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PLOS One logoLink to PLOS One
. 2023 Sep 8;18(9):e0290300. doi: 10.1371/journal.pone.0290300

Optimizing the internal phase reference to shape the output of a multimode optical fiber

Liam Collard 1,2,*, Linda Piscopo 1,3, Filippo Pisano 1,4, Di Zheng 1, Massimo De Vittorio 1,2,3,‡,*, Ferruccio Pisanello 1,2,‡,*
Editor: Yuan-Fong Chou Chau5
PMCID: PMC10490902  PMID: 37682976

Abstract

Pre-shaping light to achieve desired amplitude distributions at the tip of a multimode fiber (MMF) has emerged as a powerful method allowing a wide range of imaging techniques to be implemented at the distal facet. Such techniques rely on measuring the transmission matrix of the optically turbid waveguide which scrambles the coherent input light into an effectively random speckle pattern. Typically, this is done by measuring the interferogram between the output speckle and a reference beam. In recent years, an optical setup where the reference beam passes through the MMF has become an attractive configuration because of the high interferometric stability of the common optical path. However, the merits and drawbacks of an internal reference beam remain controversial. The measurement of the transmission matrix is known to depend on the choice of internal reference and has been reported to result in “blind spots” due to phase singularities of the reference beam. Here, we describe how the focussing efficiency of the calibration can be increased by several percent by optimising the choice of internal reference beam.

Introduction

Wavefront shaping has emerged as a powerful method to control the transmission through a multimode fiber (MMF) allowing the full modal diversity of the waveguide to be exploited [19] By pre-shaping the light prior to transmission, reconfigurable focussed spots can be generated at the distal facet of the fiber. This has enabled a plethora of imaging techniques at the tip of the fiber including in-vivo fluorescence [10, 11], Raman [12, 13], CARS [14], second harmonic generation [15] and also more complex photonic techniques such as optical tweezers [16], activation of plasmonic nanostructures [17] and also through multicore fibers [1822]. Recently, a new generation of MMF based endoscopes capable of imaging objects distal from the fiber have been developed by shaping the output light in the Fourier plane [23, 24].

All these techniques are reliant on the ability to accurately measure the transmission matrix of the waveguide. Typically, this is done by splitting the beam prior to transmission through the fiber to create an external reference beam, which is re-joined to the beam passing through the fiber after the fiber output, to create an interference pattern. The phase difference between each output mode and the reference can then be measured and re-modulated to generate constructive interference. The result is a set of diffraction limited foci scanning the output plane. The use of external reference beam has achieved considerable success, with one caveat being the need of a highly stable interferometric setup and optical alignment. Therefore, as the field develops and more complex multispectral imaging modalities, simplified transmission matrix measurements methods are of keen interest. In this respect, “reference free”, phase retrieval techniques [25, 26] and machine learning algorithms [27, 28] have both emerged as elegant alternatives, as they require no kind of interferomic measurement however they generally require larger datasets/more complex computational techniques and are generally less accurate than interferomic measurements

An internal reference beam passing through the core of the fiber is an interesting alternative. Such a system is intrinsically reconfigurable, provides high interferometric stability and is a simpler optical setup due to the common optical path. Indeed, recent works on distal imaging endoscopes have employed internal reference beams for such reasons [29]. However, it has been demonstrated that the use of an internal reference beam results in blind spots at the output due to vortex singularities in the reference beam and can also lead to an uneven focussing efficiency over the fiber core [29]. A proposed solution to this has been to use multiple internal reference beams, at the price of a greater computational complexity [30]. Thus, the merits and drawbacks of internal reference beams remain controversial. In this work we aim to provide a detailed assessment of both surface and Fourier plane calibrations (where the light is focussed on the distal facet or gets collimated in a beam of low divergence emerging from the distal facet, respectively) performed with single and serialised internal reference beams, striving at optimizing the relative focussing efficiency of the system.

Experimental methods

Optical system

The employed wavefront shaping setup is shown in Fig 1A. A continuous wave 633 nm beam was expanded by a telescope formed of lenses L1 and L2 to overfill the screen of a spatial light modulator (SLM) (ODP512, Meadowlark optics). Prior to this, the polarisation was rotated by a half wave plate for optimal modulation. The screen of the SLM was conjugated with the back aperture of microscope objective MO1 (0.65 NA, 40x, AMEP4625, ThermoFisher) by a 4f system comprised of lenses L3 and L4. MO1 focused the modulated light onto the proximal facet of the MMF (0.22 NA, 50 μm core diameter, Thorlabs FG050UGA). The numerical aperture of MO1 was significantly higher than that of the MMF. This was done so as the number of pairs (jx, jy) should roughly match the number of modes carried by the fiber [2]. The number of modes supported by the fiber is given by M=V22 where V=2πrλNA=2×π×250.633×0.22 = 54.59. Thus, M≈1490 (or 745 per polarisation). Therefore, the minimum lateral resolution at the input should be 2rMp=50μm27.3=1.83um. Meanwhile, Abbe’s diffraction formula for lateral (XY) resolution calculated with MO1 is d=λ2NA=.6332*0.65=0.48um. The transmission efficiency from MO1 to the MMF is approximately 30%”.

Fig 1.

Fig 1

A) Optical system for shaping light at the tip of a MMF using an internal reference beam. HWP- half wave plate, L- lens, SLM- spatial light modulator, M- mirror, BS- beam splitter, MO—microscope objective, MMF–multimode fiber, CCD- charged coupling device. The four focal planes (uin, vin), (xin, yin), (xout, yout), (uout, vout) correspond to the screen of the SLM, proximal fiber facet, distal fiber surface plane, distal Fourier plane and are indicated by the purple, blue, green and brown dashed lines respectively. B) Illustration of the three calibration techniques applied in this work utilising i) position of single internal reference ii) serialised internal references with optimized positions iii) an external reference.

For alignment purposes, the reflection of the input facet was monitored on CCD1. The fiber transmission was collected by MO2 (10x, 0.3 NA, MPLFLN10x—Olympus) and guided onto CCD2 (monitoring the distal fiber facet) and CCD3 (monitoring the transmission in the Fourier plane). The surface of the proximal and distal facets are defined as (xin, yin) (xout, yout) and the Fourier plane of each facet (uin, vin) (uout, vout) respectively.

Calibrating with single or serial internal references

Prior to the calibration, sawtooth gratings ϕscanjx,jy,p(uin,vin)=mod(auin+bvin+p,2π) and ϕref(uin,vin)=mod((cuin+dvin),2π) were defined, where a and b set the periodicity of the grating along xin, and yin so the beam Bscanjx,jy,p raster scans a 25 × 25 grid overlaying the input facet of the MMF indexed by (jx, jy). c and d were chosen so that the reference beam Bref was fixed. p represents the phase shift applied to the scanning points incremented in steps of π2 (for a total of 4 phase steps). ϕinterferejx,jy,p(uin,vin)=arg(exp(iϕscanjx,jy,p(uin,vin))+exp(iϕref)) could then generate both Bscanjx,jy,p and Bref simultaneously. Both sets of ϕinterferejx,jy,p and ϕscanjx,jy,p were saved as.bmp files prior to the calibration and ϕscanjx,jy,p was loaded onto the graphics processing unit (GPU—NVIDIA GeForce GTX 960) [23].

The first part of the calibration consisted of measuring the transmission of Binterferejx,jy,p(uin,vin) through the MMF on the Surface Plane (SP) and Fourier Plane (FP) of the output facet, hereafter referred to as fSP(Binterferejx,jy,p) and fFP(Binterferejx,jy,p), for each value jx, jy, p at a 30 × 30 set of points in both output planes (xout, yout), (uout, vout) which were measured on CCD2 and CCD 3 respectively. “To acquire a single speckle pattern during the calibration step, the total exposure time of the camera was 72 ms and the SLM was given 150 ms to refresh between each step to ensure no cross-talk. For each pair (jx, jy), the phase shift p generating the maximum intensity of fSP(Binterferejx,jy,p) or fFP(Binterferejx,jy,p) at each of the 30 × 30 pixels in the (xout, yout), (uout, vout) planes, denoted, popt,SPjx,jy,(xout,yout) and popt,FPjx,jy,(uout,vout), respectively, is stored. Thus, for each point in the surface or Fourier plane a phase modulation pattern was generated to maximize the intensity at each targeted pixel:

ΦSP(xout,yout)(uin,vin)=arg(jx=125jy=125exp(iϕscanjx,jy,popt,SPjx,jy,(xout,yout)(uin,vin))) (1)
ΦFP(uout,vout)(uin,vin)=arg(jx=125jy=125exp(iϕscanjx,jy,popt,FPjx,jy,,(uout,vout)(uin,vin))) (2)

This computation was then completed on the GPU for all the output foci in both SP and FP. This computational step took approximately 5 minutes for all 900 phase masks. Thus the total time for a single internal reference measurement was approximately 14 minutes. In principle it can completed faster by taking the inverse Fourier transform of the measured transmission matrix popt,SPjx,jy,(xout,yout) or popt,FPjx,jy,(xout,yout).

Serial internal reference beam calibrations were performed by calibrating multiple times with the internal reference beam focused on different regions of the input facet of the fiber, modulating the c and d parameters. The process is illustrated in Fig 1B whereby Bscanjx,jy,p raster scans the input core and Bref is indexed by jref(where 1<jref<Nref and Nref is the total number of reference beams). In this case, the measurement is governed by 4 indices jx, jy, jref, p resulting in a set of optimized phase shifts popt,SPjx,jy,jref,(xout,yout), and phase masks ΦSP(xout,yout,jref)(uin,vin). Then, the index jrefopt and ΦSP(xout,yout,jrefopt)(uin,vin) giving the highest intensity at (xout, yout) was selected and ΦSP(xout,yout,jrefopt)(uin,vin) was stored. Serialized reference calibrations were performed exclusively in the surface plane.

Results and discussion

Firstly, the impact of the choice of internal reference beam was analysed for calibrations at the output facet’s surface plane. Four calibrations were performed with the reference beam focussed on a different sub- region of the input facet of the fiber. The reference beams Brefi (imaged on CCD1) were evenly shifted (by approximately 6 μm) along the radial direction from the very edge to the centre of the fiber core (left to right in the figure), as shown in Fig 2A. CCD1 was allowed to saturate so that the reflected speckle patterns were clearly visible. Each coherent reference beam Brefi was scrambled by the turbid resulting in four different speckle patterns on the output facet on CCD2. Henceforth, they are referred to as fSP(Brefi), shown in Fig 2B. The fiber was ~ 4 cm long and parallel to the optical axis of the focussing objective. This was chosen so that, due to the short length and orientation of the fiber, each transmitted reference beam fSP(Brefi) would be well differentiated from each other in terms of its amplitude profile, thus, the dependence of the calibration quality on the amplitude pattern and as a consequence on the ith input position could be assessed. Indeed, it has been reported that for a short straight fiber, when a central mode is excited, the transmitted intensity remains largely confined to the centre [31]. In contrast, when an edge mode is excited at the input, the light is totally scrambled over a shorter length. This is also clear from the amplitude distributions of fSP(Brefi) in Fig 2B. Notably, the overall intensity transmitted by the edge aligned reference is much lower than the other 3 examples, which could potentially be accounted for as an excitation at the boundary between core and cladding. Ultimately, all reference beams resulted in the ability to focus light on the output surface of the fiber but a different quality focusing across the output facet was observed.

Fig 2.

Fig 2

Analysis of choice of internal reference beam for surface plane calibrations A) Four internal reference beams on the input facet Brefi., imaged on CCD1. The position is shifted from the edge to centre of the core to centre (left to right) (scale bar 10 μm). B) The transmitted internal reference beams fSP(Brefi) imaged on CCD2 (scale bar 10 μm). C) Focussing efficiency across the fiber core for each calibration). D) Dependence of focussing efficiency on radial position of fiber output as the reference is shifted from edge to centre (red to blue). E) Histograms of focussing efficiency (for foci within the fiber core) for measurements based on edge reference–ER, lower intermediate reference—LRI, upper intermediate reference–URI and central reference—CR. F) The fall in average focusing efficiency between edge and centre reference beam based calibrations.

The quality of the calibration was therefore analysed by estimating the focussing efficiency (FE) across the output facet, defined as FE=IfociεfociItotalεtotal where Ifoci is the intensity within the SLM-generated foci, Itotal is the total intensity transmitted by the fiber and εfoci and εtotal are the total readout electrical noise of the CMOS camera in the foci and in the entire facet, respectively. In each calibration, a 30 by 30 array of foci was targeted at the output facet. In each calibration, a 30 by 30 array of foci was targeted at the output facet. The focussing efficiency on the (xout, yout) plane, obtained by raster-scanning the output beam, is shown in Fig 2C. For a better quantification and comparison between the four investigated reference positions, Fig 2D and 2E show the dependence of the focussing efficiency on radial distance from the centre of the core and histograms of focussing efficiency (for points within the core).

The calibration quality shows a clear dependence on the choice of reference beam. Notably, the central reference generates considerably lower focussing efficiency than the other 3 examples (the average over the entire core is approximately 1.15 times less (see Fig 2F), most strikingly at the centre of the core). This can be accounted for by considering the intensity difference between fSP(Brefi) and fSP(Bscanjx,jy,p) for each input pair (jx, jy,). The intensity of an interference pattern between two plane waves I1 and I2 is given by the well known equation [32]

Iinterfere=I1+I2+2I1I2cos(φ1φ2) (3)

Thus, the strongest phase dependency will occur when fSP(Brefi) and fSP(Bscanjx,jy,p) are approximately equal in intensity. In the case of the central reference, the intensity of fSP(Brefi) is unevenly distributed over the core with a greater proportion of intensity concentrated in the centre.

Hence, most modes will poorly interfere in this region of the fiber and weaker calibrations are produced. Therefore, in this case the optimal choice for an internal reference beam is toward the edge of the fiber due to the uniformity of the speckle pattern. Indeed, even the very weak reference beam generated at the core cladding interface results in a greater focussing efficiency than the central reference beam.

The uniformity of the resultant focussing efficiency map appears to be directly related to the the intensity profile of the reference beam. Going further, we have investigated the impact of fiber length and have performed calibrations akin to that in Fig 2 for a fiber approximately 60 cm long. Results are reported in S1 Fig in S1 File. As in the previous dataset, the reference beam was evenly shifted (by approximately 6 μm) along the radial direction from the very edge to the centre of the fiber core (left to right in S1 Fig in S1 File). The resultant output speckle fSP(Brefi) are shown in S1B Fig in S1 File. Although there is still clearly some preservation, the resultant intensity distributions are much more uniform than that for 4 cm fiber. This is in turn reflected in the focussing efficiency maps which are also more uniform for the central reference beam (comparing the far right panels of Fig 2C and S1C Fig in S1 File). For both the 4 cm and 60 cm fibers, an edge reference results in higher average focussing efficiency across the fiber core, however this is significantly more pronounced for the 4 cm fiber. Overall, the average focussing efficiency over the core was slightly higher for the short fiber for all reference beams and the average focussing efficiency was much less sensitive to the choice of reference beam for the longer fiber. As well as assessing the role of fiber length and the amplitude profile of fSP(Brefi), in S2 Fig in S1 File, we also asses the role of the phase profile of fSP(Brefi). The phase profile was measured using an external reference and the phase unwrapping technique described in [3]. The phase profile is shown in S2A Fig in S1 File and the resultant focussing efficiency map is shown in S2B Fig in S1 File. No trace of the phase profile can be found in the resultant focussing efficiency map, however it is noteworthy that the output foci are no longer phase aligned (as they would be for a uniform phase profile external reference).

A single internal reference-based calibration is compared with an external reference based calibration in S3 Fig in S1 File. The optical setup was modified as shown in S3 Fig in S1 File, so that after the first HWP the laser was split by 50:50 beam splitter BS1, its polarisation was rotated again by HWP2 to match with that of the fiber transmission. The two beams were re-joined by BS3 and a linear polariser was placed in front of the screen of CCD2. Panel B shows the focusing efficiency two datasets generated with an external reference (left) and an internal reference (right) and panel C shows the histogram of focussing effiencies. The advantage of the internal reference is evident both in terms of the evenness of the calibration across the fiber core and maximum intensity attainable. Between the two datasets, the average focusing efficiency increased from 6% to 15%. Although external reference based calibrations have been demonstrated in the literature [33] to achieve significantly higher focussing efficiency, they require extensive spatial filtering, beam shaping and feedback loops to achieve the high level of interferomic stability required for this process. The data in S3 Fig in S1 File shows that, on the other hand, internal reference based calibration offers a neat solution by an inherently stable interferometric system.

We then performed the calibration in the Fourier plane denoted (uout,vout). Our previous publication has characterised Fourier plane calibrations based on an external reference beam [23] and far-field imaging through MMFs has recently gained attention as a method to image objects distal from the fiber tip [24]. In the Fourier plane the resolution limit (θwidth) of the focussed spots is given by sin(θwidth)=1.22λd=1.22×633nm50μm. As in the surface plane experiment, the calibration was performed with the reference beam in 4 different positions at the input, displayed in Fig 3A. Fig 3B shows the fourier plane transmission by the fiber fFP(Brefi) for each of the reference beams, imaged on CCD3. As in the surface plane, the central reference better preserves the structuring of the input light and in this case more intensity is concentrated at the center of the fiber. The spatial dependency of the focussing efficiency of each Fourier plane foci is shown in Fig 3C and 3D, and by a histogram in Fig 3E. Previous publications where an external reference beam was used, have demonstrated that typically the intensity of the Fourier plane foci falls for higher angles still within the NA of the fiber [23, 24]. This also appears to be the case for internal reference beam calibrations, however the data in Fig 3 shows that the picture is further complicated by the interferometric compatibility of the two beams. In the case of the central reference beam, weaker foci are generated at the centre and highest intensity foci are generated at approximately half the NA, which can be accounted for by considering the profile of fFP(Brefi) and Eq 3.

Fig 3.

Fig 3

Analysis of internal choice of internal reference beam for fourier plane calibrations A) Four internal reference beams at the input facet Brefi (scale bar 10 μm). B) The transmitted internal reference beams fFP(Brefi) imaged on CCD3, the NA limit of the fiber is shown as a white circle. C) Focussing efficiency across the fiber NA for each calibration D) Dependence of focussing efficiency on output angle of fiber facet. E) Histograms of focussing efficiency (for foci within the fiber NA) for measurements based on edge reference–ER, lower intermediate reference—LRI, upper intermediate reference–URI and central reference—CR. F) The fall in average focusing efficiency between edge and centre reference based calibrations.

For edge reference beams, the stronger foci may be generated at the half of the fibers numerical aperture, as in the external reference experiments. However, within the intensity distribution, a second shoulder peak at higher angles is observed. At higher angles, the profile of the reference fFP(Brefi) is also uneven with an increase in intensity favoured at the NA boundary which could tentatively be claimed as accounting for this behaviour.

The above results demonstrate that in both the surface and Fourier planes, the calibration quality is highly dependent on the intensity and amplitude profile of the transmitted reference beam. Therefore, selecting a reference beam with uniform speckle pattern at the output is of the upmost importance. The histograms of the focussing efficiency also exhibit shoulder peaks at lower values corresponding to “blind spots” where the reference beam has a phase singularity. An example of this is shown in Fig 4A, where a characteristic focussed spot and a blind spot are shown side by side. To solve this, multiple calibrations with different internal reference beams can be employed, as illustrated in Fig 1B(ii) and described in the recent publication [30]. Focusing efficiency maps for four surface plane calibrations are shown in Fig 4B where between 1 and 6 reference beams were used. The reference beams Brefi were evenly displaced along the radius of the core at the input facet and a calibration for each reference was performed. For each position, the calibration generating the strongest foci was saved, while the others are discarded. The calibration took approximately 30 minutes per reference beam. From the colormaps in panel Fig 4B it is clear that in all cases focussing efficiency is relatively stable across the core and independent of radial distance from the center of the core. However, the single reference-based measurement exhibits a number of blind spots that are evident at the tail of the histograms in Fig 4C (highlighted by the blue rectangle) (they correspond to blue pixels in the FE maps in Fig 4B). Based on a fitted normal distribution over the histograms, we classified points with focussing efficiency below 15% as blind spots. A final figure of merit for this experiment is shown in Fig 4D, where the number of blind spots for each measurement is shown The average number of blind spots falls from 8.67±4.16 with a single reference calibration to 0.33±0.58 for the 6 reference beam based calibration (based on n = 3 experiments). Thus, it can be assessed that at the expense of higher computational time, internal reference based calibration can be applied to improve the calibration quality. The final panel of Fig 4 shows how two foci may be generated simultaneously at two output points with both a single and multiple reference calibration following the formula:

ΦSP(x1,y1),(x2,y2)=arg(exp(iΦSP(x1,y1))+exp(iΦSP(x2,y2)))

Fig 4. Analysis of calibrations based on serialised internal reference beams.

Fig 4

A) Characteristic example of a focussed spot on the fiber surface (top) and a blind spot (bottom) B) Focussing efficiency for four serialised internal reference based calibrations based on single reference (top left), two references (top right), three references (bottom left) and six references (bottom right) C) Histograms of the focussing efficiency for the four measurements, the inset shows the highlighted region which were classified as blind spots. D) The average number of blind spots in each calibration (left y-axis) and the blind spot coverage ratio (no of blind spots/no of focussed spots) (right y-axis), based on 3 experiments. E) Multi-spot focussing through a MMF based on single (top) and multiple (bottom) internal reference calibrations.

With the focusing efficiency shared between them. As this computation discards the amplitude information, the intensity distribution is not impacted by the number of reference beams used to calibrate.

Conclusions

Consequentially, internal reference beams appear to be an attractive basis for controlling transmission through optical fibers. Chosing an internal reference beam with an even amplitude distribution in the calibration plane leads to strong interformetric contrast between fSP(Brefi) and fSP(Bscanix,iy) (or fFP(Brefi) and fFP(Bscanix,iy)). Although isolated blind spots appear in the calibration, this can be overcome (at the expense of computational time) by using a series of internal reference beams, which simultaneously increases focussing efficiency. Overall, when compared with external reference beams, the common optical path of an internal reference-based system easily achieves a high interferometric stability without the requirement of a complex optical or machine learning setup. Overall, our study demonstrates the utility of internal reference-based systems, which is inherently reconfigurable, we envisage this will be crucial in allowing the technology to reach its full potential for hyper-spectral imaging at the tip of a hair thin optical fiber.

Supporting information

S1 File

(DOCX)

Data Availability

All relevant data are within the manuscript and its Supporting Information files.

Funding Statement

M.D.V. and Fe.P. jointly supervised and are co-last authors of this work. L.C., D.Z., M.D.V., and Fe.P. acknowledge funding from the European Union's Horizon 2020 Research and Innovation Program under Grant Agreement No. 828972. L.C., M.D.V. and Fe.P. acknowledge funding from the Project “RAISE (Robotics and AI for Socio-economic Empowerment)” code ECS00000035 funded by European Union – NextGenerationEU PNRR MUR - M4C2 – Investimento 1.5 - Avviso “Ecosistemi dell’Innovazione” CUP J33C22001220001. Fi.P., and Fe.P. acknowledge funding from the European Research Council under the European Union's Horizon 2020 Research and Innovation Program under Grant Agreement No. 677683. Fi.P., M.D.V., and Fe.P. acknowledge funding from the European Union's Horizon 2020 Research and Innovation Program under Grant Agreement No 101016787. M.D.V. acknowledges funding from the European Research Council under the European Union's Horizon 2020 Research and Innovation Program under Grant Agreement No. 692943. M.D.V. acknowledges funding from the U.S. National Institutes of Health (Grant No. U01NS094190). M.D.V., and Fe.P. acknowledge funding from the U.S. National Institutes of Health (Grant No. 1UF1NS108177-01). M,D,V and Fe,P acknowledge funding from European Research Council under the European Union's Horizon 2020 Research and Innovation Program under Grant Agreement No. 966674

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Decision Letter 0

Yuan-Fong Chou Chau

1 Mar 2023

PONE-D-23-02315Determining the optimal reference beam to measure the transmission matrix of a multimode optical fiberPLOS ONE

Dear Dr. Collard,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process

Please submit your revised manuscript by Apr 15 2023 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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We look forward to receiving your revised manuscript.

Kind regards,

Yuan-Fong Chou Chau

Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Partly

Reviewer #3: Yes

Reviewer #4: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: Yes

Reviewer #4: Yes

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3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: In this paper, a reference beam optimization scheme for measuring transmission matrix of multimode fiber based on the internal reference beam is proposed through experiments and analysis. By changing the coupling position and number of the internal reference beam and analyzing the speckle distribution and Fourier transform spectral intensity of the output facet of optical fiber, the author summarizes the implementation scheme of the inner reference beam. This manuscript is written logically and clearly shown the purpose. It may be accepted for publishing in PONE after a minor revision, but there are still several problems.

1. The author said “The numerical aperture of MO1 was significantly higher than that of the MMF. This was done so that the input modes of the fiber could be fully sampled”. Please explain that in detail.

2. It seems that the choice of reference beam can be determined from the analysis of the output face of the fiber. Why do we need to do Fourier analysis?

3. Obviously, in order to record the speckle field completely, the speckle field of the reference beam must be uniform enough. So, are there other factors other than the appropriate focusing position described in the article? Such as numerical aperture of MO, quality of the reference beam, diameter of fiber, etc.

4. Can the blind spots of the inner reference beam be eliminated completely?

5. In Figures 2 and 3, the scale bar is not indicated. In Figure 3(e), the number “35” is blocked.

6. In Figure 3, what do the �u and �v refer to?

Reviewer #2: In the manuscript tilted “Determining the optimal reference beam to measure the transmission matrix of a multimode optical fiber”, the authors present the merits of using internal reference method to improve focusing through a multimode fiber. The internal reference method is a great idea in my opinion, but the work presented by the author needs to ‘dig a bit deeper’.

The best way to generate a single focus using wavefront shaping method is the step sequential method introduced at the dawn of the technique. The transmission matrix method is the most accurate but it requires determining both amplitude and phase of the transmitted signal. The most accurate method to obtained the TM uses some sort of external/stable reference beam (holographic method) or uses phase retrieval techniques (a bit more computationally intensive). All the above methods can generate/transfer images through multimode fibers.

Here the authors only demonstrate their work for a single focus. I believe Collard et al. will have to put their work more in context to justify this conclusion “Overall, our study demonstrates the utility of internal reference-based systems, which is inherently reconfigurable, we envisage this will be crucial in allowing the technology to reach its full potential for hyper-spectral imaging at the tip of a hair thin optical fiber.”

See below for further questions and concerns:

1. The authors can do a little bit more of digging in the literature. There is quite a comprehensive body of work for wavefront shaping in multimode fiber both with external reference, internal reference, and reference-less (phase retrieval). How is this method compare with the other well established endoscopic imaging method?

2. The authors should revised their references. For example Reference [24] is not a reference less method. They have an external reference going through a second fiber. I do not believe they claim AI. The references for [25] and [26] aren’t accurate either. I believe the numbers have been shifted down. Please revise.

3. The length of the fiber (4 cm) used in this experiment is of concern. As far as I know, a lot of the single fiber endoscopic method in the literature have much longer fibers, some with much larger core (more modes). How will this method presented here, work when the fiber is 10 or 20 times longer? This isn’t obvious to me; in a longer fiber the light are even more scrambled. Further, bends, temperature, vibrations, etc. will also affect both the signal and reference. How is this accounted for in this study?

4. Further, the most accurate holographic techniques use a spatially filtered external reference beam: the phase and intensity of the beam well controlled and known. The idea of an internal reference is indeed fascinating, but the internal reference will also suffer the same fate as the signal beam. Can the author discuss how is the amplitude and phase of the internal reference determined? In equation (3) in the manuscript, the intensity of the reference beam is measured accurately, but how is the phase determined?

5. How is the work presented in this manuscript improve from what has been done in reference [26]? What is novel? Different?

6. There is a discussion on a single focus. Wavefront shaping have significantly moved on to multiple foci and image transfer. How will the changes in a multiple internal reference affect a multiple foci problem? An image?

I hope the authors will find my reviews helpful and hopefully improve the manuscript

Reviewer #3: In this manuscript, the authors investigated the conditions that affect the optimization of reference beams for multi-mode optical fiber’s transmission matrix measurements based on the common-path interferometric method. The authors defined the focusing efficiency and used this as a metric to experimentally investigated the optimal parameters for determining the internal reference beam. Related experimental data can support their claims. This work helps establish the metrics and method to optimize the reference beam as well as investigate the optimal experimental scheme to this end. Since wavefront-shaping-based optical fiber transmission matrix calibrations play a central role in fiber imaging, this work can benefit related fiber-based biomedical imaging research to a large extent. Overall, I would like to recommend the publication of this work. However, The writing of this manuscript has some minor issues. The authors need to address the following minor issues in their revised manuscript.

1. In the authors’ definition of focusing efficiency, it is not quite clear how the authors define “dark currents”? The physics meaning of the “dark currents” should be clarified.

2. In Figure 1, there are supposed to be four focal planes. However, in the experimental system drawing, only three focal planes are illustrated.

3. All the figures in this manuscript use upper-case letters. However, when the authors cited the figures in their main text, they used lowercase letters, which should be corrected.

4. The authors should add the length of the scale bar in Figure 2 and Figure 3.

5. Figure 2 d is confusing. Curves with different colors indicate focusing efficiency correlated with different spatial locations. Then, what is the meaning of the horizontal axis?

Reviewer #4: In the article the authors investigate to achieve desired amplitude distributions at the tip of a multimode

fiber (MMF) by using pre-shaped light. The model is extremely simple: they use two sawtooth gratings with some phase shift between together. It allows to control a little bit the intensity and the output of short 4-cm long MMF. The work has a lot of drawbacks that should be fixed before publication:

1. The title is confusing. In fact the authors did not present a measured transmission matrix as is has been done in other works like [1-3].

2. In the abstract the authors refer to optically turbid waveguide, but a standard MMF that is absolutely transparent for light was used in the experiments. What did it really mean?

3. SP and FP planes have not denoted in the Fig.1 but mentioned in the text.

4. Why the authors use such simply expression and do not mention other more improved methods like [4] for scanning?

5. The phase shift 'p' is unclear and there is no equation how exactly it affects to the sawtooth gratings.

6. Which array of points was used, 25x25 or 30x30? Both of them can be found in the text.

7. The used MMF was only 4-cm long. Are there any applications where it would be in demand? For me such length is impractical.

8. The first paragraph on page 9 describes the figure from supplementary that is really strange... It would be better to modify Fig1 to show external reverence beam and add one more figure with the characteristics of the focal point or prepare self-consistent supplementary.

Finally, I believe that a major revision required.

References:

1. https://www.mdpi.com/2076-3417/9/1/195#

2. https://doi.org/10.1364/OL.41.005580

3. https://doi.org/10.1364/OE.389133

4. https://doi.org/10.1364/OE.15.001913

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Reviewer #1: Yes: Jianglei Di

Reviewer #2: Yes: Dr. Moussa N'Gom

Reviewer #3: No

Reviewer #4: No

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Attachment

Submitted filename: my review.docx

PLoS One. 2023 Sep 8;18(9):e0290300. doi: 10.1371/journal.pone.0290300.r002

Author response to Decision Letter 0


15 Jun 2023

Dear Professor Chou Chau,

Thank you for your efforts in reviewing our manuscript "Determining the optimal reference beam to measure the transmission matrix of a multimode optical fiber PONE-D-23-02315” Enclosed is our revised manuscript, supporting information and response to the reviewers.

We are grateful to the reviewers for their thorough reading of our manuscript and their detailed comments. In our response letter we have provided a detailed point by point response to the reviewers’ questions. In particular, a new figure has been added to the supporting information addressing the points raised by several reviewers about the dependence of our method on fiber length. As well as this, new measurements have been performed where the phase of the internal reference beam was measured and compared with the resultant focusing efficiency map.

As well as this, the introduction, discussion sections have been modified to provide a better comparison with the state of the art following the comments from all reviewers.

Following the comments from reviewer 4, we would also like to change the title of work to:

“Optimizing the internal phase reference to shape the output of a multimode optical fiber”

We look forward to hearing from you.

On behalf of all authors,

Yours sincerely,

Dr Liam Collard

Attachment

Submitted filename: ResponseLetterFINAL.docx

Decision Letter 1

Yuan-Fong Chou Chau

31 Jul 2023

PONE-D-23-02315R1Optimizing the internal phase reference to shape the output of a multimode optical fiberPLOS ONE

Dear Dr. Collard,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by Sep 14 2023 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

  • A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

  • A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

  • An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Yuan-Fong Chou Chau

Academic Editor

PLOS ONE

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Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #5: (No Response)

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #5: Yes

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #5: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #5: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #5: No

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #5: In this manuscript, the authors perform assessments for both surface and Fourier plane calibrations. In the surface calibration, they focus the light on the distal facet, and in the Fourier plane calibration, they collimate the light into a low-divergence beam emerging from the distal facet. To optimize the system's relative focusing efficiency, they employ both single and serialized internal reference beams. This work may deserve publication in my opinion.

However, several revisions, including some major ones, still need to be addressed.

1) The authors should carefully check their notations as there are several unacceptable notation errors in the manuscript. Some are listed below:

1a) On page 26, it should be "(u_in, v_in)" instead of "(u_in, y_in)."

1b) In Figure 2D, the x-axis should be labeled as "\\sqrt{x_out^2, y_out^2)}" instead of "\\sqrt{x_out^2, v_out^2)."

1c) On page 34, the correct notation for the calibration in the Fourier plane is "(u_out, v_out)" rather than "(u_out_v_out)."

2) Figure S3 has been incorrectly labeled as Figure S1 in the Supporting Information. To rectify this error, the authors must ensure that the correct label, "Figure S3," is used for the respective figure in the Supporting Information section. Moreover, it is essential to update the caption in Figure S3 to accurately correspond to the content of Figure S3.

3) The meaning of "S1B" on page 31 and "S2B" on page 32 is unclear. Should they be Figure S1B and Figure S2B?

4) Following QUESTION R3.2, the focal planes for (u_in, v_in) and (u_out, v_out) in Figure 1A are challenging to discern. To address this issue, the authors should consider modifying the colors used for these focal planes to enhance visibility and clarity.

5) Following QUESTION R3.3, the issue has not been fully addressed yet. For example, Figure 2b on page 29 and Figure 2c on page 32 still require corrections. Additionally, there are inconsistencies in the citation format within the main text, where certain figure references are bolded while others are not. Moreover, the authors sometimes use "Figure S1" and other times "figure S1."

6) The authors should include a legend to distinguish between the two different colors in the histograms for both Figure 2E and Figure 3E.

7) In addition to mentioning the information in the caption, the authors should also label the number of references in Figure 4B.

8) The "um" in the x-axis of Figure S1D should be corrected to "μm."

9) The caption of Figure 2 requires revision to include the scale bar information for Figure 2A, which is currently missing. It's important to note that the mentioned 10 m scale bar is not present in Figure 2C, despite being referenced in the caption.

10) The colorbar label in Figure 3C, Figure S2A, Figure S2B, and Figure S3B should be horizontally flipped to make them consistently.

11) The authors should be mindful of their use of abbreviations to prevent redundancy. For example, on page 28, they introduce the abbreviation "SP" for "surface plane." However, on page 29, they opt to abbreviate "surface plane" once more instead of using the established abbreviation "SP."

12) There are punctuation errors that need to be addressed. In two instances (pages 26 and 28), the authors used ".." to end sentences, but it should be corrected to use ".", instead. Additionally, on page 31, the authors used ",." which should also be fixed to "." for proper punctuation.

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Reviewer #5: No

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PLoS One. 2023 Sep 8;18(9):e0290300. doi: 10.1371/journal.pone.0290300.r004

Author response to Decision Letter 1


3 Aug 2023

Dear Professor Chou Chau,

Thank you for your continued efforts in reviewing our manuscript "Determining the optimal reference beam to measure the transmission matrix of a multimode optical fiber PONE-D-23-02315” Enclosed is our revised manuscript, supporting information and response to the reviewers.

We are grateful to the new reviewer for their thorough reading of our manuscript and their detailed comments. In our response letter we have provided a detailed point by point response to the reviewers’ questions.

We look forward to hearing from you.

On behalf of all authors,

Yours sincerely,

Dr Liam Collard

Attachment

Submitted filename: ResponseLetterTWO.docx

Decision Letter 2

Yuan-Fong Chou Chau

4 Aug 2023

Optimizing the internal phase reference to shape the output of a multimode optical fiber

PONE-D-23-02315R2

Dear Dr. Collard,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Yuan-Fong Chou Chau

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Acceptance letter

Yuan-Fong Chou Chau

31 Aug 2023

PONE-D-23-02315R2

Optimizing the internal phase reference to shape the output of a multimode optical fiber

Dear Dr. Collard:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Yuan-Fong Chou Chau

Academic Editor

PLOS ONE

Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Supplementary Materials

    S1 File

    (DOCX)

    Attachment

    Submitted filename: my review.docx

    Attachment

    Submitted filename: ResponseLetterFINAL.docx

    Attachment

    Submitted filename: ResponseLetterTWO.docx

    Data Availability Statement

    All relevant data are within the manuscript and its Supporting Information files.


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