Spatiotemporal focusing-based widefield multiphoton microscopy for fast optical sectioningof thick tissues
Li-Chung Cheng,a Chia-Yuan Chang,a Wei-Chung Yen,b and Shean-Jen Chenc,*
aDepartment of Photonics, National Cheng Kung University, Tainan 701, Taiwan
bMaterial & Electro-Optics Research Division, Chung-Shan Institute of Science Technology, Tao-Yuan 325, Taiwan
cDepartment of Engineering Science, National Cheng Kung University, Tainan 701, Taiwan
Abstract
Conventional multiphoton microscopy employs beam scanning; however, in this study a microscope based on spatiotemporal focusing offering widefield multiphoton excitation has been developed to provide fast optical sectioning images. The microscope integrates a 10 kHz repetition rate ultrafast amplifier featuring strong instantaneous peak power (maximum 400 μJ/pulse at 90 fs pulse width) with a TE-cooled, ultra-sensitive photon detecting, electron multiplying charge-coupled device camera. This configuration can produce multiphoton excited images with an excitation area larger than 200 × 100 μm2 at a frame rate greater than 100 Hz. Brownian motions of fluorescent microbeads as small as 0.5 μm have been instantaneously observed with a lateral spatial resolution of less than 0.5 μm and an axial resolution of approximately 3.5 μm. Moreover, we combine the widefield multiphoton microscopy with structure illuminated technique named HiLo to reject the background scattering noise to get betterquality for bioimaging.
Keywords:spatiotemporal focusing, widefield multiphoton microscopy, Brownian motion, second harmonic generation.
1. Introduction
Since 1990, multiphoton excited (MPE) fluorescence microscopy has been extensively used for biological imaging [1]. With superior features such as minimum invasiveness, low photobleaching, and deep penetration depth, multiphoton microscopy has proven to be particularly suitable for imaging thick tissues and living animals [1]. Furthermore, second harmonic generation (SHG), another phenomenon of nonlinear optics, can be effectively employed to acquire non-centrosymmetric contour information directly within specimens without labeling, such as collagen within tissue [2]. As has been documented, a high numerical aperture (NA) objective lens and an ultrafast laser for spatially and temporally generating an extremely high electromagnetic field are needed for the excitation of both two-photon excited fluorescence (TPEF) and SHG. TPEF and SHG occur only in a small region near the focal point of the objective lens; hence, high spatial signal-to-noise ratio (SNR) signals for three-dimensional (3D) imaging of specimens can be obtained via pixel by pixel beam scanning. In order to increase the multiphoton imaging frame rate, techniques such as the line scanning system and multifocal multiphoton microscope have been developed [3-5].
Recent studies have shown that simultaneous spatial and temporal focusing techniques can provide widefield and axial-resolved multiphoton excited imaging [6-12]. The spatiotemporal focusing microscope typically consists of a diffraction grating, a collimating lens, and a high NA objective lens [6]. When the laser pulse impinges on the grating, spatial separation of the pulse spectrum is attained. The pulse spectrum are then collected by the collimating lens, propagate along the optical axis, and focusedinto the sample from different angles by the high NA objective lens. Only in the focal plane do the different frequency components construct in phase and produce a short, high-peak power pulse, allowing effective multiphoton excitation to occur[6-7,9-12].Compared to conventional beam scanning multiphoton excitation microscopy, widefield multiphoton excitation microscopy using the temporal focusing technique detects the overall fluorescence and harmonic generation signal of the entire illumination area,which depends on the laser beam spot size and the magnification of the microscope [8]. The advantage of widefield microscopy is that less time is required to capture one frame, enabling a fast frame rate for capturing dynamic events.A video-rate multiphotonmicroscope has been realized by the combination of temporal focusing and mechanical line scanning[7]. However,the disadvantage of the mechanical scanning technique is that the entire frame is not illuminated simultaneously, limiting its maximum frame rate. When a fast, high-sensitivity camera and an ultrahigh peak power laser are employed in widefield
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multiphoton excitation microscopy, an image rate of a few hundred frames per second can be achieved. However, there is inherent difficulty for the camera-based detection to reach the same SNR, spatial resolution, and penetration depth as the photomultiplier tube-based single photon counting (SPC) technique, which effectively reduces the background noise and the scattering effect of emission light [13,14].
Differing from fixed biosample studies, interactions between living cells, the migration of cells, and the signal transition of neuron cells all require real-time microscopic imaging [15,16]. Examples of this include flagellated cells and the beating heart of embryonic zebra fish, both of which require high frame-rates per second for observation. Although widefield multiphoton excitation microscopy based on spatiotemporal focusing can in principle observe samples in real-time, factors such as the emission efficiency of the excited fluorophores, the peak power of the ultrafast laser in the focal plane, and the sensitivity of the camera can all affect the frame rate. In this paper, a 4.0 W titanium-sapphire (ti-sa) ultrafast amplifier with a 10 kHz repetition rate was used to enhance peak power.In addition, an electron multiplying charge-coupled device (EMCCD) was employed, and by cooling the EMCCD chip to -80 ˚C, background noise and dark current are reduced. Furthermore, by shrinking the number of pixels or binning the pixels, the frame rate is increased to 100 Hz with a pixel number of 256 × 256. Since typical fluorophores have a finite number of excitation emission cycles before photobleaching, shorter exposure times can provide longer fluorescence observations [17,18]. By reducing the exposure time with a mechanical shutter to 20ms the photobleaching issue was overcome. In the experimental results, Brownian motions of fluorescent microbeads, with sizes ranging from 1.0 μm to 0.5 μm, were successfully observed with a lateral spatial resolution of less than 0.5 μm and an axial resolution of 3.5 μm at the 100 Hz frame rate. Additionally, we try to make the bioimage has better quality, so we combine the widefield multiphoton microscopy with HiLo technique. HiLo can help to reject the background unwelcome signals and only keep the in focal signals we want. By rejecting background signals we could get clear and higher contrast bioimage.
2. Optical setup
A.Widefield multiphoton microscope
Figure 1 illustrates a schematic of the developed widefield multiphoton microscope based on spatiotemporal focusing. Key components include a ti-sa ultrafast amplifier (Spitfire Pro., Newport, USA), a ti-sa ultrafast oscillator (Tsunami, Spectra-Physics, USA) as the seed beam of the amplifier, an upright optical microscope (Axio imager 2, Carl Zeiss, Germany), a triple-axis sample positioning stage (H101A ProScanTM, Prior, UK), an Andor EMCCD camera (iXonEM+ 885 EMCCD, Andor, UK), and a data acquisition (DAQ) card with a field-programmable gate array (FPGA) module (PCI-7831R, National Instruments, USA). The ultrafast amplifier has a peak power of 400 μJ/pulse, with a pulse width of 90fs and a repetition rate of 10 kHz.
A half-wave plate and a polarizer adjust the polarization and power of the amplifier. The beam is spatially dispersed via a grating with 1200 lines/mm. The incident angle of the grating was adjusted to ensure thecentral frequency follows the optical axis and propagates through the 4f setup, which comprises the collimating lens and the objective lens (W Plan-Apochromat 40X/ NA 1.0, Carl Zeiss, Germany).By filtering the collectedsignal through a dichroic mirror and a short-pass filter, only nonlinear optical signals are collected through the objective lens and imaged onto the EMCCD camera. By controlling the motorized stage in the z-axis via the FPGA, sequential sectioning images at different depths can be obtained, and then assembled to reconstruct a 3D image.
After diffraction from the grating at an oblique incident angle of 69.4°, the laser’s beam attains an elliptical cross section with the two axes at200 and 100 μm in the image plane.However, refractive optical elements, such as the objective lens and collimating lens, canlead to additionalgroup velocity dispersion disturbing the overall system. To rectify this problem, the built-in grating compressor of the amplifier was adjusted to approach the optimal pulse width (<120 fs). Consequently, the efficiency of temporal focusing for excited nonlinear signals can be enhanced, allowing for a nonlinear image of a specimen to be attained with ten laser pulses at 1 ms exposure time. And as aforementioned, the20 ms mechanical shutter speed reduces photobleaching.
B.System calibration
The axial resolution of the widefield multiphoton microscopy based on the optical parameters in full-width at the half maximum (FWHM) can be expressed as [19]:
(1)
Fig. 1. Optical setup of the widefield multiphoton microscope based on spatiotemporal focusing.
where k1, k2 and k3 are system parameters depending on the wavelength and grating period, whileτ is the pulse width, M is the magnification of system, l is the beam diameter, and NA is the numerical aperture of the objective lens. To achieve superior axial resolution, these parameters were chosen: 1200 lines/mm groove density of the grating, the 240 mm focal length of the collimating lens, and the 2.5 mm focal length of the objective lens at the central wavelength of 780 nm with 100fs pulse width.To calibrate the spatial resolution of the widefield multiphoton microscope, a PMMA thin film doped with R6G dye (<200 nm thick) was first examinedto determine the axial resolution. The film was axially scanned with a range of 40 μm at a step size of 1.0 μm by controlling the motorized stageunder different laser powers. The intensity of the TPEF as a function of the scanning depth at different laser powers is shown in Fig. 2(a). It can be seen that the axial resolutionsare 2.5, 2.8, 3.1, and 3.4 μm at the respective laser powersof10, 20, 30, and 40 mW. When the laser power was increased from 10 to 40 mW, the axial resolution degraded slightly; however,an axial resolution of less than 3.4 μmcan be provided as themaximum laser poweris less than 40 mW.
The NA of the objective lens, the excitation wavelength, and the multiphoton mechanism together determine the spatial resolution [1].According to the optical parameters used in this study,thetheoreticallateral resolution is approximately 0.4 μm. Fluorescent beads with a diameter of 0.2 μm were used toexamine the lateral resolution. Figure 2(b) shows that the point spread function (PSF) of a single beadisapproximately0.5 μm. By taking the average PSF of 10 beads,the FWHM lateral resolution of the microscope was determined to be 0.5 μm, which is close to that of the theoretical result given the size of the fluorescent beads.
Fig. 2. Spatial resolution of the widefield multiphoton excitation microscope: (a) axial resolutionsunder different laser powers at 10, 20, 30, and 40 mW are 2.5, 2.8, 3.1, and 3.4 μm, respectively; and, (b) the point spread function showing 0.5 μm FWHM.
Fig. 3. TPEF intensities of the PMMA thin film doped with R6G dye as a function of time under different excitation powers.Circles: ultrafast oscillator; squares:ultrafast amplifier.
3. Experimental results and discussions
A. Photobleaching
Photobleaching of fluorophores causes decay of their emission intensity. One particular concern here is photobleaching when exposed to a high peak intensity light field for a long period of time. Figure 3 shows the behavior of TPEF for the R6G thin film described in Sec. 2.2 when continuously illuminated for 1 minute by the ti-sa ultrafast oscillatorand the ultrafast amplifier. By using the ultrafast oscillator at powers of 40, 50, and 58 mW, the florescent intensity does not decay and reveals no photobleaching. However, fluorescent intensity dramatically decayed and the photobleaching phenomenonoccurred when the ultrafast amplifier with excitation powers greater than 10 mWwas used. The average power of the ti-sa ultrafast oscillator is higher than that of the ultrafast amplifier; however, the instantaneous pulse energy of the ultrafast amplifier is 8,000 times greater than that of the ultrafast oscillator and consequently induces the photobleaching effect. Therefore, high excitation peak power with long-term exposure should be avoided in multiphoton excited fluorescence.Under the same excitation power of 10 mW, the exposure times for grabbing similar image intensityare16.5 ms and 60 s for the ultrafast amplifier and the ultrafast oscillator, respectively. The exposure time by using the ultrafast amplifier is 3,600 times faster than that by the ultrafast oscillator and hence the ultrafast amplifierprovides high efficiency for the TPEF excitation.
B. Brownian motions of fluorescent microbeads
The high frame-rate capability of the developed microscope was validated by observing the Brownian motions of fluorescent microbeads. Brownian motion is a random process involving small particles moving in arbitrary directions due primarily to particle collisions and thermal perturbations. According to the Stokes-Einstein Eq. [20], root mean square (RMS) displacement of a small particle in 3D space between a time interval can be evaluated by:
(2)
where D is the diffusion coefficient of the particle. In order to clearly observe the motion trajectory of a small particle, a multiphoton microscope must possessa sufficiently fast frame rate, which is defined as being less than the radius of the particle at the frame rate of 1/. Based on Eq. (2) with a diffusion coefficient for a 1.0 μm diameter fluorescent bead [20], the RMS displacement at a frame rate of 12 Hz is calculated as 496 nm, which is less than the bead’s radius of 500 nm. As such, a frame rate of 12 Hz should be necessary to observe the Brownian motion of 1.0 μm fluorescent beads. However, since particles will move in random directions (lateral or axial directions) and is only slightly less than the bead’s radius, a higher frame rate would be a better choice. For example, the RMS displacement at a frame rate of 100 Hz based on Eq. (2) is 172 nm for a 1.0 μm fluorescent bead. Beads with diameters of 1.0 and 0.5 μmimmersed in DI water were sealed within the cavity of a slide measuring 18 mm in diameter and 0.8 mm in depth. Figure 4(a) shows an image of the Brownian motions for the 1.0 μm diameter fluorescent beads using the higher frame rate of 100 Hz, where the excitation laser power is 40 mW, the field of view is 50 x 50 μm2, and the exposure time is 9 ms per frame.A weighted algorithm performed on the images of the 1.0 μm fluorescent beads can provide sub-pixel resolution for centroid determination. Figure 4(b) shows the displacements of the bead at the top of Fig. 4(a) along the x and y axes as a function of time at 10 msinterval from Media 1. The maximum displacements along thex and y axes are about 140 and 150 nm, respectively, whichis comparable to the simulation result of 172 nm.
A video featuring the 100 Hz frame rate clearly demonstrates the Brownian motion trajectories in the observed plane. Since the smaller the fluorescent bead’s diameter is, the faster the Brownian motion is;thus, for smaller particles, a faster frame rate is needed. For a 0.5 μm diameter fluorescent bead, the RMS displacement at a frame rate of 94 Hz is 250 nm, which is equal to the bead’s radius, and so the frame rate must be 94 Hz or faster for observing 0.5 μm fluorescent beads in 3D space without losing trajectory information. The fluorescent volume of a 0.5 μm fluorescent bead is also reduced by 23 compared to that of a 1.0 μm fluorescent bead.Figure 5(a) shows an image of the Brownian
Fig. 4. (a) Brownian motion of 1.0 μm fluorescent beads at 100 Hz frame rate (Media 1). The excitation laser power is 40 mW with a 9 ms exposure time. (b) Displacements of the bead at the top of Fig. 4(a) along thex and y axes as a function of time recorded at 10 msintervals from Media 1.
Fig. 5. (a) Brownian motions of 0.5 μm fluorescent beads at 100 Hz frame rate with an exposure time of 9 ms (Media 2). (b)Sequentially images captured from Fig. 5(a).
motion at 100 frames per second with an increased excitation laser power of 60 mW to obtain brighter fluorescence images. Figure 5(b) shows 4 sequential shots of the 0.5 μm fluorescent beads at a frame rate of 100 Hz. If the frame rate in Fig. 5(b) were lower at 33 frames/s, only the leftmost and the rightmost shots could be observed, andpertinent information between shots would be lost. The average displacement of the 0.5 μm fluorescent beads in Fig. 5(b) is 185 nm. Based on the simulation, the displacement of a 0.5 μm fluorescent bead in 3D space projected into a two-dimensional observation plane is 196 nm when the probabilities of the displacement in three directions are assumed to be equal, which is in close agreement with our observation.
C. Structured illumination-HiLo
The first bioimage, cells stained with DAPI and captured by spatiotemporal focusing-based widefield multiphoton microscopy with a ti-saultrafast oscillator as light source,hadan exposure time of 30 seconds for one image within a 140 × 140 μm2 area [6]. The excitation power was too low to excite fluorescence with such a large area, and hence the advantage of fast imaging of widefield microscopy wasn’t realized. In terms of excitation, multiphoton microscopy with spatiotemporal focusing through deep scattering tissue can maintain its axial sectioning ability [21].To demonstrate the application of widefield multiphoton microscopy in bioresearches we choose to observe the fixed neuron cell of mice. As shown in Fig. 6 (a), we scanned the fixed neuron cell axially and reconstructed a 3D image by different depth images. In the image we can figure out the neuron morphology but the detail signals are blur.The most serious problem in widefield imaging is light scattering. Different from point scanning, widefield microscopy exited whole area and receive all signals from the area. The excited wavelength is near infrared but the emitted signals is in visible range so the effect of scattering is more serious than exited light. The CCD cannot figure out where the signals come from and it makes the image blur and unclear.