Label-free microscopy of mitotic chromosomes using the polarization Orthogonality Breaking technique
R. Desapogu, G. Le Marchand, R. Smith, P. Ray, E. Gillier, S. Dutertre, M. Alouini, M. Tramier, S. Huet, J. Fade
LLabel-free microscopy of mitotic chromosomes using the polarization OrthogonalityBreaking technique
R. Desapogu, , , , G. Le Marchand , , R. Smith, , P. Ray, , E. Gillier, ,S. Dutertre, , M. Alouini, , M. Tramier, , , S. Huet, , , , ∗ , and J. Fade, , ∗∗ Univ Rennes, CNRS, IGDR (Genetics and Development Institute of Rennes), UMR 6290, F-35000 Rennes, France Univ Rennes, CNRS, Institut FOTON - UMR 6082, F-35000 Rennes, France Univ Rennes, BIOSIT, UMS CNRS 3480, US INSERM 018, F-35000 Rennes, France and Institut Universitaire de France
The vast majority of the microscopy methods currently available to study biological samplesrequire staining prior to imaging. Nevertheless, the ability to reveal specific cell structures or or-ganelles in a label-free manner remains desirable in different contexts. Polarization microscopy haslong been considered as an interesting alternative to fluorescence-based methods in order to gainspecificity on the imaged biological samples. In this work, we show how an original polarizationimaging technique, implementing micro-wave photonics and referred to as orthogonality-breaking(OB) microscopy, can provide informative polarization images from a single scan of the cell samplein a fast and sensitive way. For OB imaging, the sample is probed with a laser setup simultane-ously generating two orthogonal polarizations shifted in frequency by a few tens of MHz. If theimaged samples display some polarimetric properties, the orthogonality between the two polariza-tions is broken, leading to a beatnote interference signal that can be detected with a fast detector.The comparison of the images of various cell lines at different cell-cycle stages obtained by OBpolarization microscopy and fluorescence confocal images shows that an endogenous polarimetriccontrast arizes on compacted chromosomes during cell division. This technique paves the way tolabel-free real-time polarization imaging of mitotic chromosomes with further potential applicationsin histology and cancer diagnosis. ∗ [email protected], and [email protected] These authors contributed equally to this work a r X i v : . [ phy s i c s . b i o - ph ] O c t INTRODUCTION
Over the last decades, the relentless development of novel optical imaging techniques addressing specific issues ofthe biology or biomedical community has given rise to a huge number of unconventional imaging techniques. Severalof these were granted with worldwide commercial and scientific success such as Optical Coherence Tomography, ad-vanced confocal fluorescence microscopy, non-linear microscopy, phase imaging, etc. However, a number of interestingchallenges still remain open in this interdisciplinary research field. For some of these challenges, polarimetry caninspire novel solutions to overcome some of the remaining bottlenecks. For instance, the study of cellular biologi-cal mechanisms is overwhelmingly performed with confocal laser scanning fluorescence microscopy, which allows thestructures of interest to be imaged at high spatio-temporal resolution. Nevertheless, as for any fluorescence-basedmethod, confocal microscopy requires the labeling of the sample using either fluorescent compounds displaying spe-cific localizations whithin the cells, dyes coupled to antibodies or recombinant chimeric constructs composed of afluorescent protein fused to a protein of interest. Although this labeling step has proven to be usable in multiplecontexts, it also suffers some limitations. First, per se , it requires an additional, potentially lenghty, step in the sam-ple preparation, thus limiting the throughput of the experiments. Second, due to the crosstalk between the emissionspectra of the fluorescent dyes, it is often difficult to image simultaneously more than four different structures withinthe same sample. Finally, in the context of the observation of living samples, the labeling procedure may inducecytotoxicity and interfere with the tracked biological mechanisms. These different drawbacks justify the need for thedevelopment of label-free microscopy techniques. Among the techniques that are currently investigated (non-linearmicroscopy, quantitative phase microscopy, diffraction tomography. . . ), polarimetric approaches can provide multidi-mensional physical information on the samples, which could thus convey also some selectivity in order to identify cellconstituents [1–5], investigate the internal organization of biological structures [6–9] or discriminate unhealthy tissues[10–13] without the need for labeling. Most of the time however, polarimetric imaging is not given preference, due tothe poor sensitivity of most systems and to the complex calibration and lengthy acquisition procedures of standardpolarimetric techniques based on the sequential acquisition of several images[6, 14, 15].In this context, a direct, sensitive and fast polarimetric imaging technique, referred to as “orthogonality-breaking”(OB) polarimetric sensing has been proposed a few years ago [16–18]. It is based on a microwave photonics approach,and allows different polarimetric properties (birefringence, dichroism, and depolarization) to be identified from asingle sample scan using the appropriate detection modality [19]. In this paper, we report how such a technique hasbeen implemented on a commercial fluorescence confocal microscope set up. We demonstrate that this new imagingmodality can be used to observe mitotic chromosomes at different stages of the cell division with high contrast and atthe spatial resolution allowed by the microscope setup, i.e., approximately (cid:39)
150 nm within the focal plane. Being ableto monitor such cellular structures can prove useful to quickly identify proliferating cells within a biological sample orto identify cells displaying altered architecture of their genomic material, a feature that is shared by multiple tumorcells [20].
MATERIALS AND METHODSTheory
Principle of orthogonality-breaking polarimetric sensing
OB polarimetric imaging is based on the use of a specific dual-frequency dual-polarization (DFDP) laser illuminationto probe the sample. The frequency difference imposed between the two polarization components of equal intensitiesmust lie in the radiofrequency (RF) range, typically 10’s to 100’s of MHz, in order to match the typical bandwidth ofcommon photodetectors (PD) (photodiodes, avalanche photodiodes (APD),...), and avoid chromatic dispersion effectsduring light propagation in the setup and the sample [16]. It has been shown that the illumination polarization statesshould preferably be left/right circular [18], in which case the fast temporal evolution of the electric field producedcorresponds to a linear polarization state rotating at RF frequency. Due to the imposed orthogonality between the twocomponents oscillating at distinct frequencies, the intensity of such beam has a constant value, even when measuredwith a fast PD.When such a beam interacts with a sample, the intensity detected upon interaction with it remains constant, unlesssome polarization “orthogonality-breaking” takes place. In this configuration initially proposed in [16], such OB onlyoccurs when light interacts with a dichroic sample (absorption anisotropy): the detected intensity shows a fast RFtemporal modulation (beatnote), due to the interference of the two frequency components of the probe beam [18, 21].Interestingly, we showed previously that the normalized amplitude of the beatnote (ratio of the beatnote amplitude(AC) by the average intensity value (DC)), which we shall refer to in the following as orthogonality-breaking contrast(OBC) is a direct measure of the diattenuation rate of the dichroic sample [18, 21]. Moreover, the estimated phase ofthe detected beatnote is directly linked to the direction of the optical anisotropy (here, absorption anisotropy) whichis responsible for the OB phenomenon [18, 21].The main interest of such OB polarimetric modality is that the polarimetric information is retrieved from a singleacquisition/scan of the image, hence ensuring easy and fast implementation on existing imaging setups such asmicroscopes. However, the polarimetric information is retrieved from the analysis of a RF-modulated optical signal,which requires fast PDs and dedicated demodulation electronics (lock-in detection or quadrature demodulation board)to measure the in-phase (I) and in-quadrature (Q) signal components. The OBC and phase informations can finallybe retrieved using the following relations:
OBC = (cid:112) I + Q DC , and, ϕ = atan QI . (1)The requirement of high-speed detection/demodulation hence hinders the use of wide-field cameras, and the tech-nique so far has been implemented in a laser scanning imaging configuration, even for remote measurement applications[22]. For this reason, OB polarimetric sensing is well suited to be implemented on a confocal laser scanning microscopesetup, as will be shown in the following.
Dichroism/birefringence polarimetric imaging with induced-OB modalities
The standard OB imaging modality described above has a strong specificity since OBC contrast can only appearif the sample shows absorption anisotropy (diattenuation). This can be interesting for a number of applications.However, in biology where the samples of interest are rather transparent, dichroism is not the most likely to occuramong other polarimetric effects. To broaden the scope of application of this unconventional approach, it has recentlybeen shown that it is possible to gain sensitivity on other polarimetric effects, such as pure depolarization andbirefringence, by slightly modifying the detection setup [19].These complementary modalities have been referred to as “induced” OB as the RF beatnote carrying the polarimet-ric information is generated on an analyzing element placed ahead of the detector, after light has interacted with thesample. Two interesting modalities have been identified. The first one, called linear-induced OB (LI-OB), consists inusing a circular left/right DFDP illumination and a linear polarizer in front of the detector, and allows depolarizationcontrasts to be revealed on purely depolarizing samples [19]. The second modality, referred to as circular-inducedOB (CI-OB) uses also a circular DFDP illumination, but requires a circular analyzer in front of the detector. This iscommonly obtained by combining a quarter-wave-plate (QWP) and a linear polarizer with eigenaxes oriented at 45 ◦ from each other. This last modality has the strong potential to reveal interesting OBC contrast, not only on dichroicsamples, but also on birefringent samples [19]. In the remainder of this article, these three OB modalities will be usedand compared in terms of efficiency for polarimetric imaging of cell samples. Microscopy setup description
In this section, we describe how a standard laser scanning microscope (Leica TCS-SP2 inverted microscope setup)has been modified in order to handle OB polarimetric imaging in transmission, while maintaining the ability to performconfocal fluorescence imaging. This constraint was necessary to be able to overlay OB and fluorescence images andthus identify the cellular structures giving rise to the observed polarimetric contrasts. A sketch of the whole setup isgiven in Fig. 1.a.
Dual-frequency dual-polarization illumination
To perform polarimetric cell imaging in the visible range, we use a polarization-sensitive Mach-Zehnder based free-space architecture comprising an acousto-optic shift of 80 MHz in one of the two arms (acousto-optic modulator,MT80-A1-VIS, AA OPTOELECTRONICS). This setup is represented on the 3-D sketch of Fig. 1.b allows us toobtain a linearly-polarized DFDP beam from a 40 mW commercial blue laser (PC14584, NEWPORT, λ = 488 nm).The DFDP source has previously been extensively described in [17].In order to control the optical power deposited on the sample, a mechanically controlled optical valve has beendesigned using the association of a half-wave plate (HWP) (WPHSM05-488, THORLABS) and a Glan polarizer FIG. 1. (a) Sketch of the OB polarimetric microscope setup implemented on a standard fluorescence scanning microscope(Pol: polarizer, QWP: quarter-wave plate at 488 nm, APD: avalanche photodiode, BT: bias-tee, DFS: dual-frequency source,DM: dichroic mirror, PH: pinhole, PM: photomultiplier). (b) 3D-sketch of the dual-frequency dual-polarization laser sourceat λ =488 nm designed for the experiment (HWP: half-wave plate, PBS: polarization beam splitter, AOM: acousto-opticmodulator, GP: Glan polarizer). (c) Example of test images recorded from the fluorescence confocal microsope (Fluo), andfrom the polarimetric acquisition setup: the DC image provides the transillumination image, the I and Q channels are furtherprocessed to build the OBC and phase images displayed in next figures. inserted between the laser output and the Mach-Zehnder-like setup. The orientation of the Glan polarizer is adjustedto ensure that the intensity of the two components of the DFDP beam share equal intensities. The direction of theHWP is controlled by a rotating mount (URS50 + SMC100 controller, NEWPORT), allowing remote tuning of theoptical power from the LabView (NATIONAL INSTRUMENTS) program used to control the whole setup.In order to convey the DFDP beam into the microscope scanning head, we use a single mode polarization-maintaining optical fiber (P3-488PM-FC-2, THORLABS) whose eigenaxes have been aligned with the polarizationdirections of the DFDP beam. A mechanical shutter controlled by the LabView software is placed ahead of thefiber injection and avoids illumination of the sample before an acquisition is launched, so as to reduce fluorescencebleaching. Microscope setup
The DFDP beam conveyed by the fiber is coupled into the confocal microscope (TCS-SP2, LEICA) through theoriginal infrared port using a dichroic mirror (ZT488rdc, CHROMA, cut-off wavelength 498 nm), as sketched inFig. 1.a. This makes it possible to perform fluorescence and polarimetric imaging simultaneously, as the excitationlaser lines available in the confocal head (514 nm and 635 nm) can be used to illuminate the sample and the fluorescenceemitted in the backward direction can be detected through the confocal pinhole. The filter wheel of the invertedmicroscope stand has been equipped with a removable QWP at 488 nm (WPQSM05-488, THORLABS), allowing thelinear DFDP beam to be converted into a circular left/right DFDP illumination at the sample plane. Finally, the lightis focused onto the sample by the microscope objective. The images displayed in the following have been recordedwith one of the two following objectives: a 10 × objective for test images and polarimetric measurements on syntheticsamples (Fig. 2), whereas the images of cells have been obtained with an oil-immersion 63 × objective.Upon interaction with the sample, light is detected on the one hand in the classical confocal backscattering modefor the fluorescence emission, and the image reconstruction is handled by the TCS-SP2 system. On the other hand,the polarization information is measured in transmission by detecting the OB beatnote at 80 MHz on an avalanchephotodiode (APD) (Silicium, 400 MHz bandwidth, APD430A, THORLABS), through a custom optical arrangementcomprising the original microscope condenser lens ( f (cid:48) =28 mm), a switchable plane mirror, and an additional focusinglens ( f (cid:48) =17 mm) (See Fig. 1.b). This configuration enables full operation of the LEICA microscope in its originalmode, but hinders the ability of true simultaneous imaging since a slight tuning of the microscope focus must beensured when switching between confocal fluorescence imaging and polarimetric OB imaging.Finally, in order to implement the three OB modalities described above, removable polarization analysis elementshave been inserted after the sample, between the condenser lens and the last focusing lens. A linear polarizer allowsLI-OB to be performed, while CI-OB requires an additional QWP to be inserted before the polarizer, with eigenaxesoriented at 45 ◦ from the polarizer direction. Polarimetric data acquisition and image reconstruction
As described in the theoretical section, OB polarimetric imaging requires a specific detection/demodulation chainto recover the polarimetric information from the estimated amplitude and phase of a RF beatnote, at 80 MHz in thepresent case. For that purpose, a first homemade “bias-tee” electronic circuit separates the 80 MHz AC componentof the detect photocurrent from its continuous-wave DC component. As sketched in Fig. 1.a, the latter is directlysampled and digitized on the analog-to-digital conversion (ADC) module of a input/output board (NI-USB 6356,NATIONAL INSTRUMENTS, 16 bits, 1.25 MS/s per channel).As for the AC component, it can be demodulated either with a lock-in amplifier [17], or using a custom-made I/Qdemodulation circuit at the dedicated 80 MHz frequency. Here, we use the I/Q demodulation approach which hasbeen extensively described in previous work [22]. On our microscope setup, the RF reference (local oscillator) usedto demodulated the AC photocurrent is given by the 80 MHz RF signal that drives the acousto-optic modulator,with appropriate amplification to match the nominal operating point of the RF mixers (+7 dBm) involved in thedemodulation circuit. At the output of the I/Q demodulation circuit, the two quadratures (I and Q) are low-passfiltered (1 MHz cutoff frequency) and further amplified using two identical switchable gain voltage amplifiers (from × ×
34) so as to optimally match the input range of the ADC board which eventually samples and digitizes the Iand Q signals along with the DC component.The three digitized signals are then processed with a LabView programm on a computer to build the three rawpolarimetric images (DC, I, Q), from which the OBC amplitude and phase maps are computed using the relations ofEq. (1), taking into account the relative gain factors and input ranges of the three channels. Data acquisition andreconstruction of the images are triggered by the “frame” and “line” trigger signals from the LEICA bench whichare also digitized on the ADC board. In addition, to avoid photodamage or bleaching of the sample and to minimizeaging of the AOM, a +5V signal is output from the I/O board only when the acquisition is started, in order to openthe mechanical shutter of the DFDP source and to enable the RF high-voltage supply of the AOM.Using a 200 Hz scanning speed on the LEICA system allows polarimetric images of 256 ×
256 pixels to be recordedwithin 1.5 s. In order to cope with the acceleration/deceleration phases of the galvanometric mirrors and avoid spatialdistortion in the obtained images, the simplest way consists in acquiring an image format of 512 ×
256 pixels andremove by software the 128 first and last pixels from each line, ending up with distorsion-free 256 ×
256 pixels images.An example of raw DC, I and Q images recorded with a ×
10 objective on a stage micrometer is displayed in Fig. 1.c,along with the corresponding fluorescence image.For the sake of accuracy of the estimated OBC amplitude and phase maps, the I and Q images displayed in thefollowing figures and used to compute the OBC amplitude and phase values have been corrected by software fromslow phase drifts due to unwanted optical path changes in the DFDP source. For that purpose, we estimate the slowdrift on the quadrature images by evaluating the local average values of the I and Q signals at four locations aroundthe cell (typically, the four image corners), and by using a linear regression to calculate the slow linear trend of theI/Q signal across the image. This slow trend is finally removed to provide the I and Q images as those displayedin Fig. 3.c. Such post-processing steps could be avoided in future implementation of the system by removing theunwanted optical phase drifts in the system.
Sample cells preparation and fluorescence imaging
HeLa and U2OS cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10%fetal bovine serum (FBS), 100 µ g/mL penicillin and 100 U/mL streptomycin and maintainted at 37 ◦ C in a 5% CO incubator. Cells were seeded on coverslips 24 h prior to fixation. For DRAQ5 staining (U2OS cells), media wasremoved and cells were washed once with phosphate buffered saline (PBS) for 3 min at room temperature. Cells werefixed in 4% paraformaldehyde (PFA) for 15 min at room temperature and washed twice with PBS. Cells were stainedwith DRAQ5 (0.5 mM in PBS) for 15 minutes and washed three times with PBS before coverslips were mountedon slides using ProLong ® Gold (THERMO FISHER SCIENTIFIC). For microtubule staining (HeLa cells), mediawas removed and cells were washed once with PBS for 3 min, fixed with 4% PFA for 15 min and washed twice withPBS, all performed at room temperature. Cells were permeablised with 0.2% Triton X-100 in PBS for 5 min, washedtwice with PBS and placed in blocking buffer (5% BSA, 0.05% Tween-20 in PBS) for 60 min at room temperature.Cells were then incubated with anti-tubulin antibody (0.2 µ g/mL, sc ◦ C. Cells were washed three times with 0.1% Triton X-100 in PBS before incubation with Alexa Fluor488 anti-mouse IgG (2 µ g/mL, A11001, INVITROGEN) diluted in blocking buffer at room temperature for 1 h in thedark. Cells were washed twice with 0.1% Triton X-100 in PBS and counterstained with Hoechst (1 µ g/mL in PBS) for10 minutes. Cells were washed three times with PBS before coverslips were mounted on slides using ProLong ® Gold(Thermo Fisher Scientific). Fluorescence excitation of Alexa Fluor 488 and DRAQ5 were excited using 514 nm and633 nm lasers respectively. For fluorescence detection, we used bandpass filters adapted to the fluorophore emissionspectra.
RESULTS AND DISCUSSIONValidation on synthetic samples
Before applying the OB polarimetric imaging to cell samples, we validated the method and the setup on synthetictest samples. These samples were selected for their known anisotropic optical behaviour, such as dichroism andbirefringence, and the obtained images displayed in Fig. 2 are in good agreement with the theory presented above andin Ref. [19]. In this figure, the left column shows the transillumination image obtained with a ×
10 objective fromthe measured DC signal of the APD, while the center and right column respectively display the OBC amplitude andphase maps obtained from Eq. (1). The phase value being irrelevant when the AC amplitude is very low (i.e., lowOBC), the phase map has been thresholded and the irrelevant phase values have been represented in gray color.The images in the first two rows have been obtained on a dichroic sample, namely, the cut edges of a polaroid sheetdeposited on the stage micrometer surface, and exhibiting a rather shredded structure. The standard OB imagingmodality has been used in Fig. 2.a, whereas images of Fig. 2.b have been obtained with the circular-induced modality(CI-OB). As expected, the in-focus parts of the dichroic sample show strong OB contrast with the two OB modalities.Theoretically, the estimated phase provides an indication about the relative orientation of the absorption anisotropiesin the observed sample. The phase maps obtained here showing different values on the polarizer shreds are in fairagreement with the expected behaviour.The second synthetic sample tested corresponds to a piece of plastic (birefringent) tape sticked on the stage mi-crometer. The piece of tape is clearly visible in the upper-left corner of the transillumination image of Figs. 2.c and2.d. It has been imaged here with the standard OB (Fig. 2.c) and the CI-OB modalities (Fig. 2.d). Again, as expectedbased on the theory, such a birefringent sample does not break the polarimetric orthogonality, hence resulting in avery low OBC amplitude in standard OB. However, the birefringent nature of the sample clearly appears through thesignificant OBC amplitude obtained on the birefringent sample with the CI-OB modality. As for the phase maps, theconstant value of the phase estimated on the piece of tape is in good agreement with the expected uniform orientationof the phase anisotropy of such sample.These first imaging results on test synthetic samples thus validate the correct operation of the polarimetric OBmicroscope. It can be noted however here that the amplitude values of OBC (sometimes above 100%) lack quantitativeprecision. This would require added complexity of the system calibration and data processing [19, 23], but does notprevent qualitative evaluation of the imaging data gathered on biological samples and presented below.
OB polarimetric observations in cells
We imaged human osteosarcoma U2OS cells using the confocal fluorescence modality of the TCS-SP2 Leica system,and compared the fluorescence images with the transillumination (DC) and polarimetric images (OBC amplitude and
FIG. 2. Examples of OB polarimetric images recorded with a 10 × objective on synthethic samples. Left column: DC-transillumination image; Center: OBC amplitude map; Right: OBC phase map. (a): standard OB modality on the edge ofa dichroic sample (tears of polaroid sheets); (b): Circular-induced OB modality on a similar sample to (a); (c): standard OBmodality on cuts of a birefringent sample (plastic tape); (d): Circular-induced OB modality on the same sample as in (c). phase maps) acquired using the method and system described above. An interesting outcome of these experiments isthe observation of an intrinsic polarimetric OB contrast in mitotic cells. An example of such acquisition is displayedin Fig. 3, where a U2OS cell, stained with DRAQ5 for DNA fluorescence labeling, has been imaged with the confocalfluorescence microscope (Fig. 3.b), and with the three OB modalities (Fig. 3.c). The spatial distribution of thechromosomes within this cell as seen on the fluorescence image suggests that it is at an early mitotic stage, mostprobably prometaphase. The analysis of the raw polarimetric images (I and Q) shows that a moderate OB contrast canbe observed in standard OB imaging modality (first raw of Fig. 3.c). Such OB contrast seems to arise at compactedchromatin, as shown by the fact that the spatial distribution of the OBC signal (right column in Fig. 3.c) resemblesthe fluorescence image. The linear-induced OB modality (LI-OB) is inefficient to reveal an interesting constrast in thisstructure (middle row), as could be expected from such a thin and transparent sample for which light depolarizationinduced by multiple scattering events during light propagation is unlikely. In contrast, the circular-induced modality(CI-OB), which is able to reveal contrast on birefringent samples, displays the strongest I and Q quadrature signalcontributions, and makes it possible to retrieve a clear OBC amplitude map (lower row, right) with high signal-to-noiseratio. For this reason, we shall restrict ourselves in the following to this CI-OB modality, which seems best adaptedto the imaging of compacted chromatin in mitotic cells. FIG. 3. Fluorescence and OB polarimetric images of a U2OS cell whose DNA has been labeled with DRAQ5. Images wereobtained with a 63 × oil-immersion objective. (a) Transillumination image; (b) Fluorescence image; (c) OB polarimetric imageswith I/Q channels and OBC amplitude maps for standard OB modality (upper row); linear-induced OB (middle row) andcircular-induced OB (lower row). Label-free polarimetric contrast of mitotic chromosomes
The clear CI-OB contrast obtained in a dividing U2OS cell such as presented in Fig. 3 is very encouraging towardsthe possibility to identify mitotic chromosomes from an endogenous polarimetric contrast, without the need forfluorescence labeling. It was however important to verify that such contrast was not specific to this cell line andnot due to the DRAQ5 labeling of the DNA. Moreover, a more thorough analysis was required to confirm that thepolarimetric contrast colocalizes with the mitotic chromosomes.For this purpose, we performed some additional acquisitions in two types of cells: U2OS cells, described in theprevious section, and HeLa cells, a widely used human cancer cell line derived from cervix. These cells were labeledeither with DRAQ5, to highlight the DNA (U2OS cells), or with an anti-tubulin antibody, to display the microtubulenetwork (HeLa cells). We imaged cells undergoing mitosis in both OBC and fluorescence modalities and the twoimages were overlapped to be able to assess the colocalization between the two signals. The results are displayedin Fig. 4. It can be readily observed in Fig. 4.g that the OBC amplitude map overlaps very well with the strongfluorescence signal emitted by the DRAQ5 dye labeling the chromatin. When imaging a dividing HeLa cell whichmicrotubules were tagged by immunofluorescence, we observed in Fig. 4.g the overlay between the OBC amplitude(in green) and the fluorescence (in red) signals, a characteristic metaphase spindle composed of microtubules (seenin fluorescence) handling mitotics chromomes (seen in the polarimetric channel). This last result demonstrates that
FIG. 4. Comparison of transillumination (a-b), CI-OBC images (c-d) and fluorescence (e-f) acquired on U2OS cells labeled withDRAQ5 to highlight the DNA in fluorescence (upper row), and on HeLa cells with microtubules labeled by immunofluorescence(lower row). In each case, the superimposition of the fluorescence and OBC amplitude images (g-h) shows clear colocalizationbetween the OBC contrast and the compacted chromatin in mitotic cells. The two insets (i-j) show two examples of phasemaps extracted from the OB images revealing additional morphological contrasts on the compacted chromatin. the polarimetric contrast observed for the mitotic chromosomes is not specific to a single cell type and is not due toDNA labeling by DRAQ5, hence paving the way to label-free imaging of mitotic chromosomes in dividing cells.In addition to the OBC maps whose amplitude seems to be related to chromatin compaction, the OB polarimetrictechniques provide additional information by analyzing the estimated phase of the OB signal at each pixel of the image[19]. Two examples of phase maps (estimated only on pixels showing significant OBC amplitude) are displayed for aU2OS cell (Fig. 4.i) and a HeLa cell (Fig. 4.j). Interestingly, these two phase images reveal additional morphologicalinformation in the compacted chromatin that can be observed neither in the OBC amplitude maps nor in the fluores-cence images. Further investigation is required to possibly relate such observations with morphological/organizationalstructures in compacted chromosomes [24].As a last experiment to confirm the interest of such endogenous polarimetric contrast in condensed chromatin, weprovide in Fig. 5 a comparison of the transillumination (upper row), OBC amplitude (middle row) and fluorescence(lower row) images of U2OS cells at different cell stages. From left to right, the analysis of fluorescence images allowsus to identify cells in interphase (a), late prophase (b), prometaphase (c), late prometaphase (d), metaphase (e), andfinally late telophase (f). This figure shows that the polarimetric contrast seems to only arize at compacted chromatinin cells undergoing mitosis, while no clear OB contrast could be observed in interphase cells (Fig. 5.a). On a morequantitative basis, the magnitude of the OB polarimetric contrast seems to follow the chromatin compaction duringthe mitotic process, suggesting that OB imaging could be used to monitor the chromatin compaction state. This isconfirmed by the graph displayed at the bottom of Fig. 5. We plotted the ratio of the OBC amplitude averaged overthe regions of interest (ROIs) highlighted in blue, by its mean value in the surrounding background. The selected ROIscorrespond to the regions where labeled DNA can be identified in the fluorescence images. With this definition of a“contrast” ratio, the minimum value of 1 corresponds to the absence of any contrast with respect to the background.0
FIG. 5. Transillumination, fluorescence and OBC amplitude (circular-induced OB) images of DRAQ5 labeled U2OS cellsat different cell stages: (a) interphase; (b) late prophase; (c) prometaphase; (d) late prometaphase; (e) metaphase and (f)late telophase. Bottom plot: evaluation of the ROI-to-background contrast of the averaged OBC amplitude on the regionsdisplaying DNA in the fluorescence images (blue ROIs).
CONCLUSION
In this article, we have shown that polarized microscopy using “orthogonality breaking” approaches could providevaluable label-free information in biological samples. More specifically, it has been shown that an endogenous polari-metric “circular-induced” OB contrast could be clearly obtained at mitotic chromosomes during cell division, whichhas been confirmed by colocalization with fluorescence images recorded on the same samples. As the polarimetricmodality used in this study is very fast, only requiring a single scan of the sample, this technique has the potentialto allow real-time live cell imaging to monitor chromosome dynamics during mitosis. In this context, modifying themicroscope setup to enable imaging in a reflection configuration would ensure strict simultaneous fluorescence andpolarization live-cell imaging.Applying this label-free technique to study the chromatin compaction state could open promising perspective forhistology studies, such as the identification of abnormal chromatin compaction arising in some cancers cells [25, 26].Further investigation is being conducted to assess the interest of this approach for imaging other cellular samples suchas embryos [27, 28], or biological tissues. This next experimental work will also address the thorough and rigorouscalibration of the setup in order to provide more reliable quantitative assessments of the estimated OBC amplitudeand phase values, which can be of great interest to investigate the interesting morphological structures revealed bythese two complementary contrasts.
AUTHORS CONTRIBUTIONS
M.T., M.A., S.H. and J.F. designed the research. M.A. and J.F. designed the laser source as well as the I/Qdemodulation RF-setup. M.T., S.D., E.G., G.L.M. and J.F. designed and assembled the microscope setup. G.L.M.,E.G., P.R. and J.F. designed the experiment control & data acquisition program. R.D., R.S., G.L.M. and S.H. prepared1the cell cultures and samples. R.D., R.S., G.L.M. and J.F. performed the experiments. J.F. and S.H. performed thedata analysis and interpretation. J.F. and S.H. wrote the paper. All the authors revised the manuscript.
ACKNOWLEDGMENTS
The authors would like to acknowledge N. Ortega-Quijano, F. Parnet for their early contribution in assessingthe CI-OB modality, and to thank L. Frein, S. Bouhier, C. Hamel, and A. Carr´e for their technical help with theelectronical and mechanical design of the experiments. The authors thank C. Chapuis for her technical assistance insample preparation and X. Pinson for his technical help with the confocal microscope setup. The authors thank theMRic facility from the BIOSIT joint unit of services, the University of Rennes 1 and the CNRS “Mission pour lesInitiatives Transverses et Interdisciplinaires” (MITI) for funding this project. R.D. was supported by the Universit´eBretagne Loire, the R´egion Bretagne and the Institut Universitaire de France. R.S. was supported by the PRESTIGEprogram coordinated by Campus France [PRESTIGE-2017-2-0042], the Universit´e Bretagne Loire and the FondationARC pour la recherche sur le cancer [PDF20181208405]. We thank the GDR Imabio for funding P.R.’s and E.G.’sinternships. The MRic facility is member of the national infrastructure France-BioImaging supported by the FrenchNational Research Agency (ANR-10-INSB-04). [1] Oldenbourg, R., 1996. A new view on polarization microscopy.
Nature
Methods in Cell Biology
Annual reviewof cell and developmental biology
Micron
Opt. Lett.
Journal ofBiomedical Optics
Advances in Optics and Photonics
Journal of Biomedical Optics
Journal of Biomedical Optics
Appl. Opt.
Optics and Spectroscopy
Optics Express
Journal of biomedical optics
Journal ofBiophotonics
Biophysical Journal
Physical Review Letters
Journal of Optics
Optics letters
Optics Letters
Nature reviews cancer
Journal of the Optical Society of America A [22] Parnet, F., J. Fade, N. Ortega-Quijano, G. Loas, L. Frein, and M. Alouini, 2017. Free-space active polarimetric imageroperating at 1,55 µ m by orthogonality breaking sensing. Optics Letters In UnconventionalOptical Imaging II. International Society for Optics and Photonics, volume 11351, 113511E.[24] Kireeva, N., M. Lakonishok, I. Kireev, T. Hirano, and A. S. Belmont, 2004. Visualization of early chromosome condensation: a hierarchical folding, axial glue model of chromosome structure .
Journal of Cell Biology
Frontiers inGenetics
Scientific reports
Reproduction