The Dual-Mode Imaging of Nanogold-Labeled Cells by Photoacoustic Microscopy and Fluorescence Optical Microscopy.
Journal: 2018/November - Technology in Cancer Research and Treatment
ISSN: 1533-0338
Abstract:
Photoacoustic microscopy is dominantly sensitive to the endogenous optical absorption, while a fluorescence optical microscopy can detect the fluorescence emission to obtain the image of a sample. To some extent, the physical processes of the 2 methods are opposite, one is absorption and another is emission, but both can be used to image cells. In this article, a simultaneous dual-mode imaging system of photoacoustic microscopy and fluorescence optical microscopy is set up to image tobacco cells. Furthermore, gold nanoparticles, which have a large absorption coefficient and enough fluorescence emission with wavelength of 512 nm, are used to label certain drugs and added to the tobacco cells. Then based on the simultaneous dual-mode microscopy imaging system, the photoacoustic microscopy and fluorescence optical microscopy images of gold nanoparticle-labeled tobacco cells are obtained. The final purpose of this experimental research is to detect if the labeled drugs can enter the cells by the positions of the gold nanoparticles. This will help the experts to deliver organic pesticide more accurately and effectively. The experimental results show that by gold nanoparticle labeling technology, the imaging quality of photoacoustic microscopy and fluorescence optical microscopy can be improved, which indicates that the drugs probably enter the tobacco cells successfully.
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Technology in Cancer Research & Treatment. Dec/31/2017; 17
Published online Sep/23/2018

The Dual-Mode Imaging of Nanogold-Labeled Cells by Photoacoustic Microscopyand Fluorescence Optical Microscopy

Abstract

Introduction

Photoacoustic imaging (PAI) concentrates on the endogenous optical absorption of samples.When pathological change appears in biological tissues, their absorption coefficients changecorrespondingly. If short-pulse laser illuminates tissues, the pressure transients generatedby the tissues depend on the optical absorption distribution. So, PAI can provide a highlateral resolution for the structural and functional imaging of samples.1-9 Photoacoustic imaging technique has characteristics of deep acoustic penetrationdepth and high optical contrast. It can be used to image multiscale samples. It can beachieved by illuminating the sample from one side and detecting the acoustic pressuresignals at any directions.

Photoacoustic microscopy (PAM) is a microscopically imaging mode implemented by thedistribution of the optical energy deposition within samples.4 So far, PAM images of red blood and anemia cells have achieved their structure andfunction with high resolution.5,6 According to the principle of imaging, PAM can image most absorbent substances incells. It has been widely used in the cancer cells research because cancer cells havestronger absorbance than normal cells, so PAM can be used to image local cancer cells fortarget therapy in clinical medicine.

Fluorescence imaging is a recent development in noninvasive biomedical imaging. It bases onthe strong fluorescence intensity emitted by fluorescent material in sample. Under certainilluminating intensity of excitation, the specific fluorescence intensity is proportional tothe amount of fluorescein in a sample, so it can be used to image spatial distribution offluorescent material within sample. Imaging for specific mark proteins is its majoradvantage. But it cannot provide sample information of physiological process that hasnothing to do with fluorescent material.

As microscopically imaging modes for cells, fluorescence microscope (FOM) and PAM havesimilar systems. The differences between them are the imaging mechanisms and principle—oneis absorption and another is emission, so their reconstruction images can show differentinformation of samples. But the light source, optical transportation, and optical scanningsystems are similar to each other, which makes the dual-model imaging system of PAM and FOMfeasible. The comprehensive and complementary structural and functional information ofsamples could be obtained by different contrast mechanisms by a PAM and FOM dual-modelsystem.

In this article, PAM and FOM had been integrated together and the simultaneous dual-modemicroscopy imaging system is set up. In the system, PAM and FOM share the same source andlight transmitting path. Based on this dual-model imaging system, blank tobacco cells areimaged.

The final purpose of this experimental research is to detect if the drugs can enter thecells. Generally, drugs may not have enough absorptivity or fluorescence. To take thefurthest advantages of FOM and PAM, drugs were marked by marker, which can producephotoacoustic (PA) signals and at the same time can emit fluorescent light when bathed inlaser. As one of the 4 main immune labeling technologies, nanogold-labeling method is thefirst choice, for its strong absorptivity and fluorescence. The self-made gold nanoparticleswith 512-nm emission wavelength had been made and used to label certain drugs. These goldnanoparticles have a large absorption coefficient and enough fluorescence emission. They areused to label certain drugs and are added to the tobacco cells. Then, by the FOM and PAMimaging results of the labeled tobacco cells, we can judge if the gold nanoparticles enterthe cells, that is the same to the drugs. The experimental results show that by goldnanoparticle labeling technology, the imaging quality of PAM and FOM can be improved, whichindicates that the drugs probably enter the tobacco cells successfully.

In general, in this article, FOM and PAM are combined together and dual-mode imaging forcells is achieved. Also, nanogold labeling technology is used to improve the quality of thesample image. All these will make the tracing of drugs or nutrition in plant cells morefeasible.

Photoacoustic Microscopy and FOM Simultaneous Dual-Mode Imaging System

Both PAM and FOM have different imaging mechanisms and basic imaging principle. The formeris based on absorption and the latter emission. So, the signals in PAM and FOM system aredifferent. They are PA signals detected by PA transducer and light signals byphotomultiplier tube (PMT), respectively. As shown in Figure 1, dual-modal laser scanning imaging system withPAM and FOM setup is integrated in a same system. But to match the 2 microscopes with eachother, the fluorescence of sample is arranged to be detected by PMT backscattering in thesystem.

Figure 1.
Schematic of dual-modal laser scanning imaging system with integrated photoacousticmicroscopy imaging (PAM) and fluorescence optical microscopy (FOM) setup.

The amplification factor is determined by the size of the specific cell samples. The commonradiation source is a continuous wave (CW) laser (argon ion laser, output wavelength is514.5 nm) with the laser power about 10 mW. A chopper is used after the argon ion laser tochange the continuous light to pulse one. A field flattening lens was used as the objectivelens with the magnification of ×40 or ×20, which decide the resolution of the system.However, PA signals induced by modulated CW laser can hardly be detected by the commercialpolyvinylidene fluoride needle hydrophone or piezoelectric ceramic transducer. Besides, thePA signals induced by modulated CW laser are much weaker than that of pulse laser. Thus, aPA transducer with microcavity based on a gas detection technique is used to strengthen thePA signals of samples. The microcavity PA transducer6 can improve the signal to noise ratio of PA signals than an ordinary transducer. Itis made by the 1-mm thick polymethyl. The diameter of both the microcavity and resonantcavity is 0.5 mm; thus, the volume of the microcavity and resonant cavity is 0.2mm3. The response frequency of microcavity PA transducer is from 100 Hz to 3 kHz.6

The laser from argon ion laser (wavelength 514.5 nm, power 15 mW) becomes pulse laser aftermodulated by chopper. The modulated frequency is 2.5 kHz. Then the pulse laser is collimatedby lens L1 and L2, passes through beam splitter L3, and then is shifted by galvanometerscanner. After focused by objective, the scanning pulse laser illuminates all parts ofsample successively. At the same time, PA signals induced are recorded by microcavity PAtransducer, amplified by preamplifier (Stanford Research Systems SR550, US) and lock-inamplifier (Stanford Research Systems SR830, US), collected by data acquisition card(PCI6115, National Instrument, US), and then transferred to computer. The PAM image ofsample can be reconstructed by these PA signals.

Synchronized with the above procedure, the backscattered fluorescence from the samplebacktracks through the galvanometer scanner, reflected by beam splitter L3 to anotherobjective, focused on the pinhole and then arrives at the PMT (CR131; Beijing Hamamatsu).Then the FOM image reconstructed by these signals can be obtained.

Thus, a simultaneous dual-modal imaging technique can be achieved by one same laser sourceand optical system. Based on system shown in Figure 1, PAM and FOM images of cell samples can bothbe achieved, and such combination will provide more comprehensive information for thecytological test. Images from the 2 modes are corresponding with each other, butfunctionally complementary.

Method was used before the objective lens to scan the probe beam in the x-y direction. Withthe beam diameter of 8 mm before the objective lens, the lateral resolution of the system,that is, the diffraction-limited optical focal diameter, was calculated to be 0.42 μm at 514nm. The system lateral resolution was experimentally checked by the Guangzhou MunicipalStandard Bureau, reporting a measured value of 1.25 μm.

Experimental Results and Discussions

Tobacco cells are selected as samples to image based on the dual-model imaging system asshown in Figure 1. The tobacco cellswere divided into 3 groups.

Among the samples, 1 group remained as blank and certain drugs labeled by self-made goldnanoparticles with 512 nm (close to the irradiation beam wavelength 514 nm) emissionwavelength were added to the other 2 groups. To prevent the cells become apoptotic anddeformed during the experiment, silica was used to fix the tobacco cells.

The pictures in Figure 2 are theresults of ordinary optical microscopy imaging (OMI), photoacoustic microscopy imaging(PAMI), and fluorescence optical microscopy imaging (FOMI). The pictures shown in Figure 2(1) A to C are the OMI, PAMI,and FOMI results of the blank tobacco cells. The second and third groups in A to C of Figure 2(2) and Figure 2(3) are the OMI, PAMI, and FOMI results fortobacco cells labeled by gold nanoparticles and fixed by silica.

Figure 2.
The results of ordinary optical microscopy imaging (OMI), photoacoustic microscopyimaging (PAMI), and fluorescence optical microscopy imaging (FOMI). (1) Images for blanktobacco cells; (2) images for tobacco cells labeled by gold nanoparticles and fixed bysilica; and (3) images for tobacco cells labeled by gold nanoparticles and fixed bysilica.

Conclusion

In this article, tobacco cells are chosen as sample because tobacco cell has littlechlorophyll which will cause large noise signals. Also, its size is big enough to image.Both nanogold labeling technology and microcavity PA transducer are the effective methods toimprove the imaging quality. In addition, fixing the cells before experiment is a veryimportant aspect to make sure it will not damage because we must ensure the stability ofcells throughout the whole process. It is achieved by soaking the cells in silica.

But it can be seen from the results in Figure 2 that PAM and FOM dual-mode imaging system can obtain the sharp and exactimages of blank tobacco cells. Comparing the first group in Figure 2(1) and the latter 2 groups in Figure 2(2) and Figure 2(3), it is obvious that the PAM and FOM imagesof blank cells have low contrast because of the low absorption of the blank cell itself, butafter the tobacco cells were labeled by nanoparticles labeling technology, the quality ofthe PAM and FOM imaging results is improved, which indicates that the drugs probably enteredthe tobacco cells successfully. So, the PAM and FOM integrated imaging system can be used tojudge if the labeled drugs can combine the cells by the positions of the gold nanoparticles,or even to monitor and trace the drugs. But it is difficult to determine whether thenanoparticles enter tobacco cells or probably absorbed on the surface membrane, unless theresolution is able to distinguish the internal structure of the cells.

All these will help the experts to deliver organic pesticide more accurately andeffectively. The system and method proposed here can also be used to trace the medicine inthe cell of human resource to monitor the medicine pathway and know much of the effect.

Footnotes

Declaration of Conflicting Interests: The author(s) declared no potential conflicts of interest with respect to the research,authorship, and/or publication of this article.

Funding: The author(s) disclosed receipt of the following financial support for the research,authorship, and/or publication of this article: This work was supported by the NationalNatural Science Foundation of China under Grant Nos. 61575067 and 31401781 and the Scienceand Technology Program of Guangzhou, China under Grant Nos. 2015A020209148, 201605030013,and 201604016122.

Abbreviations

CWcontinuous waveFOMfluorescence optical microscopyFOMIfluorescence optical microscopy imagingOMIoptical microscopy imagingPAphotoacousticPAIphotoacoustic imagingPAMphotoacoustic microscopyPAMIphotoacoustic microscopy imagingPMTphotomultiplier tube.

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