Photoacoustic microscopy (PAM) is dominantly sensitive to the endogenous optical absorption compared with the confocal microscopy which images with scattering photons. PAM has similar structure such as optical transportation system, the optical scanning, and light source with the laser scanning confocal microscopy (LSCM). In order to match the PAM with LSCM, a special design microcavity photoacoustic (PA) transducer with high sensitivity is developed to detect the photoacoustic signals induced by modulated continuous wave (CW) laser. By employing a microcavity PA transducer, a PAM can be integrated with LSCM. Thus a simultaneous multimodal imaging can be obtained with the same laser source and optical system. The lateral resolutions of the PAM and the LSCM are both tested to be better than 1.25μm. Then subcellular multimodal imaging can be achieved. Images from the two modes are corresponding with each other but functionally complementary. Combining PAM and LSCM provides more comprehensive information for the cytological test. This technique is demonstrated for imaging red-blood cells and meristematic cells.
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Photoacoustic imaging (PAI), concentrated on the endogenous optical absorption of samples, provides a high lateral resolution for the structural and functional imaging [1–3]. Among all technologies in PAI, only photoacoustic microscopy (PAM) is a microscopical imaging mode for detecting laser-induced ultrasonic waves to image the distribution of the optical energy deposition and for visualizing both the structural and functional information of samples . Though the PAM has different imaging mechanisms from laser scanning confocal microscopy (LSCM) which images with scattering photons , the PAM has a similar structure such as optical transportation, optical scanning, and light source with the LSCM. Thus combining PAM with LSCM for multimodal imaging is possible . Application of multimodal imaging, especially in cells, takes the advantages of combining different contrast mechanisms to provide comprehensive and complementary structural and functional information of samples. A system that combines PAM and LSCM can eliminate the incompatibility between optical absorption and scattering. So far, high resolution PAM has been developed to image red blood cells , but it’s necessary to provide more studies on cellular or even subcellular scale with multimodal imaging to improve the function of cell analysis and shorten the inspection cycle.
LSCM usually uses a continuous wave (CW) laser but PAM uses a pulsed laser as irradiation source, in order to integrate the PAM with LSCM, a CW laser is applied as the irradiation source in the multimodal imaging system. However, photoacoustic (PA) signals induced by modulated CW laser can hardly be detected by the commercial polyvinylidene fluoride (PVDF) needle hydrophone or piezoelectric ceramic transducer (PZT). Besides, the PA signals induced by modulated CW laser are much weaker than that of pulse laser. Therefore, a transducer with high enough sensitivity must be designed and manufactured in the system to achieve cellular or even subcellular imaging. Basing on the foundational experimental work [7,8], we have developed a novel detector element, the microcavity PA transducer, which utilize a gas detection technique and a microcavity to provide high enough sensitivity to obtain weak PA signals in the subcellular scale. The microcavity PA transducer can be compactly installed on the LSCM. When the sample is illustrated by one laser source, PA signals and backscattering photons are detected by microcavity PA transducer and photomultiplier tube (PMT) respectively and simultaneously.
2. Microcavity photoacoustic transducer and multimodal imaging system setup
The microcavity PA transducer consists of microcavity, resonant cavity, microchannel and microphone, as show in Fig. 1 . In order to effectively enhance the sensitivity of PA transducer, a microcavity and a resonant cavity are employed in the PA transducer. Because the pressure variation of gas is inversely proportional to volume of gas , reducing volume of cavity can effectively enhance pressure variation under the same temperature rise. For example, while the diameter of cavity reduced to one tenth of its original size, the pressure variation becomes as one thousand times as before consequently. Besides, a resonant cavity is employed to improve the detecting sensitivity of acoustic wave. The PA transducer is made of polymethyl which is a 1 mm thick. The diameter of the microcavity and resonant cavity are both 0.5mm, thus the volume of the microcavity and resonant cavity are of the order of 0.2 mm3. The microcavity and resonant cavity are connected by the microchannel with the diameter of 0.25mm and length of 200mm. Samples lay on a cover glass which adheres to the microcavity. And a specially designed wideband microphone with the bandwidth of 2.5 kHz adheres to resonant cavity to pick up the PA signal. The sensitivity of microphone is 10mv/Pa. The whole transducer is air-tight.
The experimental setup is shown schematically in Fig. 2 . A CW laser (Argon ion laser) with wavelength 514.5nm is used as the irradiation source in multimodal imaging system. The CW laser beam, modulated by a chopper, passes through a dichromatic mirror and then is scanned by a 2D galvanometer scanner (6231C, Cambridge Technology). A field flattening objective with magnification of × 60 is used to focus the laser beam on the sample.
The backscattered photons from the sample pass through the galvanometer scanner, and then are reflected by the dichromatic mirror to another objective to focus on the pinhole. The confocal signal is detected by PMT (CR131, BEIJING HAMAMATSU) to form a LSCM image. Synchronously, the induced PA signals from sample are detected by the microcavity PA transducer. After amplified by the preamplifier and demodulated by the lock-in amplifier (Stanford Research Systems SR830), the PA and LSCM signals are acquired by a data acquisition board (PCI6115, National Instrument).
3. Results and discussions
3.1 signal-to-noise ratio of PAM
To measure the signal-to-noise ratio (SNR) of the microcavity PA transducer, red blood cells (RBCs) were used as sample. The input power of laser which was measured before focused on the sample is 15mw. The modulated frequency is 2.5 kHz. The peak-to-peak amplitude of PA signals and noise were 904.4mv and 43.9mv after amplified respectively, as shown in Fig. 3 (a) and Fig. 3 (b). Then the SNR of the PAM is 26 dB. When the input power was adjusted from 2.5mw to 16mw, the PA signals from lock-in amplifier increased from 700mv to 2600mv linearly, as shown in Fig. 3 (c).
3.2 Spatial resolution of multimodal system
To quantify the resolution of this system reliably and effectively, a resolution test target with the highest resolution (JJG 827-1993, RTA-07) was used as sample, as shown in Fig. 4 (a) . Then we chose the group 25 with the highest resolution to image, where the width of bar is 1.25µm and the space between two bars is also 1.25µm. Figure 4(b) shows a close-up view of group25 image. Figure 4(c) shows the magnitude of the PA signals measured at the location as marked in Fig. 4 (b). The full width at half-maximum (FWHM) is about 1μm. Figure 4(d) shows the PA image of the group25 in resolution test target. The result shows that the PA image agrees with its optical image well, each bar can be clearly distinguished. Hence, the lateral resolution of PAM with microcavity PA transducer is better than 1.25µm. Figure 4(e) shows the image from LSCM, which agrees well with the PAM image. Thus it is certain that the lateral resolution of LSCM is also better than 1.25μm. The resolution limit of the multimodal imaging system is determined by the numerical aperture (NA) of objective, higher NA of objective provides smaller focus spot, and then provides higher resolutions of PAM and LSCM.
3.3 complementary imaging of multimodal system
To demonstrate the complementary imaging of the multimodal imaging system, iron deficiency anemia cells and leukocyte were used as samples to image in this system. Figure 5(a) shows the PA image of iron deficiency anemia cells. Figure 5(b) shows the image of iron deficiency anemia cells and leukocyte from LSCM. Because the PAM is sensitive to optical absorption and the major source of endogenous absorption is hemoglobin in the red blood cells (RBCs), PAM can provide the distribution of hemoglobin in RBCs. For example, the hemoglobin of the iron deficiency anemia cells mainly distributes on the edge of the cell, the center of the cells is transparent and has weak absorption. Then the reconstructed profiles of cells are donut shape. However, the leukocyte with high transparency has weak absorption and cannot be imaged by the PAM. On the other hand, LSCM is sensitive to the backscattering photons. The backscattering photons from leukocyte can be detected by PMT of LSCM, thus the leukocyte can be imaged by the LSCM. Obviously, the LSCM image gives complementary information to the PAM image. Thus, combining PAM and LSCM provides more information for cytological tests.
3.4 subcellular imaging of multimodal system
To illustrate the feasibility of the subcellular imaging with multimodal imaging system, meristematic cells of a garlic root tip were used as samples. Figure 6(a) shows the image from an optical microscopy. Figure 6(b) shows the PA image of meristematic cells. Figure 6(c) shows the image of meristematic cells from LSCM. As highlighted by the arrows, the cells which are in mitosis telophase have two cell nuclei. A new nucleus is just forming, but the cell hasn't yet completely divided into two independent cells. In the image of PAM, stained cell nuclei have strong optical absorption due to their pigments, relative to transparent cytoplasm with weak absorption. But in the image of LSCM, since the cell nuclei have more optical absorption than scattering, the backscattering photons from cell nuclei can hardly be detected by PMT. Therefore, the cell nucleuses appear to be dark in the image, as shown in Fig. 6(c).
With the new detector, microcavity PA transducer, the integration of PAM with the LSCM is achieved by sharing an optical transportation system, an optical scanning system, and an illumination source. The PAM and LSCM images are obtained simultaneously. The lateral resolution of the multimodal imaging system is tested to be better than 1.25μm, imaging in the subcellular scale can be achieved. With the advantage of microcavity PA transducer in the multimodal imaging, a more comprehensive and complementary imaging is obtained.
This work is supported by the National Natural Science Foundation of China (Grant No. 60877068), the Research Fund for the Docoral Program of Higher Education of China (Grant No.20104407110008), and National 863 Program Project of China (Grant No. 2006AA02Z4B4).
References and links
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