2.1.

Custom Two-Color Stimulated Emission Depletion Microscope

The two-color custom STED microscope was constructed based on previous designs, with modifications to couple it with a commercial Olympus IX-71 microscope platform, simplifying the process of aligning the sample to the exact region of interest to be imaged. Additionally, we are able to perform widefield epi-fluorescence imaging of the two color channels or an additional GFP channel prior to STED imaging. This feature is particularly useful for sparse samples such as the olfactory cilia in dissociated olfactory sensory neurons. Because only two laser beam paths are used for alignment instead of four, the daily alignment is no more complex than for a traditional one color STED microscope. With a custom STED system we have the option in the future to make modifications, such as adding axial resolution enhancement for imaging into samples with z-structure,¹⁹ use of a spatial light modulator to correct for aberrations,²⁰ or implementing STED-fluorescence correlation spectroscopy.²¹ Another modification option is to use two-photon excitation so that we can image deeper into tissue.²² Additionally, we can incorporate future advancements in STED microscopy as they become available.

The laser source is a fiber-based laser (ALP-710-745-SC, Fianium Ltd.) operating at a repetition rate of 20 MHz as the master oscillator. The laser has two pulsed narrow-spectrum (FWHM of $\sim 3\mathrm{nm}$) outputs centered at $\sim 715$ and $\sim 745\mathrm{nm}$ (500 and 480 mW maximum average power, respectively) for the STED beams, and a pulsed supercontinuum output ranging from 400 to 2000 nm, with $<100\text{-}\mathrm{ps}$ pulses (1.64 W total average power) that generates the excitation sources. All outputs originate from the same master oscillator and are therefore time-locked. The excitation/STED wavelengths can be used with the fluorophores Atto 590/STAR 580/Atto 594/Dylight 594 and Atto 647N/STAR 635/STAR 635P (ATTO-TEC GmbH, Abberior GmbH, Thermo Fisher Scientific Inc.). Figure 1(a) shows the absorption and emission spectra of Atto 590 and Atto 647N and the excitation and STED laser wavelengths used in our system. Minimizing the crosstalk between the two-color channels requires careful selection of filters and dichroics combined with temporal gating for simultaneous two-channel imaging, to be discussed in further detail in Sec. 2.2.

Fig. 1

(a) Absorption and emission spectra of Atto 590 and Atto 647N, the two colors used in the STED, are shown, along with the excitation and STED laser wavelengths used. Emission detection bands shown are 600 to 640 nm for Atto 590 and 660 to 700 nm for Atto 647N. (b) Interleaved excitation/STED pulses for each color in the time domain and an illustration of the temporal gated collection are shown. Green boxes indicate the gates for the Atto 590 signal and the red boxes indicate the gates for the Atto 647N signal. The output signal from each gating circuit is sent to the computer.

Figure 2 shows the schematic for our STED microscope. The STED beams originate from separate output ports of the laser and are first optically filtered with a bandpass filter (Z710/10x, Chroma Technology) for Atto 590 and two filters (H745/45M and LP02-785RE-25, Chroma Technology), for Atto 647N. The supercontinuum output is split into two linearly polarized beams using a polarizing beamsplitter, after which a bandpass filter generates the excitation wavelengths, Z570/10x (Chroma Technology) and HQ647/10x (Chroma Technology). The maximum power in each excitation beam is 1.78 and 2.36 mW for Atto 590/Atto 647N. Each beam in the system passes through a half-wave plate (Thorlabs Inc.) in a computer-controlled rotation mount (ACONEX-AG-PR100P, Newport Corporation) and a polarizer for control of the power and to ensure clean polarization before coupling into the polarization maintaining (PM) fibers. Temporal interleaving of the excitation/STED pulses for the two channels allows simultaneous imaging with minimal crosstalk, shown in Fig. 1(b). Here, the STED and excitation pulses for Atto 590 arrive at a 25 ns delay from the STED and excitation pulses for Atto 647N. Extra fiber is used to achieve the correct time separation for the different beams. A single PM fiber (P5-630PM-FC-2, Thorlabs Inc.) couples both STED beams while another PM fiber (P5-488PM-FC-2, Thorlabs Inc.) couples both excitation beams from the laser to the microscope. The inputs of the fibers are polished at a small angle to avoid back reflection into the laser. At the exit of the fibers achromatic lenses (49-664, Edmund Optics Inc.) collimate the output light. In addition, both STED beams pass through the same vortex phase plate (VPP-1a, RPC Photonics Inc.). With both excitation beams aligned together in a single fiber, they are automatically overlapped spatially, similarly with both STED beams, greatly simplifying the alignment process to overlap all four beams at the focus. All mirrors and dichroics used in the beam paths from the PM fibers to the microscope objective are on 6 mm thickness substrates to ensure the preservation of the spatial phase of the STED beams.

Fig. 2

Optical schematic for the two-color STED microscope. Each beam is bandpass filtered and then sent through a half-wave plate and polarizer in succession, allowing clean polarization and individual power control. Excitation beams are combined into one polarization maintaining (PM) fiber and the STED beams are combined into another PM fiber using dichroic mirrors. STED beams pass through a vortex phase plate to generate a donut shape at the focus of the objective. A polarizing beamsplitter combines the beams and a quarter-wave plate makes the polarization circular. The beams are sent through a custom dichroic, which passes the four STED/excitation laser beams and reflects the fluorescence emission. The two emission bands are then separated using another dichroic beamsplitter. Fluorescence is focused into a multimode fiber that is coupled to an APD. The signal from the APDs goes through the custom gating circuit to counter inputs on the data acquisition (DAQ) board in the computer. The same DAQ board also controls the scanning of the piezo stage to acquire an image.

The beams pass through a custom dichroic beamsplitter designed for 15 deg angle of incidence (Chroma Technology), that has high transmission for STED/excitation wavelengths from 520 to 580 nm, 637 to 657 nm, and 700 to 770 nm and high reflection for emission wavelengths from 580 to 637 and 657 to 700 nm. After the dichroic, all beams pass through a quarter-wave plate (RAC 4.4.15, Bernhard Halle) to impart circular polarization and are then directed into the bottom port of an inverted research microscope (IX-71, Olympus, Inc.). A high NA objective (100X, 1.4 NA UPlanSApo Oil, Olympus Inc.) focuses all four beams onto the sample. The fluorescence emission is collected by the objective and separated by the custom dichroic.

The two-color channels are spectrally separated using a dichroic (T650lpxr, Chroma Technology) and emission filters remove any additional background (Atto 647N: HQ660LP, HQ675/50M and HQ620/40M, Atto 590: HQ680/40M-HC, Chroma Technology) before coupling into two $62.5\text{-}\mu \mathrm{m}$ core multimode fibers (M31L01, Thorlabs Inc.). Each multimode fiber serves as both a confocal pinhole and couples the light to an avalanche photodiode (APD) detector (SPCM-AQRH-13-FC, Perkin Elmer, Inc.). The image is constructed using a high-resolution three-dimensional (3-D) piezoelectric stage that scans the sample with a maximum $30\mu \mathrm{m}$ $x\text{-}y$ travel and $10\mu \mathrm{m}$ $z$ travel (P-733.3DD, Physik Instrumente) with an analog/digital controller (E-725.3CDA, Physik Instrumente). A $10\mu \mathrm{m}\times 10\mu \mathrm{m}$ image with 10(20) nm pixels is acquired in 79(38) s. The stage is controlled from the computer’s data acquisition board (PCIe-6353, National Instruments, Inc.). The piezo stage is mounted to a motorized stage (MD5422LOU, Physik Instrumente) connected to a controller (C-867.260, Physik Instrumente) that scans the sample at lower resolutions with a joystick, for locating the region of the sample of interest for imaging. The user is able to align the sample using widefield epifluorescence, brightfield, and/or DIC imaging. For epifluorescence imaging, we use a light source (Lumen 200, Prior Scientific Inc.) that has higher emission at red wavelengths to visualize Atto 647N. A CCD camera (Axiocam MRm, Carl Zeiss Microscopy GmbH) on the right side port is used to visualize the fluorescence that is mostly outside visible range for far-red fluorophores.

The system requires several precision alignment steps for reliable STED imaging:

2.2.

Resolution and Cross-Talk of the Two-Color Stimulated Emission Depletion Microscope

A custom time-gated detection circuit and pulse interleaving between the two excitation/STED beams for the two colors is required to minimize crosstalk between channels. It is important to image in both colors simultaneously rather than sequentially because a small spatial drift error between scans can be significant when compared to the high spatial resolution of the system. Without time gating, the 711-nm STED laser (for the Atto 590 channel) causes excitation of Atto 647N dye. The excitation wavelengths for both channels can also excite fluorescence for either dye due to overlap in the absorption spectra. Therefore, simultaneous two-color SR imaging requires electronically time-gated detection.

The laser emits pulses every 50 ns (20-MHz repetition rate), while the fluorescence lifetime of Atto 590/Atto 647N is $3.7\mathrm{ns}/3.5\mathrm{ns}$. Thus, most of the fluorescence emission photons arrive in the 25 ns between the interleaved excitation pulses. The gating circuit selectively passes photon counts that arrive within $\sim 20\mathrm{ns}$ after the excitation pulse to the corresponding counter on the DAQ board, while suppressing counts outside that time window. Electronic gating of counts is accomplished using two high-speed logical AND gates, which gate the signals from each APD with an adjustable duration and delay. The gating pulses are triggered by a common 20-MHz laser-clock signal but are otherwise independently adjustable. Each circuit has an 8-bit variable-pulse generator and a 120-ns variable delay line (3D7608 and DDU37F, Data Delay Devices Inc.). The temporal duration and delay of each gating signal are tuned while monitoring the counts from diluted dye samples to maximize the expected fluorescence signal from each channel while minimizing crosstalk.

2.3.

Computational Methods for Characterizing Resolution

Custom Matlab (Mathworks, Inc.) routines were used to analyze images. Images of bead samples were first thresholded and a peak-finding routine was used to identify the locations for each bead. ROIs were defined around each bead. A least squares fit to a two-dimensional (2-D) Gaussian function was performed and the center-of-mass position and FWHM in $x$ and $y$ for each ROI was determined. Fits with large residual values and by inspection were eliminated. For the dual-color labeled bead samples, we calculated the differences in center-of-mass positions for a given bead in the two-colors channels.

Cluster analysis of fluorescently labeled proteins on the cilia was done on user selected ROI centered on the peaks. A least squares fit using a 2-D rotated Gaussian distribution was performed. We calculated the statistical average size of the clusters using the average of the $x$ and $y$ FWHM, removing those fits with high residual values.

2.4.

Sample Preparation

Samples were prepared of $\sim 40\text{-}\mathrm{nm}$ polystyrene beads (red and dark red, Invitrogen, $n=1.59$ at 590 nm) to match the Atto 590 and Atto 647N wavelengths along with samples of custom dual-color labeled beads emitting fluorescence in both channels ($45\pm 0.6\mathrm{nm}$ diameter measured by the manufacturer with transmission electron microscopy). The beads were first attached to a coverslip coated with poly-L-lysine (Sigma Aldrich Co.). Immersion oil index matched to the objective lens was used as the medium between the slide and coverslip (Olympus immersion oil type F, $n=1.518$, coverslip #1.5, 0.17-mm thickness).

For the imaging of OSNs, olfactory epithelial tissue was dissected from adult mice (2 to 4 months). All experiments followed protocols approved by the University of Colorado Anschutz Medical Campus Institutional Animal Care and Use Committee. OSN cells were dissociated by mechanical tweezing and gentle pipetting in mammalian Ringer’s solution and allowed to settle on precoated slides as previously described in Ref. 23. Cells were fixed with 4% paraformaldehyde for 10 min, followed by washing with phosphate-buffered saline, and incubation in a blocking solution containing bovine serum albumin and 3% normal donkey serum for one hour. We then incubated the slides overnight at 4°C with two primary antibodies against CNGA2, the $\alpha \text{-}2$ subunit of the CNG channel, ACIII, and acetylated-tubulin. We then incubated the slides for 2 h at room temperature in custom secondary antibodies conjugated with Atto 647N and Atto 594 fluorescent tags (Rockland Immunochemicals Inc.) to label ACIII and CNGA2, respectively, along with Alexa Fluor 488 to label acetylated-tubulin. OSNs, identified under epifluorescence by their morphology, were imaged at $\text{9.8-}\mathrm{nm}/\text{pixel}$ resolution under both confocal and STED. Excitation power was 1 to $10\mu \mathrm{W}$ and STED power was 10 to 20 mW at the sample during imaging.