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The PUMA micro­scope is a low cost, mod­u­lar, 3D print­ed, open source micro­scope designed in FreeCAD. Its mod­u­lar nature allows re-use of many com­po­nents to recon­fig­ure the scope into a mul­ti­tude of pos­si­ble forms, from a basic mir­ror-illu­mi­nat­ed sim­ple micro­scope (mid­dle) to a com­plex scope for seri­ous research appli­ca­tions (right) – while still being ful­ly portable!

This guest arti­cle is by Dr Paul J. Tadrous, MB, BS, MSc, PhD. Dr Tadrous is a retired clin­i­cal pathol­o­gist in the UK. Although retired from clin­i­cal prac­tice he con­tin­ues to be active in research into microscopy, image pro­cess­ing, analy­sis and machine vision at Tad­Path Diag­nos­tics, Lon­don, UK. Con­tact: email, YouTube, and Twit­ter.

If you are inter­est­ed in writ­ing an arti­cle for FreeCAD News, con­tact Chris Hennes at chennes@freecad.org.

Introduction

The Portable, Upgrade­able, Modu­lar, Afford­able (PUMA) micro­scope is an advanced open source (GPL v3) microscopy sys­tem designed to be built as a DIY project by any­one with access to low cost 3D print­ing and some basic hand tools [1], [2], [3].

The scope has inter­change­able mod­ules that allow mul­ti­ple imag­ing modal­i­ties includ­ing phase con­trast, dark field, epi-illu­mi­na­tion, polar­i­sa­tion and flu­o­res­cence microscopy (fig­ure 1).

It also has some fea­tures not usu­al­ly seen on even high end com­mer­cial lab micro­scopes such as a TFT-screen based spa­tial light mod­u­lar (SLM) for Fouri­er optics effects (fig­ure 2, video 1) and a user-pro­gram­ma­ble dig­i­tal heads-up-dis­play (HUD) that is super­im­posed onto the live opti­cal image seen down the scope (not a dig­i­tal over­lay on a dig­i­tal image stream) and mul­ti-head­er capa­bil­i­ties. This HUD may be selec­tive­ly opti­cal­ly erased in real time using polar­i­sa­tion to allow advanced aug­ment­ed real­i­ty microscopy research, teach­ing and diag­nos­tic assist appli­ca­tions (video 2).

All the 3D print­able parts of the PUMA sys­tem were designed in FreeCAD and all the editable para­met­ric FreeCAD mod­els are freely avail­able to down­load on the project GitHub site [3] so any­one can cus­tomise the scope to meet their spe­cif­ic project needs.

Why FreeCAD?

One impor­tant enabling fac­tor for ground-break­ing sci­ence is the abil­i­ty of the user to under­stand their appa­ra­tus to the point where they can cus­tomise it to do their own inno­v­a­tive exper­i­ments as well as inter­pret the results cor­rect­ly (e.g. they are aware of its lim­its and poten­tial arte­facts in the results). Com­mer­cial micro­scopes are dif­fi­cult and expen­sive to cus­tomise (let alone to buy and main­tain) and the more high tech ones seem to be incor­po­rat­ing more and more ‘black box’ fea­tures full of trade secrets, pro­pri­etary mech­a­nisms and closed source soft­ware that are not fea­si­ble to cus­tomise with­out offi­cial com­pa­ny sup­port (that may be quite expen­sive and the cus­tomi­sa­tions may have cor­po­rate restric­tions placed on their use or pub­li­ca­tion to pro­tect intel­lec­tu­al prop­er­ty). Fur­ther­more the researcher only has offi­cial com­pa­ny infor­ma­tion about how (for exam­ple) a soft­ware algo­rithm works so may not be aware of cer­tain fac­tors in their results that might be due to spe­cif­ic cod­ing imple­men­ta­tion choic­es rather than nat­ur­al respons­es of the exper­i­men­tal sys­tem under study. These are some of the argu­ments in favour of the use of ful­ly open source soft­ware and hard­ware in sci­en­tif­ic research.

Low cost 3D print­ing makes cus­tom part man­u­fac­ture afford­able for any­one these days. The PUMA micro­scope project pro­vides ful­ly open source design specs for a high qual­i­ty stan­dard for­mat micro­scope and for this to be most use­ful those designs must be avail­able freely with­out such cor­po­rate restric­tions and in a for­mat that is eas­i­ly (and freely) editable by the user into 3D print­able mod­els – not just sta­t­ic STL mesh files.

FreeCAD is free soft­ware that can pro­duce such files for free dis­tri­b­u­tion. But there are oth­er well known com­mer­cial CAD pack­ages that also have ver­sions avail­able cur­rent­ly free of mon­e­tary cost for some restrict­ed uses, so why not use those instead? Well, to me, ‘cur­rent­ly free of mon­e­tary cost for some restrict­ed uses’ is not a suf­fi­cient cri­te­ri­on on which to base an open sci­ence hard­ware project because one needs also to con­sid­er the future devel­op­ment of the project and the free­dom of users to do what they need or want to do with the files and mod­els with­out undue restric­tions being placed on them. A com­mon restric­tion for the free ver­sions of some com­mer­cial CAD pack­ages is that they may only be used for non-com­mer­cial pur­pos­es, but many researchers in uni­ver­si­ties work under grants that have tech-trans­fer claus­es that seek to com­mer­cialise suc­cess­ful work. This means that either they can’t use those restrict­ed CAD pack­ages or they must upgrade to a paid sub­scrip­tion ver­sion before they make any mon­ey (which they may nev­er do if the com­mer­cial­i­sa­tion effort fails) or they must re-do all their mod­els in a tru­ly libre free CAD prod­uct if they want to progress to commercialisation.

The lack of open source code and the use of pro­pri­etary for­mats also may lim­it the use of CAD files gen­er­at­ed by such ‘free’ com­mer­cial soft­ware either now or, per­haps more impor­tant­ly, in the future should com­pa­ny pol­i­cy change. For exam­ple, if I chose a ‘free’ com­mer­cial CAD pack­age to make the designs of PUMA parts, the PUMA project could grind to a halt if an exist­ing or new CEO or board of the CAD com­pa­ny decides to change their terms for use of their free ver­sions or if they decide to revoke free use of their prod­uct or file for­mats at some stage in the future or pro­hib­it the port­ing of their CAD files into oth­er CAD pack­ages with­out obtain­ing a paid license from them, and so forth.

With FreeCAD there is no risk of this because not only is it free of mon­e­tary cost but it is also libre and open source (includ­ing the file for­mats). Some peo­ple have com­ment­ed that they tried FreeCAD some time ago and found it to be bug­gy or crashed often so they moved over to this or that oth­er pack­age. I start­ed off with FreeCAD v0.17 a few years ago and, yes, it was a bit ‘crashy’ back then but I have seen the soft­ware make great improve­ments in sta­bil­i­ty over the last few years and it has not even reached v.0.3 yet (let alone v.1.0)! So I chose FreeCAD because it fits in with the open source cus­tomis­able and libre-free nature of the PUMA project and ensures it will stay that way into the future.

What makes PUMA different to other microscopes?

Com­pared to oth­er open source micro­scopes there is no over­all ‘win­ner’ because they each have their strengths and weaknesses.

PUMA is very well doc­u­ment­ed with a whole series of detailed ‘How To’ videos explain­ing its con­struc­tion, the­o­ry and specs in addi­tion to the doc­u­ments on the project GitHub page and all editable para­met­ric FreeCAD design files are pro­vid­ed – not fixed STL mesh­es – with video tuto­ri­als on how to edit them (e.g. https://youtu.be/CPHyLTHID6g ).

PUMA is clos­er to con­ven­tion­al desk­top micro­scope design com­pared to the oth­er DIY micro­scopes and is par­tic­u­lar­ly suit­ed to ergonom­i­cal direct vision as opposed to being a ‘cam­era-only’ scope or smart­phone-attach­ment scope.

Com­pared to their com­mer­cial coun­ter­parts, PUMA sys­tems are ultra low cost , easy to cus­tomise, open source (no trade secrets) and high­ly portable. All the cus­tom parts are designed in FreeCAD and 3D print­able with a low cost print­er such as the Ender 3 and all the oth­er parts are gener­ic fix­tures and optics that can be bought from acces­si­ble online stores like AliEx­press, eBay and Ama­zon. For exam­ple, to build a com­plete epi­flu­o­res­cence PUMA sys­tem, includ­ing 3D print­ing, fix­tures, fit­tings, elec­tron­ics and optics, the cost is typ­i­cal­ly around $100 USD if full DIY is used. Although com­mer­cial flu­o­res­cence micro­scopes would have a stur­dier met­al con­struc­tion with more pre­cise mechan­i­cal mech­a­nisms and more con­ve­nient adjust­ments com­pared to the PUMA equiv­a­lent, if you are short of the $5000 (and some­times con­sid­er­ably more) such a scope would cost you brand new then PUMA pro­vides a func­tion­al alter­na­tive at a frac­tion of the price – great for home users or labs on a tight bud­get, esp. if you need mul­ti­ple scopes for a prac­ti­cal teach­ing class or spe­cial research project.

In addi­tion to diverse func­tion­al­i­ty and cost sav­ings, PUMA boasts com­plete porta­bil­i­ty weigh­ing just a few hun­dred grams e.g. 875 g (31 oz) for the flu­o­res­cence scope (includ­ing bat­tery pow­er sup­ply and lamp), and even lighter – about 540 g (19 oz) for the basic trans­mit­ted light mir­ror-illu­mi­nat­ed ver­sion of the scope (which can be seen in the mid­dle of the fron­tice pic­ture at the top of this article).

Porta­bil­i­ty of PUMA is fur­ther enhanced by the fact that it is designed as a pri­ma­ry vision micro­scope – i.e. one where the user looks down an eye­piece. This allows the user to get the full opti­cal res­o­lu­tion and a com­plete wide field of view of their obser­va­tions with­out the need for a smart­phone or oth­er com­put­er-cam­era-mon­i­tor-pow­er-sup­ply com­bo (or ‘cam­era-scopes’ for short). Oth­er 3D print­ed open source cam­era-scopes may look small and ‘portable’ until you take all these nec­es­sary acces­sories into full con­sid­er­a­tion at which point ‘lug­gable’ might be a more apt descrip­tion. One of the biggest issues when using those cam­era-scopes for mak­ing obser­va­tions out in the field (as opposed to record­ings – we’ll come back to that lat­er) is that what the user can see at the time is restrict­ed to a small field of view on a small portable screen such as a smart­phone (if you want to boast porta­bil­i­ty) that is fur­ther lim­it­ed by avail­able pow­er sup­ply issues. If cam­era-scopes want to boast great res­o­lu­tion and wide field of view then they need large high res­o­lu­tion dis­play mon­i­tors and wide field of view optics – which coun­ters any claim to afford­abil­i­ty let alone porta­bil­i­ty. Remem­ber – for now I am only talk­ing about mak­ing real-time obser­va­tions out in the field – not stitch­ing togeth­er many pic­tures record­ed on a portable com­put­er for study lat­er, back at base, with a large desk­top mon­i­tor. Hence PUMA was designed as a direct vision scope with the abil­i­ty to use day­light or any suit­able exter­nal light source so it can give high qual­i­ty wide field of view real time opti­cal micro­vi­sion via an eye­piece in a tru­ly portable way. Even the option­al LED lamp and on-board micro­proces­sor (an Arduino Nano) for the option­al advanced TFT screen func­tions like the HUD are pow­ered from one or two small domes­tic 9V bat­ter­ies or solar recharge­able bat­tery packs. Video 3 shows the deploy­ment in the field of a ful­ly func­tion­al com­plete PUMA sys­tem ready to make observations.

All this ‘direct vision’ empha­sis does­n’t mean that PUMA can’t do image record­ings – on the con­trary, the ‘M’ in PUMA stands for ‘Mod­u­lar’ and there are sev­er­al mod­ules designed to allow high qual­i­ty cam­era record­ings – actu­al­ly with up to three sep­a­rate cam­eras recod­ing in dif­fer­ent modal­i­ties simul­ta­ne­ous­ly (fig­ure 3, video 4). This is some­thing that, to the best of my knowl­edge, has not been demon­strat­ed to date with oth­er 3D print­ed open source micro­scopes. PUMA can accept stan­dard C‑mount and CS-mount cam­eras as well as eye­piece cam­eras (includ­ing smart phone cam­eras if suit­able adapters are used).

Anoth­er fea­ture that sets PUMA apart from some oth­er attempts at open source micro­scopes is its high qual­i­ty illu­mi­na­tion sys­tem. Men­tion has already been made of the Abbe con­denser (fig­ure 2). In addi­tion PUMA has it’s own portable full Köh­ler illu­mi­na­tor (fig­ure 4) for even illu­mi­na­tion and access to the Köh­ler field stop for more advanced pho­tomi­crog­ra­phy. Fur­ther­more PUMA has a co-axi­al epi-illu­mi­na­tor mod­ule with sev­er­al options for epi-polar­i­sa­tion and epi-flu­o­res­cence microscopy (see results shown in fig­ure 1 and this YouTube video for details: https://youtu.be/cAEB10K8PqI ).

Unlike some com­mer­cial portable micro­scopes that use their own spe­cial mini objec­tives that can only be used with their scopes, PUMA uses Roy­al Micro­scop­i­cal Soci­ety (RMS) stan­dard optics and mechan­i­cal spec­i­fi­ca­tions so can accept a wide range of stan­dard objec­tives and eye­pieces either gener­ic or from major micro­scope man­u­fac­tur­er brands.

So is PUMA the perfect microscope for all?

Of course not! It does have its own lim­i­ta­tions and pecu­liar­i­ties. These include that it can only accept one objec­tive at a time – there is no rotat­able objec­tive tur­ret. It also suf­fers from some ‘wob­ble’ and focus mech­a­nism hys­tere­sis which makes aut­o­fo­cus imprac­ti­cal­ly slow. These lim­i­ta­tions are part­ly mat­ters of cur­rent design and also due to it being con­struct­ed of 3D print­ed plas­tic instead of pre­ci­sion machine-milled met­al mech­a­nisms. The cur­rent stage of the PUMA has lim­it­ed space – it  can han­dle a full stan­dard micro­scope slide but noth­ing much big­ger than that. It is also cur­rent­ly only of con­ven­tion­al ‘upright’ design. How­ev­er, the mod­u­lar, open source and ful­ly cus­tomis­able nature of PUMA mean that these obsta­cles can be addressed by future improve­ments in the designs and by the devel­op­ment of new mod­ules – some­thing which I am active­ly work­ing on.

What does the future hold for PUMA?

New Hardware

I am cur­rent­ly work­ing on a sta­bil­i­ty brack­et to reduce vibra­tions (fig­ure 5a) and I used this with some suc­cess to take the oil immer­sion pic­tures of plant chro­mo­somes seen in fig­ure 1. Anoth­er recent devel­op­ment is the ‘Day­light Köh­ler adapter’ (fig­ure 5b) which allows the full Köh­ler sys­tem to be used with nat­ur­al day­light for enhanced portable pow­er-sav­ing use. I am also in the ear­ly stages of devel­op­ing a pre­ci­sion motorised XYZ stage for auto­mat­ed track­ing and scan­ning microscopy. All of these are not yet ready for release until thor­ough­ly test­ed and doc­u­ment­ed. Fur­ther pos­si­bil­i­ties for the future include a Michel­son inter­fer­om­e­ter mod­ule, new illu­mi­na­tion mod­ules (such as struc­tured illu­mi­na­tion, light sheet, etc.) and an invert­ed stage sys­tem to make PUMA use­ful for cell cul­ture stud­ies – but work on those mod­ules has not begun at the time of writ­ing this article.

New Software

One of my aims is to help peo­ple do advanced microscopy with PUMA. I already start­ed the PARDUS micro­scope con­trol sys­tem for auto­mat­ed microscopy [4] and hope to devel­op that in in con­junc­tion with PUMA once the PUMA robot­ic XYZ stage is finalised. I also would like to cre­ate more ded­i­cat­ed soft­ware for Fouri­er aper­ture manip­u­la­tion with the SLM of the scope but have not start­ed work on that yet. Fur­ther soft­ware devel­op­ment will occur in con­junc­tion with the edu­ca­tion­al out­reach dis­cussed next.

Microscopy and image processing education

I would like to make a series of tuto­ri­als on the YouTube chan­nel about image pro­cess­ing in microscopy using my free BiaQIm Image Pro­cess­ing Suite [5] as a basis for work-along exam­ples and the first video in the series has already been pub­lished [6].

Most of the basics of microscopy the­o­ry have been cov­ered already as ‘the­o­ry sec­tions’ in the PUMA YouTube ‘How To’ videos pub­lished to date. In addi­tion I would like to make a series of more advanced microscopy the­o­ry tuto­ri­als on top­ics such as Fouri­er optics, super-res­o­lu­tion, live cell imag­ing and decon­vo­lu­tion microscopy using PUMA as the hard­ware plat­form to exem­pli­fy these subjects.

Funding for PUMA

All the above future devel­op­ment plans remain aspi­ra­tional. If and when they can be deliv­ered depends part­ly on the inter­est shown by peo­ple in the project and a lot on secur­ing the nec­es­sary fund­ing. To date (up to Jan­u­ary 2023) PUMA has been a total­ly self-fund­ed project. I intend to keep the PUMA resources free and open source for all but, of course, they are not free to devel­op and main­tain. Buy­ing com­po­nents for pro­to­typ­ing, mak­ing doc­u­men­ta­tion, videos and main­tain­ing web­sites get more expen­sive all the time so I have recent­ly set up a Patre­on page [7] and Pay­Pal links [8] to help make the project viable and sus­tain­able into the future and com­plete all the aspi­ra­tional devel­op­ments men­tioned above.

References

[1] Tadrous, P. J. (2021). PUMA–An open-source 3D-printed direct vision micro­scope with aug­ment­ed real­i­ty and spa­tial light mod­u­la­tor func­tions. Jour­nal of Microscopy, 283(3), 259–280. https://onlinelibrary.wiley.com/doi/10.1111/jmi.13043

[2] The PUMA Micro­scope YouTube Chan­nel https://youtube.com/@PUMAMicroscope

[3] The PUMA Micro­scope GitHub page https://github.com/TadPath/PUMA

[4] The PARDUS motor con­trol GitHub page https://github.com/TadPath/PARDUS

[5] My BiaQim Image Pro­cess­ing suite web­site https://www.optarc.co.uk/bialith/

[6] First PUMA image pro­cess­ing tuto­r­i­al video https://youtu.be/MZOCSkbz3ko

[7] PUMA Micro­scope Patre­on page: https://www.patreon.com/PUMAMicroscope

[8] PUMA Pay­Pal dona­tion: https://www.paypal.com/donate/?hosted_button_id=NPMYJKJATDLQ4