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Abstract
In this manuscript, we describe the development of a single shot, self-referencing wavefront division, multiplexing digital holographic microscope employing LED sources for large field of view quantitative phase imaging of biological samples. To address the difficulties arising while performing interferometry with low temporally coherent sources, an optical arrangement utilizing multiple Fresnel Biprisms is used for hologram multiplexing, enhancing the field of view and increasing the signal to noise ratio. Biprisms offers the ease of obtaining interference patterns by automatically matching the path length between the two off-axis beams. The use of low temporally coherent sources reduces the speckle noise and the cost, and the form factor of the setup. The developed technique was implemented using both visible and UV LEDs and tested on polystyrene microspheres and human erythrocytes.
© 2022 Optica Publishing Group under the terms of the Optica Open Access Publishing Agreement
1. Introduction
Digital Holographic Microscopes provides thickness distribution of living cells, which the conventional brightfield microscopes fails to provide [1–9]. It also provides important bio-physical and bio-mechanical information of living cells based on their morphology and its time variation [2,3,7–9]. These parameters could act as discriminators in identification and classification of cells, which may lead to disease diagnosis [4,5,9].
Digital Holography usually employs a laser source for the ease of obtaining interference fringes and arrangement of optical components. But laser being a high coherent source, induces various unwanted effects such as formation of parasitic interference fringes due to multiple reflection/transmission from different location of the system and speckle noise due to scattering [10–12]. The signal to noise ratio can be improved by controlling the spatial coherence of the laser source with the use of a moving diffuser to tailor the spatial coherence according to the requirement but it becomes impractical to have a miniaturized and portable setup with high temporal stability with this arrangement which is essential while examining the dynamic properties of the sample. Use of a partially coherent source may solve the problem of coherent artefact noise [13,14] in Digital Holographic Microscopy, but due to the low temporal coherence of the source, it becomes extremely difficult to use it in the case of two beam off-axis geometries, as it becomes difficult to adjust the path length differences to produce high contrast fringes over a large field of view [15]. For the ease of producing high contrast fringes over the full field of view there are various common path self-referencing geometries which can be used as well as it provides high temporal phase stability [4,8,16–23]. The low temporal coherence of the source can be utilized using certain specific self-referencing geometries while the spatial coherence of LED source can be enhanced by decreasing the lateral extent of the effective emitting area by using a spatial filter assembly. However, it can appreciably reduce the operational intensity as there is a trade-off between the pursued degree of coherence and the usable intensity [14].
The use of Fresnel Biprism for performing phase contrast microscopy by digital holography employing a high coherent source is presented by V. Singh et al. [24] and S. Ebrahimi et al. [25], which used laser sources as well as spatial filtering or sparse object distribution to generate a separate reference beam. However, it is difficult to use low coherent sources, which offers many advantages over laser sources like higher signal to noise ratio and exotic wavelengths, in holographic imaging owing to their lower coherence length leading to lower field of view (regions where superposition of object and reference waves give rise to interference fringes). To address the difficulties arising while performing interferometry with low temporally coherent sources, an optical arrangement utilizing multiple self-referencing modules for hologram multiplexing, enhancing the usable field of view and increasing the signal to noise ratio can be used. Here we report the development of a LED based single shot, self-referencing wavefront division, large field of view digital holographic microscope, using the multiplexing aspect of parallelly placed Fresnel biprisms, which acts as self-refencing interferometer modules. Spatial multiplexing has been demonstrated using grating for interferometric microscopy, where multiple duplicate images of the same sample is formed on imaging sensor [26,27]. On the contrary, in the presented work multiple interference regions containing information about different samples are formed on the same sensor array, allowing imaging of large number of samples in a single shot, increasing the field of view. The large field of view is because of simultaneous recording of multiple spatially separated small field of view self-referencing holograms on the same imaging array. Also, in the presented device, for interferometric imaging, the spatial coherence of the low coherent source is enhanced without affecting the output intensity, by using a microscope objective lens which reduces the effective emitting area of the source and increases the spatial coherence area at the sample plane. The developed device required only few optical elements and was converted into a field-portable prototype. It is tested on polystyrene microspheres and red blood cells using a visible as well as a UV LED.
2. Experimental setup
The schematic of the of the microscope setup is as shown in Fig.1a. LED (Lumilux, λ=627nm, maximum power output of 2W, Δλ=20nm, Coherence length∼8.7µm) with emitting area 1mm2 is employed as a source of light. This size of the source is reduced by creating a demagnified real image of it with the help of 10x Microscope Objective lens (MO) to increase the spatial coherence area of the source. The de-magnified source illuminates the sample (a thin smear of human RBC on a microscope slide) which is imaged using a microscope objective (20X, 0.40NA). The diameter of the area of spatial coherence is 450 µm at the sample plane. A pair of Fresnel biprisms (176°) with the same specification is placed which splits the incoming light and recombines it to form multiple self-referencing holograms on the same sensor (Fig. 1(b)). These holograms are recorded using a CCD sensor (Thorlabs, 8bit dynamic range, 4.65 µm pixel pitch) at the rate of 10z for 30s. Supplementary Figures S1 and S2 provides details about the table top version of the microscope.
Fig. 1. (a) Schematic of the Fresnel Biprism interferometer employing LED. (b) Multiplexing of holograms. (c) Portable 3D printed device.
The use of biprism allows path length matching leading to high contrast fringes even while using LED sources. In Digital holographic microscopy, the field of view (from which phase information can be extracted) is determined by the region in which interference pattern exist. For off-axis, low coherence digital holographic microscopy field of view, is limited by the temporal coherence (coherence length) of the source [15]. For a source with low coherence length, interference fringes will result only when the path length difference between the object beam and the reference beam is less than the coherence length of the source (Fig. 2). In the case of off-axis Digital Holographic Microscopy, which offers single shot phase retrieval capability, the angle between the object and reference wavefronts should be high enough to generate interference patterns with enough fringe density to separate out the spectral information of different components (undiffracted component, virtual image, and real image) (Fig. 2). Larger angle between the wavefronts results in higher fringe density suitable for single shot phase retrieval. However, it also results in lesser area in which interference fringes appear as the path length difference reaches the critical value (equal to or more than coherence length) faster (Fig. 2). In the proposed setup the use of a pair of parallelly placed biprisms increase the field of view, by creating two spatially separate holograms (multiplexed interference patterns) of different sample regions on the same sensor array (Fig. 2 and Fig. 1(b)). The CCD array records two holograms simultaneously. This is equivalent to having two separate self-referencing field of views on the same sensor array.
Fig. 2. Creation of holograms (interference patterns) in Self-referencing digital holographic microscope using LED sources. Larger angle between the object and reference wavefronts leads to higher fringe density suitable for single-shot phase retrieval, but results in smaller area in which interference fringes appear. Field of view is increased by the simultaneous use of two self-referencing components (biprisms), which results in creation of two holograms of different regions of the sample on the same sensor array (hologram multiplexing).
Figure 3(a). shows the holograms recorded using the proposed microscope without sample (only microscope slide as the object) using a single biprism whereas Fig. 3(b). shows the hologram recorded using two biprisms. It is quite evident that the field of view is increased using set of two parallelly placed biprisms. With the use of a single biprism the interference fringes covered 1024×320 pixels of the camera sensor and with the use of two biprisms the interference fringes covered 1024×566 pixels in the camera equivalent to 73.7% of the camera pixels. This is similar to the values reported in the literature [15], where an AOTF was used as the low coherence source. It should be noted here that the technique can be extended to larger sized arrays, by using more than two biprisms.
Fig. 3. (a) Hologram recorded with a single biprism. (b) Hologram recorded with a two biprisms (Multiplexed holograms).
The recorded holograms are reconstructed by the numerical implementation of angular spectrum diffraction integral [1], leading to computation of the complex amplitude distribution of the wavefront interacting with the sample. The sample phase information can be extracted from the complex amplitude and used for mapping of sample thickness distribution. The recent boost in 3D printing [28] and the simplicity of the presented geometry combined have also facilitated the fabrication of a portable and sturdy 3D printed device. The developed 3D printed self-referencing quantitative phase microscope using biprisms is shown in Fig. 1(c). Supplementary figures S3 to S5 provides details about the 3D printed version of the microscope.
3. Results and discussion
3.1 Spatial stability
It is expected that the spatial stability, which depends upon variation in background phase distribution to be improved due to the use of a low temporally coherent LED source, leading to the smoother background and lower speckle noise. The standard deviation of the optical path length/phase variation acts as a quantifier of spatial stability. Holograms using both Laser (He-Ne, λ=632.8nm, power = 0.8mW, Δλ=0.0012nm) and LED (λ=627nm) were recorded. Figure 4(a) and 4(c) show the 3D rendering of the reconstructed optical path length distribution without any object in the field of view with a laser source and the LED source, respectively. The optical path length variation is higher for laser source compared to LED source. From the histograms (Fig. 4(b) and 4(d)) of the spatial variation of optical path length, it can also be seen that the spatial stability with LED source is more than that using laser source. The spatial stability (measure of signal to noise ratio) improves by more than 2.4 times in the case of LED source compared to laser source, which will lead to improved thickness measurement.
Fig. 4. Spatial Stability (a) Spatially varying optical path length using laser source (b) Histogram of the spatial thickness variation along with the standard computed standard deviation value for laser source. (c) Spatially varying thickness using LED source. (d) Histogram of the spatial thickness variation along with the standard computed standard deviation value for LED source. σ in the histogram represents the standard deviation of the optical path length.
3.2 Temporal stability
The temporal stability of the system was measured by recording a time series of holograms (10Hz, 30 s). Each hologram is numerically reconstructed and the phase distribution at each time instance is extracted. The standard deviation of the time variation of phase at each spatial location acts as the measure of fluctuation at that point. The spatial average of the fluctuation across the field of view acts as the spatial stability quantifier. Compared to a two-beam setup, which provides a temporal stability of 4-5nm over the period with vibration isolation, the presented common path setup provides higher temporal stability measured to be 0.91nm without the need of vibration isolation demonstrating that it is more immune to external mechanical noise as shown in Fig. 5. The measured temporal stability is comparable to those reported in literature [29–30].
Fig. 5. Temporal stability of the system. Histogram represents the measured counts for thickness fluctuation at each spatial point (standard deviation of time varying thickness). The mean of these values represents the temporal stability of the device. Inset shows the time varying thickness at a spatial point in the field of view.
3.3 Calibration of the developed system
The developed system is calibrated using polystyrene beads with 15µm diameter (refractive index no= 1.58) immersed in microscope oil (refractive index nr = 1.52). A reference hologram of the medium surrounding the object (oil) is also recorded to extract the object phase. The reference phase obtained from the hologram of the surrounding medium is subtracted from the phase obtained from the hologram recorded with the object in the field of view. The phase subtraction nullifies the phase that remained constant between the exposures (due to aberration etc.) bringing out the phase due to the object alone as shown in Fig. 6(a). This wrapped phase distribution is converted into continuous phase distribution using Goldstein branch cut phase unwrapping algorithm [6] and is shown in Fig. 6(b). This continuous phase distribution along with the refractive index values of the polystyrene bead and microscope oil provides the thickness distribution of the object [8] using equation $\Delta \phi = 2\pi (no - nr)h/\lambda$ as shown in Fig. 6(c). The cross-sectional profile shown in Fig. 6(d) shows that the reconstructed thickness variation is very close to the manufactured specified value. The measured thickness was 14.86 ± 1.04µm, indicating that the technique is suitable for accurate thickness measurement of transparent micro-objects. The lab setup used finite conjugate microscope objective lens for imaging (20X, 0.4NA). The sensor was kept 160mm from the collar of the objective lens. The system magnification also was calibrated using 15µm polystyrene microspheres to determine the experimental magnification of the system. It was found to be 20.35. For the field portable system, a DVD pickup lens of focal length 3.07mm was used for imaging. The sensor was kept 10cm from the imaging lens yielding a magnification of 31.6.
Fig. 6. (a)Reconstructed wrapped phase distribution of polystyrene micro-spheres. (b) Continuous phase distribution obtained after phase unwrapping. (c) Calculated thickness profile (d) Line profile of the thickness distribution
3.4 Quantitative phase imaging of human erythrocytes
After calibrating the system, the developed technique was employed for the quantitative phase imaging of RBC. A thin smear of human RBC is prepared on a microscope glass slide and holograms are recorded (at the rate of 10Hz for 30s) with and without RBCs in the field of view. The reference phase obtained from hologram containing only blood plasma is subtracted from the phase obtained with object in the field of view which brings out the phase due to the object alone, nullifying the constant phase between exposures. Figure 7(a) shows the hologram of the human erythrocytes recorded with red LED source. Quantitative phase images were obtained after phase subtraction (Fig. 7(b)). As explained previously, the region where interference fringes exist represents the field of view. This is identified from the hologram recorded with only the microscope slide (Fig. 3) as sample by examining its line profile along the direction of intensity change. A binary mask of the regions with interference fringes is then created excluding the region of overlap (central portion in the hologram). The continuous phase distribution obtained after phase subtraction is multiplied with the binary mask to create the phase profile of the sample and the phase at the non-zero regions of the binary mask is then stitched together to extract the final phase profile of the sample (Fig. 7(b)). Extracted phase image (Fig. 7(b)) was used along with constant average refractive index of red blood cells (1.42) and that of blood plasma (1.34) [31], to reconstruct the thickness profiles of the red blood cells shown in Fig. 7(c). The cross-sectional thickness profile of the red blood cell is shown in Fig. 7(d). From the reconstructed phase profile, the measured diameter of RBCs was 7.86 ± 0.95µm and the mean of the maximum thickness was 2.42 ± 0.43µm. Numerical focusing capability of the technique is demonstrated in Fig. 8, which shows the region inside the white rectangle in Fig. 7(b) for different propagation distances.
Fig. 7. Quantitative Phase imaging of Human erythrocytes using LED source and multiplexed holograms (Illuminating source – LED working at 627 nm). (a) recorded multiplexed hologram. (b) Quantitative phase image corresponding to the two fields of views marked in Fig. 7(a). (c) 3D rendering of thickness distribution of red blood cells inside the area of interest marked by the white rectangle. (d) Cross-sectional thickness profile of red blood cell along the dashed line in Fig. 7(b).
Fig. 8. Numerical focusing capability of the microscope. Reconstructed intensity profile (of the region inside the white rectangle in Fig. 7(a)) at (a) 15µm inside focus (b) at focus, (c) 15µm outside focus
3.4 Quantitative phase imaging using UV LED
The advantage of the technique is that the path length of the object and reference beams are automatically matched for all the multiplexed holograms. This makes the method ideal to be used along with short wavelengths, where the coherence length is further restricted. The use of short wavelengths also leads to higher lateral as well as axial resolution. In order to improve both the lateral and axial resolution in the system, we have explored the use of exotic UV wavelength (Luxeon UV LED, λ=385 nm, maximum power output of 425mW, Δλ=9nm, Coherence length∼7.2µm). Figure 9(a) shows the portion of the self-referencing hologram of red blood cells recorded with UV LED as the illuminating source. Figure 9(b) shows the optical thickness distribution of red blood cells obtained after numerical processing of the recorded hologram and phase subtraction.
Fig. 9. Quantitative Phase imaging of Human erythrocytes using UV LED source and multiplexed holograms (Illuminating source – LED working at 385 nm). (a) Portion of the recorded multiplexed hologram. (b) 3D rendering of thickness distribution of red blood cells obtained after numerical processing of the hologram.
The improvement in lateral resolution of the digital holographic microscope at UV wavelength was tested by recording holograms of USAF resolution targets (Thorlabs R1DS1P). Figure 10(a) shows the recorded hologram of the USAF resolution target using Red LED source. Figure 10(b) and 10(c) shows the portion of the holograms of the resolution target (Group 7, Element 6, Linewidth 2.2µm) recorded with LEDs working at 627nm and 385nm respectively (region inside the white rectangle in Fig. 10(a)). Figure 10(d) and 10(e) shows the reconstructed intensity profile obtained from the numerically processed holograms. Figure 10(f) shows the line profile of the intensity variation along the solid lines shown in Fig. 10(d) and 10(e), indicating improvement in lateral resolution with the UV illuminating source. The resolution improvement is due to the use of short wavelength (385nm).
Fig. 10. Improvement in lateral resolution at shorter wavelengths. (a) Hologram of the resolution target recorded with Red LED source (b) and (c) Portions of recorded hologram (region inside the white rectangle in Fig. 10(a)) showing lines structures of Group 7, Element 6 of the resolution target, using 627nm and 385 nm LED sources respectively. (d) and (e) reconstructed intensity patterns for 627 nm and 385 nm LED sources. (f) Line profile of the intensity variation.
4. Conclusions
A single shot, off-axis, self-referencing, large field of view digital holographic microscope ideal for low temporally coherent sources is developed, exploiting the multiplexing properties of parallelly set Fresnel biprisms. Use of multiple biprisms, created multiple off-axis holograms on the same digital array, enabling us to examine large number of micro-objects in a single shot even with a low temporally coherent source. The spatial stability of the system is measured to be 1.92 nm in the case of LED as opposed to 4.68 nm in the case of Lasers which leads us to the conclusion that LED reduces the speckle noise and phase associated with high coherent source (due to parasitic interference) thereby enhancing the image quality of the reconstructed images. This noise reduction can also be seen from the reduction of contrast of the speckle pattern [32] from 0.193 in the case of He-Ne laser source to is 0.028 in the case of UV LED. This can also be seen from the contrast of the speckle recorded with the UV LED source The temporal stability of the system is found to be 0.91nm which enables accurate investigations of the dynamic events occurring in the biological cells. The device is also demonstrated for short wavelength source (UV LED at 385nm), where the technique was found to yield accurate 3D reconstructions of red blood cells. The use of short wavelength source also improves the lateral resolution of the system. From Fig. 3, it can be seen that contrast of the interference fringes is highest where the path length difference between the refence and object wavefronts are almost zero (central part of each individual field of view – See Fig. 2(b)). As one move away from the central portion, the path length difference approaches the coherence length and the contrast decreases leading to low contrast interference fringes. In digital holographic microscopy of phase objects, the phase information is stored as the modulation (shift) of the resulting interference patterns. Even low contrast fringes store the phase information of the object. In the developed method, phase information is extracted by subtracting the phase recovered using the reference hologram from phase recovered using object hologram. The spatial frequency component arising due to change in fringe contrast (slowly varying envelope) and diffraction effects gets nullified as they remain same for the object and reference holograms, thereby bringing out the phase due to the object alone, making it ideal for large field of view quantitative phase imaging of cells using exotic LED sources. The microscope can provide bio-physical and biomechanical properties of cells, which may be used for disease diagnosis. Moreover, the simplicity of the arrangement makes the microscope easy to implement, requires fewer components and thus has a small form factor. The device was converted into a compact, field portable, standalone device, that can be deployed in places where easy diagnostic is not available, by 3D printing its structure and then integrating the optical components. Since Fresnel biprisms offers automatic path length matching, the device might be useful even with very broadband sources including sunlight, by employing cheap bandpass filters. The technique is ideal for converting a clinical microscope, which utilizes a broadband illumination source into a holographic microscope by integrating the multiple self-referencing module.
Funding
Department of Science and Technology, Ministry of Science and Technology, India (DST-FIST, DST-PURSE); Board of Research in Nuclear Sciences (2013/34/11/BRNS/504); Science and Engineering Research Board (EMR/20l7/002724).
Acknowledgements
The work was supported by research grants SERB (EMR/20l7/002724), DAE-BRNS (2013/34/11/BRNS/504), DST-FIST and DST-PURSE. AA and VC would like to acknowledge Abdus Salam International center for Theoretical Physics (ICTP), Trieste, Italy for Regular Associate fellowship.
Disclosures
The authors declare that there are no conflicts of interest
Data availability
Data underlying the results presented in this paper are not publicly available at this time but may be obtained from the authors upon reasonable request.
Supplemental document
See Supplement 1 for supporting content.
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