2.1.

Video-Rate Line-Scanned Dual-Axis Confocal Microscope

Figure 1 provides a schematic of the optical setup of the high-speed (15 and 30 fps) LS-DAC microscope. In brief, a single-mode fiber-coupled diode laser (658-nm from Coherent Inc., 488-nm from Coherent Inc., or 405-nm from SFOLT Co., Ltd.) was collimated and focused to a $\sim 500\text{-}\mu \mathrm{m}$-long [full width at half maximum (FWHM)] line in the sample without magnification (Gaussian $\mathrm{NA}=0.12$). A solid immersion lens (SIL, $n=1.45$) was used to index match the illumination and collection beams into the sample, increasing the NA of the illumination beam from 0.12 to 0.17. The light from the sample was collected off-axis from the illumination path (with a half-crossing angle of 30 deg), transmitted through a long-pass fluorescence filter (Semrock, LP02-488RU-25 or LP02-664RU) and imaged with $5\times$ magnification ($L2$) onto a sCMOS camera (ORCA flash 4.0 v2, Hamamatsu). A galvanometric scanning mirror (Cambridge Technologies), driven by a custom LabVIEW program (National Instruments), was used to scan the confocal line along the $x$-axis. Individual confocal image frames were stitched together in a line-by-line fashion using a custom MATLAB^® script (Mathworks), and the image stacks were rendered into videos using ImageJ (NIH).

Fig. 1

(a) Schematic of the video-rate line-scanned dual-axis confocal (LS-DAC) microscope. The scan mirror rotates about the $y$-axis and scans the confocal line in the $x$-direction. C1: cylindrical lens. (b) Zoomed-in view of the video-rate LS-DAC microscope near the sample. (c) Photograph of the LS-DAC tabletop implementation. SMF: single-mode fiber. (d) Photograph of the LS-DAC microscope near the focus.

The sCMOS camera used as a detector in this study was able to acquire $\sim \mathrm{10,000}$ raw acquisitions (confocal lines) per second at a $100\text{-}\mu \mathrm{s}$ exposure time for a thin rectangular region of interest of $8\times 2048\text{pixels}$. The center 3-pixel width of this strip corresponded to an imaging dimension of $\sim 2.7\mu \mathrm{m}$ in the tissue sample (slightly larger than the width of our diffraction-limited focal line) and was binned to create a digital confocal slit.

For each two-dimensional (2-D) image, the scanning mirror was programmed to translate the focal line in the $x$-direction (see Fig. 1). Serially acquired confocal lines were stitched together, enabling a LS-DAC video imaging rate of 15 fps over a $\sim 500\times 500\mu \mathrm{m}$ FOV, or a 30 fps video imaging rate over a smaller FOV of $\sim 250\times 500\mu \mathrm{m}$. A consistent sampling pitch of $\sim 0.75\mu \mathrm{m}\mathrm{per}\text{pixel}$ (at $100\text{-}\mu \mathrm{s}$ line-acquisition rate) was utilized in the $x$-direction. According to diffraction theory, the FWHM width of the focal line in tissues is $1.4\mu \mathrm{m}$.³² Thus, we were sampling the $x$-direction at approximately the Nyquist frequency.

The sampling density in the $y$-direction was determined by the pixel pitch in the sCMOS camera, which is $6.5\mu \mathrm{m}$. Taking into account the magnification of the SIL sample holder ($n=1.45$), as well as the magnification of the collection-side optics ($5\times$), the sampling pitch within tissue was approximately $0.9\mu \mathrm{m}\mathrm{per}\text{pixel}$. The diffraction-limited FWHM resolution in the $y$-direction is $1.2\mu \mathrm{m}$.³² Therefore, we were slightly undersampling the $y$-direction per Nyquist.

2.2.

System Characterization with Reflective Targets

The axial response of the video-rate LS-DAC microscope was measured with a flat reflective mirror in a homogeneous scattering phantom, 5% Intralipid (${\mu }_s\sim 11{\mathrm{mm}}^{-1}$), or a nonscattering water sample. To obtain the axial response as a function of defocus (mirror distance from the focal plane), a chrome mirror was translated axially away from the microscope’s focal plane ($z=0$) using a computer-controlled actuator (Newport Corporation, CMA-12CCCL) at a velocity of $0.1\mu \mathrm{m}/\mathrm{s}$. Here, the imaging depth is expressed as a nondimensional quantity, “the perpendicular optical length” ($L_P$), which is the total number of scattering mean free paths on a round-trip path between the tissue surface and the mirror target, $L_P=2{\mu }_{s}d$, where $d$ is the physical depth of the mirror in the imaging media, and ${\mu }_s$ is the scattering coefficient of the media. The axial resolution is defined as the FWHM of the axial-response curve (approximately Gaussian in shape).

The lateral resolution of the video-rate LS-DAC microscope was assessed by imaging a high-resolution reflective 1951 USAF bar target (Edmund Optics, 58-198).

2.3.

In Vivo Video-Rate Fluorescence Imaging

This study was performed in accordance with an animal use protocol approved by the Institutional Animal Care and Use Committees at Stony Brook University. For in vivo imaging, fluorescein isothiocyanate (FITC)-conjugated dextran (Sigma Aldrich, FD2000S, 2000 kDa, $10\mathrm{mg}/\mathrm{mL}$) was injected retro-orbitally into an anesthetized mouse to highlight its vasculature. The mouse was anesthetized with Avertin (Sigma Aldrich, 2,2,2-tribromoethanol, $20\mathrm{mg}/\mathrm{mL}$) via intraperitoneal injection at $250\mathrm{mg}/\mathrm{kg}$ body mass. The animal was placed on a custom platform that allowed for the imaging of the vasculature in its ears. The animal remained under anesthesia during the imaging experiments and was immediately euthanized upon the completion of the experiments.

In order to image blood cells trafficking within the capillaries of mouse at various depths beneath the ear skin surface, a computer-controlled actuator (Newport Corporation, CMA-12CCCL) was used to translate the sample stage along the $z$-axis in $25\mu \mathrm{m}/\mathrm{s}$ steps. Three-dimensional images of mouse-ear vasculature were constructed by acquiring a stack of horizontal image sections [see Figs. 2(a) and 2(b)]. The laser power for in vivo fluorescence imaging of red blood cells trafficking within the capillaries of a mouse ear was $\sim 1.8\mathrm{mW}$.

Fig. 2

Axial response of the video-rate LS-DAC microscope to a flat reflective mirror in 5% Intralipid (or water) over a range of optical lengths ($L_P$) plotted with: (a) a linear scale and (b) a logarithmic scale. The measured half width at half maximum is $1.10\mu \mathrm{m}$. (c) Reflectance image of a 1951 USAF bar target. (d) Zoomed-in view of groups 8 and 9 of the USAF bar target. The video-rate LS-DAC microscope has the ability to resolve group 8, element 5 ($\text{line width}=1.23\mu \mathrm{m}$) of the bar target.

2.4.

Ex Vivo Video-Rate Fluorescence Imaging

To show that the camera exposure times ($100\mu \mathrm{s}$) used for these video-rate in vivo imaging experiments could also provide sufficient sensitivity to image both topically applied and systemically delivered fluorescent contrast agents in animal and human tissues, we imaged excised mouse tongue tissues topically stained with methylene blue, as well as PpIX-expressing brain tumors from glioma patients that had been administered 5-ALA prior to surgery. The laser power for ex vivo imaging of both mouse tongue and PpIX-expressing human brain tumor was $\sim 1.3\mathrm{mW}$.

Excised mouse tongue tissues were topically stained with 0.5 mL of 1% methylene blue (MB) for 10 min and then rinsed thoroughly with $1\times$ PBS. Images were obtained from the top surface of the tongue.

Thick (unsectioned) human brain tumor specimens (low-grade glioma) were obtained during surgery and fixed in 4% paraformaldehyde before being imaged. Prior to surgery, 5-ALA, a prodrug currently in clinical trials (phase II) for image-guided resection of malignant brain tumor, was orally administered to glioma patients at the Barrow Neurological Institute in Phoenix, Arizona, under an IRB-approved protocol and IND approval for the use of 5-ALA. 5-ALA is intracellularly metabolized by the mitochondria to form PpIX, which has been shown to preferentially accumulate in various tumor cells (including glioma cells).³³ Low-grade gliomas typically do not generate enough PpIX to enable detection using wide-field low-resolution fluorescence imaging, but optical-sectioning confocal microscopy has the resolution and sensitivity to visualize the sparse generation and punctate PpIX expression in these tissues.³⁴ Here, thick low-grade glioma tissues were imaged with the video-rate LS-DAC microscope to visualize the subcellular PpIX expression (405-nm excitation and 625-nm collection).

3.1.

Experimental Characterization with Reflective Targets

Axial-response measurements of the microscope, to a flat reflective mirror in a 5% Intralipid scattering phantom (or water), were performed over a range of depths [Figs. 2(a) and 2(b)], where depth is expressed here in terms of a nondimensional perpendicular optical length ($L_P$) as described in Section 2.2. The contrast (SBR: signal-to-background ratio) of the video-rate LS-DAC microscope in scattering media can be approximated as the ratio between the infocus signal (mirror located at $z=0$) and the background signal (mirror located 15 to $20\mu \mathrm{m}$ away from $z=0$). For example, the SBR is approximately 100 at a depth of $L_P=4.4$, which is equivalent to a physical depth of $d=200\mu \mathrm{m}$ in our 5% Intralipid scattering phantom where ${\mu }_s\sim 11{\mathrm{mm}}^{-1}$. At shallow depths, the FWHM axial resolution for the microscope is $2.2\mu \mathrm{m}$ [Figs. 2(a) and 2(b)]. The reflectance images [Figs. 2(c) and 2(d)] demonstrate that the microscope can resolve group 8, element 5 of a USAF bar target, in which the line widths are $1.23\mu \mathrm{m}$. The galvanometric scanning mirror introduces a slight field curvature (approximately $7\mu \mathrm{m}$ of axial displacement over a FOV of $500\mu \mathrm{m}$ in diameter) that results in the vignetting seen in Fig. 2(c). However, this slight curvature is not a significant problem when imaging within thick tissues.

3.2.

In Vivo Imaging of Blood Cells Trafficking within Capillaries

Figure 3(a) shows a maximum-intensity projection of a $z$-stack of images collected at 30 fps (FOV of $250\times 500\mu \mathrm{m}$) over a depth span of 125 to $200\mu \mathrm{m}$ beneath the surface of the mouse ear. The maximum-intensity projection displays the highest intensity from each $z$-column of pixels when projecting the entire $z$-stack of images onto a 2-D image (the projection is in the axial or $z$-direction). At an imaging rate of 30 fps, the motion of individual red blood cells within the capillaries of the mouse ear can be visualized (see Video 1, in which the microscope imaging depth is varied over time). Retro-orbitally injected FITC-dextran (2000 kDA molecular weight) illuminates the blood plasma while red blood cells can be observed as shadows trafficking through the capillaries. Serially acquired images can be analyzed to determine the velocity of the erythrocytes by observing a line profile centered along the length of a vessel.^35–37 For example, Fig. 3(d) shows 30 line profiles obtained over a total duration of 1 s (assuming a frame rate of 30 fps). The velocity is the ratio of the distance, $d_x$, traveled by a single red blood cell over a certain time interval, $d_t$, which is equivalent to the slope of the shadows seen in Figs. 3(d) and 3(f). The red blood cell velocities within the selected capillaries were calculated to be $\sim 0.110\mathrm{mm}/\mathrm{s}$ [Fig. 3(d)] and $\sim 0.150\mathrm{mm}/\mathrm{s}$ [Fig. 3(f)]. To image an FOV of $500\times 500\mu \mathrm{m}$, the imaging frame rate was decreased to 15 fps [Fig. 3(b)]. However, the trafficking of red blood cells in capillaries could still be visualized and quantified (typical red blood cells velocity in the mouse ear capillaries is $\sim 0.1$ to $2\mathrm{mm}/\mathrm{s}$)³⁸ [see video 2 and Figs. 3(e) and 3(f)].

Fig. 3

A maximum-intensity projection (in the axial or $z$-direction) of a stack of images collected at depths ranging from $z=125$ to $200\mu \mathrm{m}$ from the surface of a mouse ear at a frame rate of (a) 30 fps over a field of view (FOV) of $250\times 500\mu \mathrm{m}$ (Video 1) and (b) at 15 fps over a FOV of $500\times 500\mu \mathrm{m}$ (Video 2). (Video 1, MPEG, 389 KB [URL: http://dx.doi.org/10.1117/1.JBO.20.10.106011.1]; Video 2, MPEG, 2.3 MB [URL: http://dx.doi.org/10.1117/1.JBO.20.10.106011.2]). (c) and (e) Single-vessel images cropped from regions indicated in (a) and (b). The red lines indicate the locations where line-profile data are analyzed in panels (d) and (f) to quantify the velocity of the erythrocytes. The arrows indicate the direction of flow. The line profiles are arranged in the order of increasing time to calculate the velocity of the blood cells (see text for details). (d) Thirty line profiles from serially acquired images (with a frame rate of 30 fps). (f) Fifteen line profiles from serially acquired images (with a frame rate of 15 fps).

3.3.

Ex Vivo Imaging of Mouse Tongue

Excised mouse tongue stained with methylene blue was fluorescently imaged at 15 fps over an FOV of $\sim 500\times 500\mu \mathrm{m}$. The ex vivo image shown in Fig. 4(a)—acquired with a $100\text{-}\mu \mathrm{m}$ line-acquisition rate, or 15-fps imaging rate—contains similar detail and contrast compared to the image of the same tissue in which a 1-ms line-acquisition rate (a $10\times$ longer integration time) was utilized along with slow-stage scanning [Fig. 4(b)]. However, as shown in the example line profiles, there is a slight increase in noise for the high-speed image frame, as is expected due to lower photon counts at a $100\text{-}\mu \mathrm{s}$ line-acquisition rate. The fungiform papillae (red arrow) and the numerous surrounding filiform papillae (blue arrows) are clearly visualized at a depth of $50\mu \mathrm{m}$. Figure 4(c) shows a H&E stained histology section of a mouse tongue exhibiting similar structural details.

Fig. 4

(a) Excised mouse tongue stained with methylene blue and fluorescently imaged at (a) 15 fps over a FOV of $\sim 500\times 500\mu \mathrm{m}$, or imaged at (b) 2 fps over a FOV of $\sim 500\times 500\mu \mathrm{m}$. The normalized intensities of the pixels along the red band in (a) and (b) are plotted on the bottom. (c) H&E stained histology section of a mouse tongue. $\text{Scale bars}=100\mu \mathrm{m}$.

3.4.

Ex Vivo Imaging of Protoporphyrin IX-Expressing Human Brain Tumor

Fluorescence imaging of thick (unsectioned) glioma tissues with LS-DAC microscopy at 15 fps [Fig. 5(a)] demonstrates adequate sensitivity to visualize subcellular PpIX expression (405-nm excitation and 625-nm collection), with similar structural details in comparison to fluorescence histopathology of PpIX, as shown in red in Fig. 5(b). Previous studies have already indicated the potential benefit of intraoperative confocal microscopy for guiding glioma resections.^39–41 However, the frame rates of previous devices have been insufficient to mitigate motion artifacts during handheld intraoperative use. Therefore, a miniature LS-DAC microscope, with a fast frame rate of 15 fps or higher, would be beneficial.

Fig. 5

(a) Surgically resected glioma (brain tumor) specimen fluorescently imaged at 15 fps over a FOV of $\sim 500\times 500\mu \mathrm{m}$. The patient was administered 5-aminolevulinic acid (5-ALA) prior to surgery. (b) A histological section of a similar glioma specimen from a patient administered with 5-ALA prior to surgery. The image shows DAPI-stained nuclei (blue), 5-ALA-induced protoporphyrin IX fluorescence (red), and the expression of glial fibrillary acidic protein (green). $\text{Scale bar}=100\mu \mathrm{m}$.