A detailed instruction is described on how to build a highly inclined swept tile (HIST) microscope and its usage for single-molecule imaging.
Single-molecule imaging has greatly advanced our understanding of molecular mechanisms in biological studies. However, it has been challenging to obtain large field-of-view, high-contrast images in thick cells and tissues. Here, we introduce highly inclined swept tile (HIST) microscopy that overcomes this problem. A pair of cylindrical lenses was implemented to generate an elongated excitation beam that was scanned over a large imaging area via a fast galvo mirror. A 4f configuration was used to position optical components. A scientific complementary metal-oxide semiconductor camera detected the fluorescence signal and blocked the out-of-focus background with a dynamic confocal slit synchronized with the beam sweeping. We present a step-by-step instruction on building the HIST microscope with all basic components.
Single-molecule fluorescence imaging plays an important role in many biological studies that reveal ultrastructures, dynamics and the quantity of biomolecules1,2,3. However, it has been challenging to study single-molecules inside cells or tissues. While confocal microscopy provides high sectioning capability4, it is not suitable for single-molecule imaging due to severe photobleaching by the high excitation intensity or slow imaging speed. Widefield microscopy uses weaker illumination but suffers from a poor signal to background ratio (SBR)5. Light-sheet microscopy, on the other hand, could show good sectioning and low photobleaching6; however, the available numerical aperture (NA) is greatly limited by the requirement of orthogonally placed objectives7. Alternatively, it requires special illuminators and sample chambers8,9.
For these reasons, highly inclined and laminated optical sheet (HILO) microscopy has been widely used for 3D single-molecule imaging10. When an inclined beam encounters an interface of two media (glass and water, for example), the beam is refracted according to Snell's law. Importantly, the refracted beam gets thinner, and its thickness is described as dz = R/tan(θ) where R is the diameter of the inclined beam and θ is the refraction angle of the transmitted beam. This simple implementation results in a good sectioning capability. Nevertheless, this relation indicates that a thin illumination (i.e., high sectioning capability) requires a small R and/or a large θ. For example, when R = 20 µm and θ = 72 degrees, one can obtain dz = 6.5 µm. Since there is a practical limit to increasing the refraction angle in order to image deep inside cells and avoid total internal reflection, there is a strong coupling of the illumination diameter and the beam thickness. For this reason, HILO imaging shows a relatively small field-of-view (FOV) that greatly restricts its applications in multicellular imaging.
Recently, we have overcome this problem by highly inclined swept tile (HIST) microscopy where the FOV is decoupled from the beam thickness in a very simple way11. First, a beam elongated in one direction is generated via a pair of cylindrical lenses. This beam, termed as a tile, produces a thin illumination with dz ~ 4 µm while its FOV is 130 x 12 µm2. Then, the tile is swept across the sample using a rotating galvo mirror. Meanwhile, the fluorescence image is recorded on a scientific complementary metal-oxide semiconductor (sCMOS) camera that filters efficiently out-of-focus background by operating in a rolling shutter mode which serves as tunable confocal slit detection. In this way, HIST microscopy enables single-molecule imaging with a larger field of view (~130 x 130 µm2) and a thinner illumination than HILO imaging. We applied this new imaging technique to detect RNA transcripts with a single probe in cells or with a few probes in mouse brain tissues, which has significant potential for studying gene expression and diseases. Unlike other approaches, HIST employs only a single high numerical aperture objective without an additional illuminator or remote detection objectives and is fully compatible with inverted microscopes. These advantages along with a large FOV and high contrast will make HIST microscopy a prominent tool in biology and medicine. We present detailed instructions regarding instrumentation of the HIST microscope, and how to test and calibrate its performance as below.
1. Setting up the microscope, lasers and alignment tools
2. Setting up the detection path
3. Setting up the excitation path
4. Setting up the cylindrical lenses
5. Testing tile imaging
6. HIST imaging
As an example, single-stranded DNA labeled with Atto647N was imaged with an excitation wavelength of 638 nm in a 3D hydrogel. DNA was anchored to the hydrogel network via an acrydite moiety during gel polymerization. The images were taken at 5 µm above the surface as shown in Figure 5a. The HIST image showed much less background compared to the Epi image, from which the signal to background ratio was calculated to be 1.9 ± 0.7 for the HIST image while most of the single molecule spots could barely be detected by Epi.
Single-molecule RNA fluorescence in situ hybridization (smFISH) was performed with 4 FISH probes. Figure 5b displays smFISH images of EEF2 (eukaryotic translation elongation factor 2) labeled with AlexaFluor 647 on A549 cells in an imaging buffer (refer to our previous work regarding the sample preparation11). A maximum intensity projection was performed on 20 z-stacks corresponding to 5 µm thickness. The HIST image showed not only much improved SBR but also more uniform illumination compared to Epi image. For Epi imaging, the exposure time was 400 ms while for HIST imaging the integration time per line was 32 ms, both of which had the same illumination power of 7.5 mW measured before the objective. The imaging speeds of Epi and HIST were 2.5 fps.
Figure 1. Microscope body, lasers and alignment tools. (A) Objective and sample holder. (B) Photo of laser systems. LP, long-pass dichroic mirror; λ/2, half-wave plates; PBS, polarizing beamsplitter. (C) Collimated light source. (D) Beam alignment tool with two insertable pinholes. (E) Double pinhole system. Please click here to view a larger version of this figure.
Figure 2. Detailed setup for highly inclined swept tile (HIST) microscopy. Photo (A) and schematic (B) of HIST microscope system. BF, multi-band pass filter; CL1-2, cylindrical lenses; DM, dichroic mirror; GM, galvo mirror; BF, band-pass filter; M1-7, mirrors; L1-4, lenses; SMF, single mode fiber; TL, tube lens; cIP, conjugated image plane; cBFP, conjugated back focal plane. Please click here to view a larger version of this figure.
Figure 3. Tile illumination with a compression ratio of 8. (A) Fluorescence image of 20 nm beads in a 3D hydrogel. Scale bar, 20 µm. (B) Standard deviation projection along the y direction of A, smoothed by 10 data points. The red arrow indicates an effective illumination width of 12 µm. Please click here to view a larger version of this figure.
Figure 4. Control and imaging software front panels. (A) A custom-made LabView program synchronously controls the scanning of the galvo mirror, the starting acquisition of sCMOS camera and the movement of the piezo stage. (B) Camera acquisition setting control panel. Please click here to view a larger version of this figure.
Figure 5. (A) Images of Atto647N-labeled DNA in a 3D hydrogel with Epi and HIST illumination. (B) smFISH images of EEF2 using 4 FISH probes on A549 cells by Epi and HIST microscopy. DAPI stain is shown in blue. Scale bars, 20 µm. Please click here to view a larger version of this figure.
There are two critical steps in this protocol. The first one is the proper placement of L4 in step 3.3, ensuring that the incident beam passes through the center of the lens and a perfect Airy disk pattern is formed on the ceiling. The position of L4 determines the placement of all the other optical components, including M5, L3, GM and L2. The second critical step is the synchronization process. To reject the out of focus background, active pixels whose effective detection width is equal to the tile width should be synchronized with the beam sweeping. Therefore, it is necessary to measure the effective illumination width of a tile beam (step 5.6) and set camera parameters accordingly in step 6.4.
When imaging with very large FOV, the presented method shows an increased background at one side compared to the other side. This is attributed to slightly altered angles of illumination at different imaging positions. Implementing a second galvo mirror instead of M5 alleviates this problem as demonstrated before by synchronously adjusting the position and the scanning angle11. Instead of off-the-shelf achromatic doublets, a telecentric scan lens will be also helpful. However, for imaging an area of <8,080 µm2, single galvo mirror sweeping was sufficient. HIST microscopy has a limit of the imaging depth, however, it is able to obtain a good SBR when imaging up to ~15 µm with a 12 µm tile beam and a NA 1.45 oil immersion objective lens11.
In this protocol, we used a beam compression ratio of 8 to make a tile beam. A thinner illumination can be used in HIST microscopy to achieve higher SBR, which might be powerful for single-molecule tissue imaging11. However, in this case, photobleaching effect should be considered by an increased excitation intensity while the current beam compression ratio showed reduced photobleaching in 3D imaging compared to Epi11. Compared to light-sheet microscopes with two orthogonally placed objectives, HIST microscopy is simple to implement and compatible with conventional sample preparations. The enhanced SBR and large FOV of HIST microscopy is suitable for studying the interactions and dynamics of single biomolecules in multiple cells and can be used further in super-resolution imaging and single-molecule tracking.
The authors have nothing to disclose.
This work was supported by Defense Advanced Research Projects Agency (DARPA) (HR00111720066) and National Science Foundation (NSF) (1805200). We thank Michael Serge in Andor Technology for generously loaning the sCMOS camera.
1" Achromatic doublet | Thorlabs | AC254-060-A-ML | Collimator |
1" Achromatic doublet | Thorlabs | AC254-100-A-ML | L1,L2 |
1" Achromatic doublet | Thorlabs | AC254-300-A-ML | TL |
1" Broadband Dielectric Mirrors | Thorlabs | BB1-E02-10 | M1~M7 |
1" Cylindrical Lenses | Thorlabs | LJ1363RM-A | CL1 |
1" Cylindrical Lenses | Thorlabs | LJ1695RM-A | CL2 |
1" square kinematic mount | Edmund Optics | 58-857 | For dichroic mirror mounting |
1" Threaded Cage Plate | Thorlabs | CP02 | For holding other lenses |
2" Achromatic doublet | Thorlabs | AC508-150-A-ML | L3 |
2" Achromatic doublet | Thorlabs | AC508-400-A-ML | L4 |
2" Threaded Cage Plate | Thorlabs | LCP01 | For holding L4 |
2" Threaded Cage Plate | Thorlabs | LCP01T | For holding L3 |
2% Bis Solution | Bio Rad | 64085292 | hydrogel component |
20 nm fluorescent beads | Thermo Fisher | F8782 | For testing imaging |
30 mm Cage Right-Angle Kinematic Mirror Mount | Thorlabs | KCB1 | For objective & camera mounting |
30mm Cage System Iris | Thorlabs | CP20S | |
3-Axis NanoMax Stage | Thorlabs | MAX311D | |
40% Acrylamide Solution | Bio Rad | 64148001 | hydrogel component |
405 nm laser | Cobolt | Cobolt 06-MLD | |
50x TAE buffer | Bio-Rad | 161-0743 | hydrogel component |
561 nm laser | Cobolt | Cobolt 06-DPL | |
638 nm laser | Cobolt | Cobolt 06-MLD | |
Ammonium persulfate | Sigma | A3678-25G | hydrogel component |
Beam alignment tool | custom made | ||
BNC terminal blocks | Natural Instruments | BNC-2110 | |
Cage plate with M9 x 0.5 internal threads | Thorlabs | CP1TM09 | For holding aspheric lens |
Cage System Rods | Thorlabs | SR series | |
Cell culture & smFISH | See a reference [11] | ||
Double side tape | Scotch | 515182 | Flow chamber |
Epoxy | Devcon | 14250 | Flow chamber |
Galvo mirror | Thorlabs | GVS211 | GM |
Galvo System Linear Power Supply | Thorlabs | GPS011 | |
Half wave plate | Thorlabs | WPH10M-405/561/633 | Power adjustment |
long-pass dichroic mirror | Chroma | T550lpxr | For combining lasers |
Microscope slides | Fisherbrand | 12549-3 | Flow chamber |
Mikroskopische Deckglaser | Hecht Assistent | 990/5024 | Flow chamber |
Mounted Frosted Glass Alignment Disk | Thorlabs | DG10-1500-H1-MD | For double pinhole system |
Mounted rochester aspheric lens | Thorlabs | A230TM-A | |
Multi-band dichroic mirror | Semrock | Di03-R405/488/561/635-t3 | DM; 3 mm thickness |
Multi-band filter | Semrock | FF01-446/523/600/677-25 | BF |
Multimode fiber | Thorlabs | M31L02 | MMF |
N,N,N',N'-tetramethyl ethylenediamine | Sigma | T7024-25ML | hydrogel component |
NI-DAQ board | Natural Instruments | PCI-6733 | |
Ø1" Kinematic Mirror Mount | Thorlabs | KM100 | For holding mirrors |
Objective lens | Olympus | PLANAPO N 60X | 60X 1.45NA oil |
Pedestal Base Clamping Forks | Newport | 9916 | |
Pedestal Pillar Posts | Thorlabs | RS1P8E | |
Piezo controller | Thorlabs | BPC303 | |
Polarized beam splitter | Thorlabs | PBS251 | For combining lasers |
RMS-SM1 adapter | Thorlabs | SM1A3TS | For objective lens |
Rod holder | custom made | ||
Rotation cage mount | Thorlabs | RSP1/CRM1/CRM1P | For HWP & cylindrical lens mounting |
sCMOS camera | Andor | Zyla-4.2P-CL10 | |
Shearing interferometer | Thorlabs | SI100 | Beam collimation test |
Single mode fiber | Thorlabs | P5-405BPM-FC-2 | SMF |
SM1 Lens Tubes | Thorlabs | SM1S25 | For double pinhole system |
SM1 Slotted Lens Tube | Thorlabs | SM1L30C | For double pinhole system |
Stage mount | custom made | ||
threaded fiber adapter | Thorlabs | SM1FC | |
Z-Axis Translation Mount | Thorlabs | SM1Z | Fiber coupling |