WO2016027229A1

WO2016027229A1 - Apparatus and methods for simultaneous multimodal nonlinear optical microscopy for label-free bio-imaging

Info

Publication number: WO2016027229A1
Application number: PCT/IB2015/056273
Authority: WO
Inventors: Zhiwei Huang
Original assignee: National University Of Singapore
Priority date: 2014-08-18
Filing date: 2015-08-18
Publication date: 2016-02-25
Also published as: GB201414631D0

Abstract

A simultaneous multimodal nonlinear imaging apparatus of imaging a cell sample to identify changes caused by the acetowhitening phenomenon; the apparatus comprising: an illumination source capable of producing a plurality of excitation wavelengths; an optical element for directing the or each excitation wavelength to the sample to thereby illuminate the sample; a first photodetector for capturing a first image related to a first excitation wavelength; and a second photodetector for capturing a second image related to a second excitation wavelength; wherein the first and second images are used to identity changes in the cell caused by the acetowhitening phenomenon.

Description

Apparatus and methods for simultaneous multimodal nonlinear optical microscopy for label-free bio-imaging.

FIELD OF INVENTION The present invention relates to an apparatus and method simultaneous multimodal nonlinear optical microscopy for label-free bio-imaging. In particular, but not exclusively, for any applications of label-free imaging in tissue and cells at submicron resolutions.

BACKGROUND OF THE INVENTION The tissue acetowhitening effect of an acetic acid instillation procedure is a simple and economic method for neoplasia detection and has been clinically utilized since 1925. The acetowhitening effect is widely used during colposcopy for enhancing disparities of the optical properties, such as scattering, between the intraepithelial neoplastic lesion and the non-neoplastic tissues. In recent years, the acetowhitening effect has been applied to pre-neoplasia and early neoplasia detection during gastroscopic inspection in the upper gastrointestinal tract and has achieved promising diagnostic sensitivity and specificity of approximate 95.0% and 100.0%, respectively. Some research work has been carried out to study the tissue acetowhitening process; however, the fundamental epithelia kinetic mechanism involved in the acetowhitening process at the subcellular level remains largely elusive.

Nonlinear optical (NLO) microscopy is a label-free imaging technique and has attracted much attention for biomedical imaging (bio-imaging) due to its biochemical selectivity and optical sectioning ability at sub-micron resolution. For example, third-harmonic generation (THG) signals can only be generated in regions with optical heterogeneities, such as third-order nonlinear susceptibility, refractive index and dispersion, inside biological samples. It is suitable to map the subcellular origin of light scattering such as: Two-photon excited fluorescence (TPEF) imaging of tryptophan which excels in providing protein information [10]; and TPEF microscopy of Nicotinamide adenine dinucleotide (NADH) which can effectively probe proteins (enzymes) associated with metabolic activities. Concomitant TPEF imaging of these endogenous biochemicals can elucidate proteostasis in cells, such as transient aggregation of ubiquitinated proteins and etc.

In this invention, a unique simultaneous multimodal nonlinear optical microscopy platform is developed. This is based on a femtosecond laser pumped photonic crystal fiber to systematically investigate the mechanistic details of acetowhitening phenomenon at a molecular level in living mammalian cells using THG and TPEF imaging without labeling. Human cervical squamous carcinoma cell lines (SiHa) are specifically employed as the mammalian cellular model for studying the acetic acid induced epithelial tissue aceto whitening effect.

One object of the present invention is to overcome at least some of the problems associated with the prior art and the present techniques.

A further object of the present invention is to provide apparatus and method simultaneous multimodal nonlinear optical microscopy for label-free bio-imaging for any applications of label-free imaging in tissue and cells at submicron resolutions.

SUMMARY OF THE INVENTION

A unique simultaneous multimodal nonlinear optical microscopy imaging (e.g., third-harmonic generation (THG) and two-photon excited fluorescence (TPEF)) platform is developed. This is based on a femtosecond laser pumped photonic crystal fiber to investigate the acetowhitening phenomenon induced by acidic acid in live mammalian cells without labeling. After treatment with acetic acid at concentrations of higher than 0.2%, THG images show that light scattering is unusually increased inside the nucleus and cytoplasm in cells. Co-localized TPEF and THG imaging on tryptophan and Nicotinamide adenine dinucleotide (NADH) in cells indicates that the change of scattering property is largely originating from the morphological change of metabolic proteins induced by acidic acids. Further TPEF imaging on NADH and flavin adenine dinucleotide (FAD) in cells confirms that this change is irreversible when acidic acid concentration is higher than 1.2%. These subcellular-level THG/TPEF imaging results reveal that the acetowhitening phenomenon is highly related with proteins involved in metabolic pathways in the nucleus and cytoplasm in live cells.

According to one aspect of the present invention there is provided a simultaneous multimodal nonlinear imaging apparatus of imaging a cell sample to identify changes caused by the acetowhitening phenomenon; the apparatus comprising: an illumination source capable of producing a plurality of excitation wavelengths; an optical element for directing the or each excitation wavelength to the sample to thereby illuminate the sample; a first photodetector for capturing a first image related to a first excitation wavelength; and a second photodetector for capturing a second image related to a second excitation wavelength; wherein the first and second images are used to identity changes in the cell caused by the acetowhitening phenomenon.

In an implementation the first and second images are used to identify abnormal growths.

In an implementation the optical source is a laser. In an implementation the laser is a femtosecond titanium sapphire laser.

In an implementation the optical elements include guiding optics to take the first excitation wavelength through a first optical path to the sample and then to the first photodetector.

In an implementation the optical elements include guiding optics to take the second excitation wavelength through a second optical path to the sample and then to the second photodetector.

In an implementation the first excitation wavelength produces a third harmonic generation image.

In an implementation the second excitation wavelength produces a two photon exited fluorescence image.

According to a second aspect of the present invention there is provided a method of simultaneous multimodal nonlinear imaging of a cell sample to identify changes caused by the acetowhitening phenomenon; the method comprising: tuning a source of illumination to a predetermined wavelength range; splitting the illumination into a plurality of excitation wavelengths; illuminating the sample with the plurality if excitation wavelengths; capturing a first image from a first photodetector related to a first excitation wavelength; and capturing a second image from a second photodetector related to a second excitation wavelength; wherein the first and second images are used to identity changes in the cell caused by the acetowhitening phenomenon.

An Implementation may further comprise using the first and second images to identify abnormal growths.

An Implementation may further comprise illuminating the sample with a laser.

In an implementation the laser is a femtosecond titanium sapphire laser. An Implementation may further comprise guiding a first excitation wavelength through a first optical path to the sample and then to the first photodetector.

An Implementation may further comprise guiding a second excitation wavelength through a second optical path to the sample and then to the second photodetector.

DETAILED DESCRIPTION OF THE DRAWINGS

Reference will now be made, by way of example, to the accompanying drawings to provide a better understanding of embodiments of the present invention. The drawings should not be interpreted to be limitative and dimensions may not be to scale.

Figure 1 is a schematic diagram of the multimodal nonlinear optical imaging (TPEF/THG) platform for label-free imaging of live cells, in accordance with an embodiment of the invention;

Figure 2 is an example label-free images based on third-harmonic generation (THG) signals arising from live mammalian cells in monolayer culture after (a) 5- minutes acetic acid treatment, and (b) 5-minute acetic acid treatment followed by 5-minute washout using fresh culture medium, in accordance with an embodiment of the invention;

Figure 3 is (a) a THG image, (b) a TPEF of tryptophan image and (c) an NADH image taken from live mammalian cells before and after 5-minute treatment by acetic acid, in accordance with an embodiment of the invention; Figure 4 is representative normalized cellular TPEF images of NADH and FAD taken (a, b) after 5-minute acetic acid treatments, and (c, d) after 5-minute acetic acid treatment followed by 5-minute washout, in accordance with an embodiment of the invention;

Figure 5 shows THG (a, d), TPEF (b, e) and overlay images (c, f) of normal liver tissue where the image sets (a, b, c) and (d, e, f) are taken from the two identical tissue locations, respectively, in accordance with an embodiment of the invention;

Figure 6 shows THG (a, d), TPEF (b, e) and overlay images (c, f) of diseased liver tissue at week 2 (a, b, c) and week 4 (d, e, f) after BDL surgery where the image sets (a, b, c) and (d, e, f) are taken from the two identical tissue locations, respectively, in accordance with an embodiment of the invention;

Figure 7 shows representative 3-D co-localized THG (a) and TPEF (b) images of normal liver tissue where the overall thickness of liver tissue for 3-D sectioned imaging is ~25 μηι with a scanning interval of 0.2 μηι. Image size: 37 x 37 x 25 μηι, in accordance with an embodiment of the invention; Figure 8 shows a processed 3-D THG images through de-convolution of the THG images in Figure 7a, where the image size is 37 x 37 x 25 μηι, in accordance with an embodiment of the invention;

Figure 9 is a graph of normalized THG intensities of the cell nuclei in the centre of figure 7a and figure 8 at different imaging depths where the signal intensity is calculated by integrating intensity value of each pixel within a fixed circle enclosing the nucleus and the arrows indicate the estimated boundary position of the nucleus in liver cell, in accordance with an embodiment of the invention; and

Figure 10 is a bar chart showing the fractal dimensional analysis results of co- localized NADH and FAD TPEF images at baseline and 5-minute acetic acid treatment conditions, in accordance with an embodiment of the invention. DETAILED DESCRIPTION OF THE INVENTION

Figure 1 shows the schematic of the simultaneous multimodal nonlinear optical microscopy imaging (e.g., THG/TPEF) system developed for label-free imaging of live cells. In figure 1, the nomenclature is as follows: · FM is a flip mirror;

• L is a lens;

• M is a mirror;

• OPO is an optical parametric oscillator;

• MO is a microscope objective; · PCF is a photonic crystal fiber;

• Fl is a first filter set which comprises a 600±20nm bandpass filter;

• F2 is a second filter set comprising 730 nm excitation: a 650 nm shortpass and a 460±30nm bandpass filter; 860 nm excitation: a 750 nm shortpass and a 540±20nm bandpass filter; 600 nm excitation: a 532 nm shortpass and a 355±20nm bandpass filter;

• F3 is a third filter set comprising a 650 nm shortpass filter and a 390±20nm bandpass filter;

• DM is a dichroic mirror; and

• PMT is a photomultiplier tube. An illumination source, such as a 120-femtosecond (fs) Ti: sapphire laser, for example the Mira 900 from Coherent Inc., was tuned to 730 and 860 nm to correspondingly produce excitation energy to excite TPEF of NADH and FAD, and adjusted to 800 and 835 nm to act as a pump source for a photonic crystal fiber (PCF), such as the Femtowhite from CARS, Newport and an optical parametric oscillator, such as the OPO Mira from Coherent Inc., respectively. The super- continuum generated from the PCF was filtered using for example a FF01-600/37 from Semrock for the optimal TPEF excitation of tryptophan; while the excitation energy at 1170 nm from the OPO was employed for THG imaging. With the use of flip mirrors, different excitation wavelengths can be separately generated and uniquely delivered to a pair of galvanometer scanning mirrors, and subsequently focused onto the sample through a water immersion microscope objective, such as the UPlanSApo 60x, N.A. 1.2, from Olympus, for high contrast cellular THG/TPEF imaging. The generated TPEF signal in the backward (epi-) direction was reflected by a dichroic mirror, such as the FF580-FDi01 from Semrock, and filtered by different filters for different excitation wavelengths (730 nm excitation: FF01-650SP and FF01-460/60, Semrock; 860 nm excitation; FF01-750SP and FF01-540/40, Semrock; 600 nm excitation: SP01-532RU and FF01-355/40, Semrock), and then detected by a photomultiplier tube (PMT), such as the R3896 from Hamamatsu. The forward propagating THG signal was collected by a condenser, such as the U-TLD, NA 0.9 from Olympus, and filtered, using a filter such as the FF01-650SP FF01-390/40 from Semrock, before being detected by a PMT such as the R3896 from Hamamatsu. Note the maximum powers used on the biological samples for TPEF of NADH, FAD, tryptophan, and THG were about 3, 4, 2, and 15 mW, respectively. A total integration time of approximately 60-180s per image was used for collection of the THG/TPEF cellular images.

It should be noted that the various optics used to direct the illumination to the cell sample may be replaced by and suitable optical elements and the examples shown in figure 1 are just one possibility. Similarly, other elements many be replace by equivalent elements and those shown in figure 1 are not intended to be limitative.

Human cervical squamous cell carcinoma (SiHa) cells such as the American Type Cell Collection from VA were removed from cryopreservation and cultured at 37°C in a 5% CO2 humidified incubator. On the day before the analysis, the cells at density of approximately 1 x 10⁶ cells were placed in lysine coated petri dishes with cover glass bottom such as size, 35 mm; P35G-0-41-C dishes from MatTek, Ashland, MA, and immersed in a Dulbecco's modified Eagle's medium for example from Gibco, Invitrogen, supplemented with a 10% fetal bovine serum for example from Gibco, Invitrogen and a 1% penicillin/streptomycin for example also from Gibco, Invitrogen. It should be noted that the present invention relates to one or two specific cancer cells or carcinomas, however it may equally apply to any other types of abnormal cells or abnormal growths found in the body.

To investigate the cellular acetowhitening process, acetic acid treatment and washout procedures on the 35 mm-diameter size petri dishes were carried out. For the acetic acid treatment procedure, the original growth medium was replaced with the growth medium containing acetic acid with seven different concentrations varying from 0.0% to 3.0% with pH values from 7.52 to 3.22. THG/TPEF microscopy imaging of cells was carried out after 5 minutes of acetic acid-growth medium immersion. In the acetic acid washout procedure, before the THG/TPEF imaging, cells were immersed in the acetic acid-growth medium for 5 minutes, and then immersed in a fresh growth medium for another 5 minutes.

Figure 2 compares the THG images between live mammalian cells treated by acetic acid with different concentrations and those under washout conditions. Images labeled with (i) to (v) are taken at different acetic acid concentrations from 0% to 0.5% (pH values from 7.56 to 4.42) for treatment and washout conditions. All images are reconstructed from raw data without any processing and image sizes are 60 μηι ^χ 60 μηι. The mammalian cells under 0.0 and 0.1% acetic acid treatment (Figure 2(ai), 2(aii)) and washout condition (Figure 2(bi), 2(bii)) have similar homogenous optical properties indicated by the weak THG signals. Cells treated by acetic acid with 0.2% or higher concentrations show strong THG signals from the nucleus as well as some weak signals from the cytoplasm (Figure 2(aiii)-2(av)). It is also observed that the signal from nucleolus is most prominent (indicated by arrows in Figure 2(av)). These enhanced THG signals due to acetic acid treatment disappear in the cells under washout conditions (Figure 2(biii)-2(bv)). These results indicate that acetic acid with 0.2% or higher concentrations induces significant optical scatterings within the nucleus and especially the nucleolus, which may be due to the result of refractive index increase in these regions; and the change of cell scattering properties is reversible by the wash of fresh culture medium. The observed concentration threshold is in agreement with light reflectance and scattering spectroscopy of cells in acetowhitening effect that have been seen before, and is also consistent with the clinical observation of epithelial tissue acetowhitening phenomena.

To identify which kind of biochemicals is affected by acidic acid and which contribute to the scattering property change in cells, co-localized THG and TPEF images of tryptophan and NADH are compared in cells which have been untreated and cells which have been treated with 0.3% acetic acid. Figure 3 shows before and after 5-minute treatment by acetic acid with 0.3% (pH value 4.84) concentration. Image sizes are 100 μηι ^χ 100 μηι. It can be seen that with 0.3% acetic acid treatment, the cell nuclei show a stronger THG signal (Figure 3(aii)) as compared to the untreated cells (Figure 3(ai)); while tryptophan and NADH TPEF signals are also stronger inside the nuclei (Figs. 3(bii) and 3(cii)) compared with those of untreated cells (Figs. 3(bi) and 3(ci)). Moreover, the images of all three modalities become strikingly similar. As TPEF imaging of tryptophan excels in providing proteins information; while TPEF imaging of NADH can effectively probe proteins such as enzymes, associated with metabolic activity, the TPEF images indicate that acetic acid induces coagulation of metabolic proteins inside cells. This is particularly the case inside the nuclei. The high resemblance of tryptophan and NADH images to the THG image after acetic acid treatment indicates that the metabolic proteins are responsible for the scattering property changes of cells in the acetowhitening process. The relationship between spatial distribution changes of metabolic proteins with respect to the concentration of acidic acid used to treat cells, TPEF images of NADH and FAD were taken at different acidic acid concentrations is now described with reference to figure 4. Figure 4 shows the representative TPEF images of NADH and FAD in live mammalian cells treated by acetic acid with different concentrations and also in those under washout conditions. Images labeled with (i) to (iii) are taken at different acetic acid concentrations from 0% to 1.2% (pH 7.56 - 3.86) for treatment and washout conditions, respectively. Note the excitation power used on the samples was less than 5 mW, and a total integration time of 60s per image was used. Image sizes are 50 μηι x 50 μηι. The TPEF images demonstrate significant spatial distribution changes of metabolic proteins in cells treated by acetic acid with 0.3 and 1.2% concentrations (Figs. 4(aii), 4(bii), 4(aiii), and 4(biii)) compared with those in untreated cells (Figs. 4(ai) and 4(bi)). On the other hand, the images under washout conditions show that the spatial distribution changes of metabolic proteins are reversible in cells treated by acetic acid with 0.3% concentration (Figure 4(cii), 4(dii)) but not in cells treated by acetic acid with 1.2% concentration (Figure 4(ciii), 4(diii)). The TPEF imaging result indicates that cellular changes induced by acetic acid become permanent at high acetic acid concentrations from approximately 1.2%, which is consistent with known practices.

The spatial distribution changes of metabolic proteins in cells treated by acidic acid with different concentrations are quantified by analyzing NADH and FAD TPEF images using fractal dimension analysis. The analysis data are shown in figure 10. It can be seen that after cells have been treated by acetic acid with 0.2% or higher concentrations, the fractal dimensions significantly increases (p<0.05, Wilcoxon matched pairs test). This is especially the case for FAD. The fractal dimensional analysis result affirms that the spatial distributions of metabolic proteins are considerably disturbed during the cellular acetowhitening process.

In summary, a unique simultaneous multimodal nonlinear optical microscopy (THG/TPEF) platform to study the acetowhitening process induced by acidic acid in live mammalian cells at the subcellular level without labeling has been developed. THG microscopy demonstrated the threshold effect of approximately 0.2% acetic acid concentration to induce acetowhitening process in the cytoplasm and nucleus. Additionally, the integrated THG/TPEF imaging of tryptophan and NADH in mammalian cell reveals that the increased light scattering property during the acetowhitening process in cells is highly associated with metabolic proteins coagulation induced by the acidic acid. TPEF imaging on NADH and FAD confirms that the resulting morphological change becomes permanent when the concentration of acidic acid is higher than 1.2%. Further fractal dimension quantitative analysis of TPEF images on NADH and FAD affirms that significant light scattering changes are due to proteins perturbations involved with metabolism by acetic acid with 0.2% and higher concentrations. The simultaneous multimodal nonlinear optical imaging reveals that the acetowhitening phenomenon is highly related to proteins involved in metabolic pathways in the nucleus and cytoplasm in live cells.

A further embodiment of the present invention is now described with reference to Wistar rats and the study of liver disease. To induce liver fibrosis and steatosis, a bile duct ligation (BDL) was performed under general anesthesia with ketamine and xylazine. A midline abdominal incision was performed to explore the liver and intestines. The bile duct was then ligated near the porta with silk. The wound was then closed with a double layered tissue closure with vicryl sutures. A total of 18 rats were ligated and sacrificed each week from week-1 to -6 (n = 3 per week). Three control rats were also sacrificed at week-0. The fresh liver tissues harvested were immediately frozen in liquid nitrogen, and sectioned into a series of 20-μηι tissue slides in a microtome cryostat such as the HM 500 OM from, Carl Zeiss, Germany for THG and TPEF imaging. The system of figure 1 is used in this analysis to obtain the images described below.

Figure 5 shows the THG (a, d) and TPEF (b, e) images of normal control liver tissues at different zoom scales. In the large-scale images (Figs. 5(a, b, c)), it can be seen from the THG imaging (Figure 5(a)) that the nuclei of hepatocytes are bright and the cell membranes are also visible; while in the corresponding TPEF image taken from the same location (Figure 5(b)), only cytoplasm of hepatocytes are visible. The overlay image (Figure 5(c)) confirms that the bright spots in the THG images reside in the dark region of TPEF images, where the nuclei are located. In the zoom-in images (Figs. 5(d, e, f)), several individual hepatocytes are observed more clearly. In the THG image (Figure 5(d)), one nucleus in the center is clearly identified. The nuclear membrane, chromatin structures can also be observed inside the nucleus. The brightest spots may be chromo-centers, which are large heterochromatic clusters, congregations of repetitive DNA, and particularly prominent in mouse cell nuclei. These are shown to have a higher refraction index than their nuclear environment and thus have stronger THG intensity. Two other nuclei can also be seen at the lower part of THG image with a lower intensity, which may be due to the THG signal originating from different cell depths out of light focus. In the zoom-in TPEF image (Figure 5(e)), the boundary of nucleus is roughly visible but little signal is generated from the inside. In the overlay image (Figure 5(f)), it can be seen that THG imaging shows much clearer cell and nuclear membranes than that TPEF imaging. These results indicate that the THG imaging is capable of imaging the hepatocyte nuclei as well as the cell and nuclear membranes, providing complementary information that is missing by TPEF cellular imaging. Figure 6 shows THG and TPEF images of diseased liver tissue at week 2 (Figs. 6(a, b, c)) and week 4 (Figs. 6(d, e, f)) after BDL surgery. THG images (Figs. 6(a, d)) shows a much stronger signal intensity than that of normal liver tissue (Figure 5(d)); however, the patterns become distorted and the cell morphology is virtually not recognizable. The TPEF images (Figs. 6(b, e)) also shows blurred cell morphologies compared to that of the normal liver tissue (Figure 5(e)); while the individual hepatocyte is still distinguishable as indicated by the arrows. In the overlay image (Figure 6(c)), it can be seen that the nucleus size as indicated by the arrow is larger than that of the normal liver tissue, and the fragmentation is obvious. This may be the result of the chromatin condensation and nuclear fragmentation, indicating the early stage of apoptosis. With the progression of liver disease as shown in Figure 6(f), the cell nuclei are totally destroyed due to continuous chromatin condensation and nuclear fragmentations in THG imaging (indicated by the arrow). These results confirm that THG is a unique NLO modality, particularly suited for label-free imaging of cell nucleus at a sub micron-resolution. The 3-D imaging of liver tissue can be readily realized by the combined THG/TPEF microscopy due to its intrinsic optical sectioning ability. Figure 7 shows an example of 3-D intensity distributions of THG and TPEF signals in a 20-μηι sectioned normal (control) rat liver tissue. Two adjacent nuclei can be clearly identified from the THG images (Figure 7(a)) and they have regular round shapes representing normal cell states. Moreover, the cell membranes are also visible. While in the TPEF images (Figure 7(b)), the nuclear region is shown as an empty cavity since signal only comes from the cytoplasm. These results demonstrate that THG imaging is able to provide the 3-D morphological architectures of the cell nuclei. One notes that both 3-D images seem to be elongated in the longitudinal direction. This is because the point spread function (PSF) of the laser focus has a larger full-width at half-maximum (FWHM) in the longitudinal direction than that in the transverse direction. Since the image formation in laser-scanning optical microscope is the result of convolution between laser PSF and the sample, the non- spherical shape of laser PSF results in the image elongation.

The image distortion could be minimized by characterizing the PSF structure through de-convolution process. De-convolution is applied to the 3-D THG images shown in Figure 7(a) using an Iterative Deconvolve 3-D ImageJ plugin and the processed images are shown in Figure 8. It can be seen that the spatial resolution is improved and the image elongation in the longitudinal direction is greatly reduced. To quantify the image distortion, the size of the nucleus is measured at the center in the original (Figure 7(a)) and processed (Figure 8) THG images. The nucleus diameter in the transverse plane is around 7.2 μηι in both images. To measure the nuclear size in the longitudinal direction, its signal intensity is integrated in each transverse plane and the intensity values are plotted shown in Figure 9. From the curves, the nuclear size in the longitudinal direction estimated from the 3D THG images is ~12.3 μηι and ~8.5 μηι, respectively, before and after the image de-convolution. Since the shape of a normal cell nucleus is close to spherical, the nuclear size proved by de-convoluted 3-D THG image is more reliable. The relative volume occupied by the chromatin revealed by 3-D THG. A threshold cut-off was also calculated and was applied based on analysis of the images in order to determine which intensity levels were considered as chromatin signal. After analyzing the 3-D THG intensity values inside the nuclear region pixel by pixel, the nuclear volume occupied by chromatin is ~36.7%, which is close to the value achieved by scanning electron microscopy.

In conclusion, THG together with TPEF microscopy have been applied to investigate the morphological changes of cell nucleus in liver tissue. The results show that THG microscopy is able to provide 3-D images of cell nucleus as well as reveal nuclear morphological changes associated with liver disease progression, which is not achievable by TPEF imaging. Moreover, the nuclear size and volume occupied by chromatins in cells can also be estimated using 3-D THG imaging. This work suggests that 3-D THG microscopy has the potential for quantitative analysis of nuclear morphology in cells at a submicron-resolution without the need for DNA staining. The simultaneous multimodal nonlinear optical microscopy imaging technique developed can be used to analyze the changes of intrinsic biochemicals (e.g., FAD, NADH, and tryptophan) and morphologic structures associated with acetowhitening effect in live cells without labeling.

This invention will greatly accelerate the process of rapid and label-free imaging of tissue and cells at the molecular, cellular and tissue levels.

The Nonlinear optical microscopy (NLO) techniques described above are not limited to TPEF, but may also include second-harmonic generation (SHG) and coherent anti-Stokes Raman scattering (CARS) which also possess many attractive features such as three-dimensional (3-D) sectioning capability at submicron scale resolutions, deep tissue penetration depth, and non-destructiveness which can be applied to biomedical imaging due to their biochemical specificity.

This method has no limitations given that ultrafast laser sources, photonic crystal fibers and other optics have similar specifications.

The invention is not confined to the label-free tissue and cells imaging, but can also have applications in other areas. In general, this includes any applications of analyzing intrinsic chemicals and morphological structures and compositions in samples with different responses to different nonlinear optical microscopy imaging modalities.

Claims

1. A simultaneous multimodal nonlinear imaging apparatus of imaging a cell sample to identify changes caused by the acetowhitening phenomenon; the apparatus comprising: an illumination source capable of producing a plurality of excitation wavelengths; an optical element for directing the or each excitation wavelength to the sample to thereby illuminate the sample; a first photodetector for capturing a first image related to a first excitation wavelength; and a second photodetector for capturing a second image related to a second excitation wavelength; wherein the first and second images are used to identity changes in the cell caused by the acetowhitening phenomenon.

2. The apparatus of claim 1, wherein the first and second images are used to identify abnormal growths.

3. The apparatus of claim 1 or claim 2, wherein the optical source is a laser.

4. The apparatus of claim 3, wherein the laser is a femtosecond titanium sapphire laser.

5. The apparatus of any preceding claim, wherein the optical elements include guiding optics to take the first excitation wavelength through a first optical path to the sample and then to the first photodetector.

6. The apparatus of any preceding claim, wherein the optical elements include guiding optics to take the second excitation wavelength through a second optical path to the sample and then to the second photodetector.

7. The apparatus of any preceding claim, wherein the first excitation wavelength produces a third harmonic generation image.

8. The apparatus of any preceding claim, wherein the second excitation wavelength produces a two photon exited fluorescence image.

9. A method of simultaneous multimodal nonlinear imaging of a cell sample to identify changes caused by the acetowhitening phenomenon; the method comprising tuning a source of illumination to a predetermined wavelength range; splitting the illumination into a plurality of excitation wavelengths; illuminating the sample with the plurality if excitation wavelengths; capturing a first image from a first photodetector related to a first excitation wavelength; and capturing a second image from a second photodetector related to a second excitation wavelength; wherein the first and second images are used to identity changes in the cell caused by the acetowhitening phenomenon.

10. The method of claim 9, further comprising using the first and second images to identify abnormal growths.

11. The apparatus of claim 9 or claim 10, further comprising illuminating the sample with a laser.

12. The apparatus of claim 11, wherein the laser is a femtosecond titanium sapphire laser.

13. The apparatus of any of claims 9 to 12, further comprising guiding a first excitation wavelength through a first optical path to the sample and then to the first photodetector.

14 The apparatus of any of claims 9 to 13, further comprising guiding a second excitation wavelength through a second optical path to the sample and then to the second photodetector.

15. The apparatus of any of claims 9 to 14, wherein the first excitation wavelength produces a third harmonic generation image.

16. The apparatus of any of claims 9 to 15, wherein the second excitation wavelength produces a two photon exited fluorescence image.

17. A method of using the apparatus of claims 1 to 8 to bio-image an abnormal growth.

18. An apparatus substantially as hereinbefore described with reference to the accompany drawings.

19. A method substantially as hereinbefore described with reference to the accompany drawings.