is no smaller than 1/3 of the diffraction limit of the system:

Finally, it is also important to consider that the max frame rate of the camera will set the max scanning speed of the system if one is looking to push the limits of the scanning, as described by Bouchard et al.6

2.5 Excitation laser: Choose a wavelength as close as possible to the peak of emission curve of fluorophore to be imaged. Common choices include 532 nm or 561 nm (green) for red emission fluorophores and 405 nm or 488 nm (blue) for green emission fluorophores. An excellent tool to investigate good laser and fluorophore pairs is the free fluorescence spectra-viewer26. In terms of laser power, we recommend at least 100mW for a single mode laser. For reference, our laser was 100mW and achieved a maximum of 40mW past the pinhole at TEM00 mode. 

2.6 Dichroic: This is a wavelength-dependent mirror that functions to block the excitation light from entering the camera. Choose a dichroic that reflects all excitation wavelengths (T < 1%) and transmits the majority of the emitted signal (T > 90%) for most of the emission wavelengths of the chosen fluorophore.  

2.7 Galvanometer: To achieve volumetric scans, this system incorporates a scanning mirror galvanometer (galvo), as pioneered by Bouchard et al. in 20156. This galvo interacts with both the excitation light sheet and the de-scanned emission light, coupling the movement of the imaging plane to that of the excitation sheet at the sample. Taking inspiration from the developments made by Yang et al. and Kumar et al.8, 24, the galvo in this system is aligned to provide tilt invariant scanning in the y-plane (Figure 3), optimal for distortion free 3D imaging. As the only moving part in the system, the use of the galvo to allows for high volumetric scanning limited only by the frame rate of the systems camera2, 6, 8, 13.

2.8 Tube Lenses: Most objectives are designed to be used with a particular length tube lens to form the correct objective compound lens system with the intended magnification. As such, the three tube lenses in the system should be chosen to the correct specifications, or, if changed, with the understanding the this will affect the performance of the optical system as a whole. When selecting tube lenses, one might also consider the use of telecentric scan lenses, which provide minimal distortion as the angle of incoming light is varied. Compared to all other lenses in the system, the two tube lenses will see the largest angles of incoming light.  

2.9 Scan Lenses: The scan lenses are the two lenses in the system that create 4f systems with the tube lenses for the objectives (SL1 and SL2). These lenses must be chosen in combination with the specific tube lens to meet the Herschel condition. The Herschel’s condition sets requirements for the focal lengths of scanning lenses in order to minimize spherical optical aberrations and is given by the equation:

where TL1 and TL2 are the tube lenses, SL1 and SL2 are the corresponding scan lenses, and FL refers to the focal length of each lens. 

2.10 Other Lenses in the System: The other lenses in the system include two standard plano-convex lenses and three cylindrical lenses, all of which serve to control the size and shape of the excitation light sheet. The cylindrical lenses function to stretch the excitation beam in the correct direction, while the plano-convex lenses collimate the stretched light along the correct axis then focus the sheet onto the galvo. If users wish to adjust the shape of the light sheet using different lenses, a thorough discussion of shaping the excitation light can be found in the articles published by Olarte et al. and Wolff-Madrid et al.27, 28 

2.11 Custom Hardware: 3D printing parts can be an effective method to save funds and also allows for system-based customization. For example, some lasers may have built in mounting plates, while others that do not may need a more custom solution to be mounted. Such was the case with the 561nm green laser used in this setup; because there were no mounting holes built into the laser, OpenSCAD software was used to design a custom 3D printed mount to securely attach the laser to the optical table at the correct height. Beyond custom mounting components, systems that use an immersion medium at the O2-O3 relay interface may require custom water chambers or other custom components to allow for proper immersion of the horizontally mounted objectives.  

3. Layout Considerations: Several aspects should be considered when designing the overall system.   

3.1 Height of Components: When mounting the laser, an important consideration is the height of the illumination beam above the table. Given the optical components already in the possession of this lab (many 4" posts and post mounts), a height of 6” made sense for our build. However, it is commonplace in high-resolution optics to build microscope system as close to the table surface as possible in order to increase the stability of individual components. As such, a user may consider purchasing optical components with this shorter height in mind, rather than following the height of our system. This will not affect any capabilities of the system beyond the long-term stability of the components. 

3.2 Physical Footprint: Another consideration when building on an optical table is the system footprint. While our design does not optimize the footprint of the SOLS microscope as a whole, it would be simple to do so by adding in reflective elements between the different 4f systems to create more 90° bends. For users that are interested in building multiple setups on a single optical table, this may be of more concern. 

3.3 Alignment Guides: If building on an optical table, utilizing the different lines of holes to align different segments of the overall build. This can be achieved through the intentional placement of different reflective elements to direct the laser light directly above a line of holes on the table. If achieved correctly, optical components can be mounted directly onto those lines of holes for easy centering, rather than going through a tedious alignment process to center optical components to the laser light. On a related note, it may be helpful to design the system so that different segments of the system are 90° to each other, ensuring that the laser light follows a particular line of holes along the entirety of the design.

3.4 Mounting Issues: When designing the physical system, it is not uncommon to run into mounting issues (i.e. two optical components are so close that there is not room for both base plates on the optical table). This should be taken into account when designing the physical layout of the system by aiming to maximize the space between components that must be mounted individually; often, this is a matter of where the reflective elements are placed in relation to the 4f systems of lenses. If these issues can not be solved through the design, users can purchase different mounting accessories to extend the position of a mounted component from the top of the mount itself. 

References

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