Thesis
Reference
Intrinsic Optical Imaging: Major limitations in the perspective of the local clinical experience at Geneva University Hospital
CORNIOLA, Marco Vincenzo
Abstract
L'imagerie optique intrinsèque a été présentée à la communauté neuroscientifique comme une solution de visualisation directe, rapide et peu coûteuse de l'anatomie fonctionnelle physiologique et pathologique du cerveau, en salle d'opération. En utilisant les propriétés optiques du cortex cérébral, la délimitation de processus pathologiques durant la résection permettrait ainsi de sauvegarder des zones corticales éloquentes et ainsi d'améliorer le pronostic fonctionnel post-opératoire des patients. Au moyen d'une revue de la littérature, d'expériences animales et du recul accumulé au sein du service de neurochirurgie des Hôpitaux Universitaires de Genève, l'utilisation concrète de l'imagerie optique intrinsèque et la réalité du terrain ont été éprouvées et évaluées de façon objective et indépendante. Il en ressort que l'imagerie optique intrinsèque, présentée par certains groupes comme une solution viable et prometteuse, comporte des limitations techniques importantes et n'est pas, en tout cas pas encore, adaptée à une utilisation clinique robuste.
CORNIOLA, Marco Vincenzo. Intrinsic Optical Imaging: Major limitations in the
perspective of the local clinical experience at Geneva University Hospital. Thèse de doctorat : Univ. Genève, 2018, no. Méd. 10883
DOI : 10.13097/archive-ouverte/unige:106939 URN : urn:nbn:ch:unige-1069396
Available at:
http://archive-ouverte.unige.ch/unige:106939
Disclaimer: layout of this document may differ from the published version.
1 / 1
1 Section de médecine Clinique
Département des Neurosciences Cliniques Service de Neurochirurgie
Thèse préparée sous la direction du Docteur Shahan Momjian et du Professeur Karl Schaller
Imagerie Optique Intrinsèque
Limitations majeures et expérience clinique aux Hôpitaux Universitaires de Genève
Thèse
présentée à la Faculté de Médecine de l'Université de Genève
pour obtenir le grade de Docteur en médecine par
Marco Vincenzo CORNIOLA de
Genève
Thèse n° 10883 Genève (Genève)
2017
2
3 Table of contents
1. Résumé (en français) 2. Introduction (en français) 3. Foreword
4. Introduction
5. Common neurovascular brain imaging applied to the surgical field 5.1 Context
5.2 Pre-operative investigations 5.3 Intra-operative investigations 6. Fundamentals
6.1 General concepts of brain mapping and basics of intrinsic optical imaging 6.2 The rule of five
6.3 Neurovascular coupling 6.4 Sources of intrinsic signals 7. Practical aspects
7.1 Context
7.2 Temporal and spatial evolution of intrinsic signal 7.3 Applications
7.3.1. Mapping the somatosensory cortex 7.3.2 Mapping Broca’s and Wernicke’s areas
7.3.3 Functional and interventional study of epilepsy
7.3.4 Cortical stimulation mapping and intrinsic optical imaging;
comparison isn’t reason
4 8. Review of the literature
9. Technical note: the use of a circular illuminating device to improve the signal- to-noise ratio
9.1 Background and purpose 9.2 Methods
9.3 Results 9.4 Discussion 9.5 Conclusion 10. Discussion
10.1 Context
10.2 Brain movements 10.3 Glare effects 10.4 Acquisition time
10.5 Spatiotemporal acquisition and point-spread 10.6 Intrinsic optical imaging and electrophysiology
10.7 What’s next? The emergence of functional ultrasonography 11. Conclusion
12. Bibliography
5 1. Résumé (en français)
L’imagerie optique intrinsèque a été présentée à la communauté neuroscientifique comme une solution de visualisation directe, rapide et peu coûteuse de l’anatomie fonctionnelle physiologique et pathologique du cerveau, en salle d’opération. En utilisant les propriétés optiques du cortex cérébral, la délimitation de processus pathologiques durant la résection permettrait ainsi de sauvegarder des zones corticales éloquentes et ainsi d’améliorer le pronostic fonctionnel post-opératoire des patients. Au moyen d’une revue de la littérature, d’expériences animales et du recul accumulé au sein du service de neurochirurgie des Hôpitaux Universitaires de Genève, l’utilisation concrète de l’imagerie optique intrinsèque et la réalité du terrain ont été éprouvées et évaluées de façon objective et indépendante. Il en ressort que l’imagerie optique intrinsèque, présentée par certains groupes comme une solution viable et prometteuse, comporte des limitations techniques importantes et n’est pas, en tout cas pas encore, adaptée à une utilisation clinique robuste.
6 2. Introduction (en français)
La visualisation et la délimitation de zones corticales fonctionnelles avant, puis durant une procédure neurochirurgicale, est un point-clé dans la prise en charge chirurgicale des patients. L’identification du cortex fonctionnel et des pathologies adjacentes à des zones éloquentes est réalisée au moyen de techniques diverses, dont la connaissance des bases scientifiques, l’utilisation et l’interprétation font désormais partie de la formation de base des neurochirurgiens. Par ailleurs, de nouveaux outils sont continuellement développés et mis sur le marché par l’industrie, afin d’augmenter la panoplie diagnostique et le traitement chirurgical des patients.
Le tout visant à rendre meilleurs les résultats fonctionnels post-opératoires et à renforcer la sécurité du patient. Face à la variété de l’offre disponible sur le marché et face aux tendances successives présentées par la communauté neuroscientifique, le neurochirurgien se doit de garder un esprit critique, afin de recourir au meilleur examen au moment le plus adapté au patient et à sa pathologie. Pour cela, la compréhension des phénomènes sous-jacents à la pratique d’une technique ainsi que la connaissance pragmatique de ses limitations sont d’une importance capitale.
Aux Hôpitaux Universitaires de Genève, l’expérience gagnée sur plusieurs années dans les domaines de la neuronavigation, de la réalité augmentée, l’angiographie per-opératoire, de l’électrophysiologie et, bientôt, de l’imagerie par résonnance magnétique intra-opératoire, apporte une plus-value dans le traitement de pathologies cranio-spinales complexes (et moins complexes). Il est désormais clair et établi, au sein de la communauté neurochirurgicale, que les outils diagnostiques intra-opératoires, tels que décrits ci-dessus, font partie de la panoplie standard de prise en charge chirurgicale des patients. Par ailleurs, l’application, le
7 développement, l’expérimentation et la mise à jour de techniques radiologiques et électrophysiologiques destinées à l’aide opératoire font partie du cahier des charges de l’équipe genevoise de neurochirurgie et, plus communément, des neurochirurgiens en général.
Pour cette raison, l’exploration de l’imagerie optique intrinsèque a été initiée au début des années 2010 par le Dr. Shahan Momjian. Lorsque j’ai rejoint l’équipe en 2013, j’ai intégré le Laboratoire des Neurosciences Chirurgicales et, étant intéressé par l’essor de nombreuses technologies d’aide à la navigation chirurgicale, l’évaluation de l’imagerie optique intrinsèque comme technique fondamentale en neurochirurgie a été un sujet d’étude approfondi. Nous avons donc commencé par revoir la littérature sur le sujet, nous permettant ainsi de nous faire une idée précise du « state of the art », notamment dans son application en neurochirurgie. Nous avons également établi des contacts avec le service de neurochirurgie de Dresde, au sein duquel j’ai passé quelques jours pour voir et comprendre leur utilisation systématique de l’imagerie optique intrinsèque. Leurs conditions d’utilisation étaient très spécifiques, car leur centre travaille en collaboration avec une école d’ingénieurs et un ingénieur est rattaché à leur service, spécifiquement pour le développement de l’imagerie optique intrinsèque.
Enfin, nous avons utilisé un illuminateur bien spécifique, circulaire en l’occurrence, afin d’étudier l’influence de cette géométrie particulière sur la qualité du signal obtenu par imagerie optique intrinsèque. Ce protocole local, à notre connaissance jamais étudié dans le détail comme nous l’avons fait, nous a permis d’élaborer solidement un contre-argument à une idée répandue dans le milieu : l’illuminateur circulaire ne permet pas la diminution du rapport signal sur bruit, tant recherchée.
8 L’élaboration d’algorithmes spécifiques dédiés à l’imagerie optique intrinsèque et le substrat mathématique on été précédemment décrits par Pierre Bouillot et Shahan Momjian [43] ; ces concepts ne seront pas développés plus avant dans ce manuscrit. Ce travail constitue ainsi la seconde partie du projet, décrivant l’application clinique concrète de l’imagerie optique intrinsèque à la pratique neurochirurgicale quotidienne aux Hôpitaux Universitaires de Genève.
Les neurochirurgiens confrontés à une lésion se trouvant au sein ou à coté d’une région cérébrale adjacente doivent identifier précisément les limites séparant le cortex éloquent de la région à réséquer, afin de parvenir à la résection chirurgical de la façon la plus sûre et aussi, la plus complète possible. Ceci représente un véritable défi, qui dépend d’une part de la nature de la lésion ainsi que de la difficulté à discriminer, macroscopiquement, le tissu cérébral sain du tissu pathologique, en particulier pour les neurochirurgiens peu expérimentés. Ainsi, la règle est à l’utilisation d’outils opératoires tels que l’électrophysiologie et l’imagerie intra-opératoire (en pratique, des systèmes de navigation chirurgicale), guidant l’opérateur durant la procédure chirurgicale et empêchant des lésions de régions corticales et sous-corticales saines et fonctionnelles. L’imagerie optique intrinsèque fait partie de ces outils opératoires.
Le terme « imagerie optique médicale » est un générique employé afin de définir diverses techniques utilisant la lumière et ses longueurs d’ondes afin d’améliorer l’identification intra-opératoire de processus lésionnels. Plus spécifiquement, l’imagerie optique en neurosciences utilise les photons afin de délimiter les zones corticales fonctionnelles ou anatomiques. Ce concept général de mesure des propriétés optiques du cerveau a été introduit à la fin des années 80 par Grinvald et Frostig [1, 2]. À l’époque, l’application directe sur le cortex de colorants électro-
9 sensitifs, rapportée en tant que « extrinsic optical imaging» ou «voltage-sensitive dye imaging» était réalisée sur l’animal afin de détecter de minimes variations électrophysiologiques dans les couches corticales superficielles, survenant en réponse à la stimulation sensitive à laquelle l’animal était soumis (stimulation d’une moustache). Deux limitations avaient alors été identifiées : 1) la toxicité intrinsèque des colorants électro-sensitifs et 2) la pénétration limitée de ces colorants, rendant l’analyse des sous-couches corticales impossibles. Ces deux facteurs ont rendu la technique inapplicable à la recherche humaine.
Pour surmonter ces obstacles, il a été proposé d’utiliser les propriétés optiques intrinsèques du cerveau afin de délimiter les limites de tissus pathologiques de lésions expansives contournant ainsi l’utilisation de colorants et autres marqueurs électro- sensitifs [1, 2]. Les signaux optiques détectés (absorption et diffusion de la lumière) et leurs variations peuvent ainsi être quantifiés ; leur interprétation est une source d’information renseignant sur l’activité corticale régionale et la limite fonctionnelle de résection à ne pas franchir lors d’une résection chirurgicale [1]. Ce concept repose sur l’utilisation des propriétés optiques basiques du cerveau et interviennent lors de la résection de tumeurs cérébrales primitives, de métastases, de malformations vasculaires et de foyers épileptiques néocorticaux. Cette techniques a pour avantages de 1) ne pas utiliser de marqueurs extrinsèques potentiellement dangereux ; 2) supprimer une étape électro-chimique chronophage ; 3) d’augmenter la profondeur de mesure des signaux. Depuis sa première description, le concept d’imagerie optique intrinsèque du cerveau a généré un grand intérêt au sein de la communauté scientifique et, tout au long des années 90, de nombreux groups ont travaillé au développement et à l’utilisation systématique de l’imagerie optique intrinsèque chez l’animal et l’humain. Des exemples d’avancées
10 importantes dans la cartographie fonctionnelle du cerveau, rendues possibles grâce à l’imagerie optique intrinsèque, seront présentés plus loin.
Bien que les bases techniques et scientifiques soient robustes, l’application clinique systématique et fiable de l’imagerie optique intrinsèque présente des limitations qui seront discutées et critiquées. Celles-ci restreignent l’utilisation de cette technique et rendent sa démocratisation peu probable, en tout cas dans l’état actuel des connaissances. Par ailleurs, le développement continu d’autres modalités d’imagerie fonctionnelle mettent sérieusement en jeu le futur de l’imagerie optique intrinsèque. Enfin, face à la rationalisation des coûts et à l’optimisation financière continue et accrue du secteur de la santé, l’investissement en temps et en moyens dans une technique dédiée à l’investigation d’un seul secteur du système nerveux central, sous certaines conditions, n’ayant que partiellement fait ses preuves les derniers trente ans, doit être sérieusement remis en question.
11 3. Foreword
The visualization of pathologic processes of the central nervous system before and during surgical procedures is a long-standing key to patient’s management. As this is done by various investigations, the knowledge of their nature and fundamentals, their use and interpretation is nowadays part of neurosurgeon’s education.
Moreover, new tools are constantly developed to improve diagnosis armamentarium and surgical management of patients, leading to better outcomes and increased patients’ safety.
At Geneva University Hospital, experience gained over the years with the use of neuronavigation, augmented reality, intra-operative angiography and, soon, intra- operative magnetic resonance imaging, is of great value in the treatment of complex (and less complex) cranio-spinal pathologies. As part of our scientific activity, the application, development, experimentation and update of radiological and electrophysiological monitoring modalities are major objectives. For that reason, the exploration of intrinsic optical imaging at Geneva University Hospital was initiated in early 2010 by Dr. Shahan Momjian; as I had interest for the technique, I studied the topic since I joined the clinic in early 2013.
The elaboration of specific algorithms dedicated to intrinsic optical imaging and mathematical substrate were previously described by Pierre Bouillot and Shahan Momjian [43]; these concepts will not be further developed in this manuscript. The present thesis constitutes the second part of the project, describing the clinical application of intrinsic optical imaging to daily practice.
12 4. Introduction
Neurosurgeons being confronted to a lesion in or adjacent to an eloquent brain area have to precisely identify borders separating functional and non-functional areas, to achieve surgical resection as safely – but as well as radically – as possible. This aim represents a challenge, depending on the lesion type and given the difficulty in macroscopically differentiating normal brain parenchyma from abnormal tissue, particularly for inexperienced neurosurgeons. Thus, it is the rule to use auxiliary means such as electrophysiology and intra-operative imaging to guide surgeons during surgical procedures and to avoid lesions of functional healthy cortical and sub- cortical areas. Being an intra-operative mapping tool, optical imaging is frequently described as one of these aids.
Medical optical imaging is a general term employed to define various techniques using light and its wavelengths to gain visual access to a specific processes inside the body. More specifically, optical imaging in neurosciences uses photons to delineate functional or anatomic cortical zones. The general concept of measuring optic properties of the brain was first described in the late eighties, by Grinvald and Frostig [1, 2]. At that time, direct application of electro-sensitive dyes on the cortex, referred as extrinsic optical imaging or voltage-sensitive dye imaging was performed in animal studies to detect minimal electrophysiological variations in superficial cortical layers, occurring in response to a sensory stimulus to which the animal was submitted (e.g., the stimulation of cats’ whisker). Back then, two major issues to the application to human brain were identified: 1) the intrinsic toxicity of the voltage-sensitive dyes and 2) the restricted depth of penetration, rendering the technique useless in the recording deep cortical layers activity. This so-called extrinsic optical imaging technique was therefore not applicable to human research.
13 To overcome these major limitations, it has been proposed to make use of brain intrinsic optical properties to delineate pathological tissue of cerebral expansive lesions, bypassing the use of dyes and other electro-sensitive markers [1, 2].
Local optical signals of the brain (absorption and diffusion of the light) and their variations can be quantified; their interpretation is a source of information concerning regional cortical activity and, in theory, could be an aid in delimitating non-functional pathological processes [1]. The concept of intrinsic optical imaging relies on the use of basic optical properties of brain parenchyma to delineate functional borders of primary brain tumors, metastases, vascular malformations and epileptic foci. In comparison to voltage-sensitive dye imaging, advantages are: 1) absence of potentially harmful drugs, 2) suppression of a time-consuming electro- chemical step of the procedure and 3) increased depth of measurement of the signal.
Since its first introduction, the idea generated a great interest in the scientific community and, throughout the nineties; many groups worked on the development and use of intrinsic optical imaging in animal models and in humans. Examples will be provided further in this manuscript and, as we will detail, major discoveries in functional studies were done with the aid of intrinsic optical imaging.
The technique is meant to be reproducible and reliable. However, even if the technical background and the scientific basis are strong, intrinsic optical imaging presents major practical limitations to be discussed and criticized, restricting its use in the daily clinical practice and probably rendering its democratization unlikely, at least in the actual state of the knowledge. Moreover, the continuous development of other functional imaging techniques (functional magnetic resonance imaging, functional ultrasonography) jeopardize the future of intrinsic optical imaging.
14 Figure 1: Original publication of Grinvald et al. in Nature, 1986 entitled “Functional architecture of cortex revealed by optical imaging of intrinsic signals [1].
5. Common neurovascular brain imaging applied to the surgical field
15 5.1 Context
For clarity purpose, diagnostic and monitoring tools are here separated into radiological investigations and electrophysiology. Pre-and intra-operative direct and indirect visualization of pathological processes, by the mean of magnetic resonance imaging, and electrophysiology, together with post-operative radiological control of the extent of resection are complementary; therefore, certain techniques may be used in both pre-, intra-, and post-operatively. Electrophysiology requires interpretation of electric signals (surface electroencephalogram, stereotactically implanted electrodes, motor evoked potentials, sensitive evoked potentials, electroneurmoyography). Radiology allows static or dynamic architectural interpretation, requires the intervention of a radiologist and a technician, whereas a specialist is needed when using electrophysiology (setup, data collection and interpretation).
In the neurosurgical perspective, brain mapping is useful in determining the dominant hemisphere in cases where a surgical procedure is taking place next to eloquent language cortex or when a pathologic process is adjacent to motor or sensory areas. Hence, functional mapping helps reducing the potential for iatrogenic damage during the resection. It is also a useful adjunct to maximize the resection of the pathologic tissue.
Mapping studies can be divided in pre-operative and intra-operative. Each mapping modality has a characteristic intrinsic sensitivity/specificity in detecting functional signals, with inter-and intra-group variations.
5.2 Pre-operative investigations
16 Nowadays, pre-operative magnetic resonance imaging is the main source of information in the planning of surgical tumor resection. A common source of failed total tumor resection is the peri-interventional brainshift, due to the opening of the skull and the sagging of the brain on itself, increased by the creation of an empty space, due to the resection of the tumor. This results in the loss of accuracy of the pre-surgical planning, because the accuracy of pre-operative images applied to intra-operative situation might be sub-optimal.
We consider three groups of interventions concerning the brain; 1) removal of intra- cranial expansive process (low/high grade glioma, metastasis, and cerebral abscess), 2) vascular neurosurgery (aneurysm clipping, arteriovenous malformation, dural arteriovenous fistula) and 3) epilepsy surgery (anterior temporal lobectomy, cortical resection, functional hemispherectomy, posterior quadrantectomy). For any of these surgeries, a pre-operative 3D T1 gadolinium magnetic resonance imaging study is performed, in order to better identify the pathologic process and for surgical planning purposes.
17
Surgery MRI CT-Scan Other
Vascular neurosurgery
3D T1
gadolinium TOF1
sequences
Angio-CT scan Skull bone CT scan
Pre-operative angiography Augmented reality
Neuronavigation Electrophysiology Oncology
surgery
3D T1 gado.
DWI2 studies
None Neuronavigation
Electrophysiology if lesion adjacent to eloquent areas (motor, sensitive, visual)
Awake surgery if lesion adjacent to language area
Intraoperative fluorescence
monitoring (5- ALA;
aminolevulinic acid) Epilepsy
surgery
3D T1 gado.
T2 sequences
None Electrophysiology (Visual evoked potentials) if temporal lobe surgery.
Electophysiology or awake surgery if cortical resection next to eloquent area
Table 1: Three classes of brain surgeries and their respective studies performed before, during or after the surgery.
In vascular surgery, the use of pre-operative and per-operative angiography, 3D T1 gadolinium magnetic resonance imaging and augmented reality are used as
1 Time Of Flight
2 Diffusion Weighted Imaging
18 regular, protocol studies and guide the surgeon during the procedure, together with electrophysiology. The development of augmented reality is also a major aid in the surgical planning and also in the procedure itself, because it maintains a constant overview of the vascular architecture and guides the surgeon. Thus, however described, intrinsic optical imaging has no place in vascular surgery and is nowadays not used in our clinic, for that application.
In epilepsy surgery, pre-surgical planning includes standard magnetic resonance imaging studies, ictal positron emission tomography and inter-ictal single-photon emission computed tomography; when a extra-temporal cortical focus is in an extra- temporal location, functional studies such as functional magnetic resonance imaging can be performed if the cortical focus is adjacent to a functional area.
Moreover, surgical evaluation of epilepsy together with invasive monitoring (stereotactically implanted recording electrodes) is routinely performed in our clinic, in collaboration with neurology department and epileptology unit.
Regarding oncology surgery, whenever the lesion is adjacent to a cortical eloquent region, functional magnetic resonance is performed and eventually awake surgery can be proposed to the patient. During the surgery, the use of aminolevulinic acid as a fluorescent tracer of the ruptured blood-brain barrier, indicating the site of tumor infiltration and proliferation, is a valid adjunct to neuronavigation and electrophysiology [3].
Functional magnetic resonance imaging has an excellent depth of penetration, shows a temporal resolution of 1 to 10[s] and a spatial resolution of 10⁻³ to 10⁻² [m].
It is safe, reliable and the access to machines is provided by numerous university hospitals. The study displays a functional map of the brain, dependent of what the investigator wants to observe: language, motor, visual and sensitive areas. Moreover,
19 diffusion tensor imaging techniques are available to identify and design white matter tracts (associative, ascending and descending). These studies can virtually establish a brain model focused on eloquent motor, sensitive and visual functions for a specific patient. Restrictions are applicable to patients with cardiac pacemakers, ferromagnetic aneurysm clips, metallic implants or foreign bodies, which are not compatible with this technique. Also, claustrophobic patients and obese patients may have difficulties with functional magnetic resonance.
We also provide transcranial magnetic stimulation procedures. By creating an electric field by the mean of coils placed over the patient head, there is a modification of the pattern of activity of neurons “entrapped” in the magnetic field.
Indeed, cortical motor area was first described using transcranial magnetic stimulation induced action potentials, propagated through the spinal cord to the muscle [4]. Later on, transcranial magnetic stimulation allowed motor cortex mapping [5-8]. In the case of a lesion next to the central region, a non-invasive mapping of the motor cortex by navigated transcranial magnetic stimulation is performed preoperatively, to find motor evoked potentials; then, images of the stimulation are fused to the pre-operative MRI and used during the surgery with the neuronavigation. These data can be used together with per-operative cortical and sub-cortical mapping and with functional magnetic resonance imaging. For lesions adjacent to the left frontal opercular cortex, a repetitive transcranial magnetic stimulation is performed before the operation to find the so-called “speech arrest”.
This language mapping may be used intra-operatively during awake surgery and confronted to the cortical/subcortical direct electrophysiological mapping together with functional magnetic resonance imaging.
20 Electroencephalogram is a non-invasive electrophysiological investigation; it is essential in the diagnosis and the characterization of epileptic seizures and, more largely, the epileptic disease. Its limitations are the difficulty to interpret results and the need to often co-registrate with a 24/7 video, requiring hospitalization of the patient.
Magnetoencephalography is a popular non-invasive recording technique of epileptic foci. It allows the detection of magnetic field variations generated by the electrical activity of the brain. This is for instance useful in the diagnosis and the anatomo-functional characterization of epileptic foci. Recent publications report the use of real-time magnetoencephalography as a reliable monitoring tool and a feasible approach to best, with rather low signal-to-noise ratio10 [9].
5.3 Intra-operative investigations
The use of intra-operative functional magnetic resonance imaging has been largely reported in the literature[10] [11, 12]. The development of intra-operative magnetic resonance imaging allows direct visual monitoring of extent of resection of an intra- cranial expansive lesion. In Geneva, we are building an operating room with magnetic resonance imaging equipment, which will be used during the surgery, giving direct feedback of the extent of resection to the surgeon. The first surgery with intra-operative magnetic resonance imaging will be in 2018, according to our previsions. However, the concept of direct radiological control of an ongoing surgery is already available in vascular neurosurgery, as intra-operative cerebral angiography are routinely performed in our hybrid theater, during aneurysm clipping, arteriovenous malformations and dural arteriovenous fistulas exclusion. Ideally, the use of intra-operative magnetic resonance imaging would be the best option for mapping and monitoring surgical resections. However, the spread and
21 democratization of intra-operative magnetic resonance imaging might be impeded by its relatively important size, the need for a dedicated infrastructure and the budget to purchase it and to make its daily use possible (dedicated personal, infrastructure adaptations, cost of maintenance). In the era of healthcare resources rationalization and cost management, intra-operative magnetic resonance imaginge could suffer of those disadvantages. Moreover, in a medico-surgical point of view, the time of acquisition and the relative non-dynamic characteristics of this imaging modality are serious breaks to its application in surgery.
Intra-operative cortical stimulation mapping is intended to preserve cortico-sub- cortical functions and is at the basis of a good post-operative functional outcome. It represents the gold-standard of intra-operative functional investigations. Cortical stimulation mapping was first mentioned by Ferrier and Horsley[13] and is now used daily in brain surgery to map and assess functions by direct stimulation of the cortex and sub-cortical region. Most of the time, electrophysiologists are present in the operating room to guide the surgeon, but it can be used without them, by a specifically trained neurosurgeon. Figure 2 represent and compare temporal and spatial resolutions of different brain mapping techniques.
Figure 2: Comparison of SR and TR of different mapping techniques [14]
22 6. Fundamentals
6.1 General concepts of brain mapping and basics of intrinsic optical imaging
Brain mapping techniques need to be spatially and temporally accurate. As functional imaging is a mean of quantification of biochemical processes occurring in the cortical layers, specific marker to be monitored needs to be as sensitive and specific as possible, conveying accurately the physiologic processes to be monitored, i.e. the neuronal activation in the case of brain mapping. By measuring optical variations of the light absorption during cortical activation, intrinsic optical imaging allows functional and electrophysiological cortical mapping and. Moreover, pathologic processes harboring modified metabolism, altered blood-brain barrier or diffuse peri-nidal hypervascularity such as low-grade gliomas and respectively, arteriovenous malformations can be monitored.
By quantifying the dynamic evolution of the hemoglobin concentration and due to its chromophore activity, intrinsic signals can be obtained and converted into interpretable and reliable functional maps. This is done by exposing the cortical surface to the light of the microscope, after performing a craniotomy and opening the dura mater. The reflected light passes through a bandpass filter, which has been previously set at a relevant wavelength. A charge coupled device camera is mounted on the microscope; images taken during rest and activation phases are analyzed and compared by specific software. In comparison to conventional pre- operative functional mapping tools, there is no limitation due to the intra-operative brainshift and this technique can be used during the surgery and it gives almost real- time information of neuronal activation.
23 6.2 The rule of five
Five parameters are considered when building a useful mapping tool applicable to the clinical field: 1) spatial resolution; 2) temporal resolution; 3) signal-to-noise ratio; 4) time of acquisition and 5) invasiveness of the procedure.
The spatial resolution, also named “power of separation”, is the distance separating two contiguous - but not superimposed - points, allowing the monitoring system to differentiate them. Figure 3 shows an example of different spatial resolutions of a picture.
Figure 3: Pictures of different spatial resolution and effects on object perception. In this case, a satellite view of an hamlet in the Sahel desert [44]
Temporal resolution is the ability of a system to differentiate two events separated by a time interval. This parameter determines the detection of specific movements of the examined structures. To obtain a sufficiently high temporal resolution, a high spatial resolution is required. Furthermore, the afterglow (time taken by a pixel of a screen to switch from the “on” state to the “fully off” state) of the detector must be as low as possible. Figure 4 shows an example of a low spatial resolution charge- coupled device (CCD) camera.
24 Figure 4: A picture of a moving object, taken with a low temporal resolution sensor (CCD scanning line by line, slowly) [45]
The signal-to-noise ratio is a general concept used in telecommunication engineering; it compares the level of an emitted signal to the level of the background noise. Substantially, it represents the amount of useful data extracted from the non interpretable background information. This concept is also applicable to optics, in which signal-to-noise ratio can be used to refer to the ratio of useful information to irrelevant optical data. The basic equation of signal to noise ratio is shown below:
The time of acquisition is defined as the time-lapse (expressed in milliseconds, seconds or minutes) required to obtain a useful and interpretable signal. Invasiveness represents the degree of invasiveness of a procedure and thus indirectly indicates the probability of potentially harmful complications related to the measure (e.g.
hemorrhage, infection, air embolism).
25 6.3 Neurovascular coupling
This concept was first described by Roy and Sherrington in a publication of 1809 [46]
and is at the basis of the mapping of neuronal activity. As neuronal activation leads to a micro-vascular dilation and locally augmented perfusion, (release of glutamate by activated neurons into the synaptic cleft); this process leads to an increased level of metabolic by-products, such as nitric oxide, protons, carbon dioxide and potassium [15] [16]. Hence, cerebral consumption of oxygen increases21 [17], leading to increased level of hemoglobin in the erythrocytes situated in the local capillary beds22[18]. Vasoactive by-products (NO, K+) are released, and endothelial cells, pericytes and smooth muscle cells change their permeability and contractility, leading to a change in overall cerebral blood flow, cerebral blood volume and total amount of hemoglobin, resulting in the so- called functional hyperemia. The net result is an increased delivery in oxy-hemoglobin. The phenomenon is at the basis of functional imaging of the brain, mainly represented by functional magnetic resonance imaging, positron emission tomography and Single Positron Emission Computed Tomography, that measures this hyperemia.
The brain is considered as an inhomogeneous object compounded of various elements (neurons, vessels, glial tissue), each having specific optic properties. As these elements compounds interact in a tight fashion in a very little time-lapse, IOS to be monitored directly depends on what one would like to observe, and the biomarker has to be chosen according to this principle. In the specific case of neurosurgery, the aim is to spare important structures such as functional grey matter.
Thus, the focus is to monitor healthy grey and white matter with axo-neuronal compounds.
26 In the case of cerebral grey matter, light absorption properties are mainly determined by the hemoglobin and its chromophore activity [2] [19] [20]. Thus, quantification of hemoglobin and the measure of its dynamics allow the indirect quantification of neuronal activity, hence functional mapping.
27 6.4. Sources of intrinsic signals
The Wavelength λ (in [nm]) dependence of the absorbance coefficients of the oxy- and deoxyhemoglobin has been described by many authors [21] (Bouillot et Momjian 2012). Haemoglobin (Hb) is divided in two forms; Oxidized Hb (HbO, Hb bounded to oxygen) and reduced hemoglobin (HbR, Hb free of oxygen). The proportion of each fraction varies depending on the degree of neuronal activation.
The Beer-Lambert law modelizes the light absorption in a liquid. For a monochromatic light of wavelength λ, a local temporal variation of oxy- and deoxyhemoglobin concentrations ( and ( , respectively) induces a linearly dependent variation of the reflected light intensity :
∝ - ελ
HbO2 · ∆[HbO2](r, t) − ελ
Hbr · ∆[HbR](r, t)
Light reflectance at rest;
absorbance coefficient of HbO2;
absorbance coefficient of HbR
Figure 5: When HbR and HbO absorb light at the same wavelength (this is the isosbestic point), it is possible to determine functional hyperemia, hence functional activation of the cortex. Reproduced with permission of Drr. Bouillot and Momjian [43].
When HbR and HbO absorb light at the same wavelength, it is possible to determine local cerebral blood volume, cerebral blood flow and cerebral consumption of
28 oxygen; functional hyperemia and activation of the cortex can thus be detected.
This represents the so-called isosbestic point; at 550 and 570 nm; = , and the variation of light intensity is as follows :
∝ - ελ
HbO2 · ∆[HbO2](r, t) + ελ
Hbr · ∆[HbR](r, t)
measuring the total hemoglobin concentration variation.
The light spectrum can be categorized into green-yellow spectrum, red spectrum and near-infrared spectrum [16]. According to the specific spectrum considered, various intrinsic optical signals are obtained. Table 2 summarizes the characteristics of these spectra and their application in IOI [2, 16, 22].
Spectrum Wavelength (λ), [nm]
Main source of signal
Signal Pattern Interpretation
Green-yellow 500-599 Hbtot Monophasic
signal, peaking 3- 5 [s] after stimulation
Functional hyperemia
Red 600-699 HbR Biphasic signal;
first peak after 0.5-1.5 [s].
Second peak 3-5-
[s] after
stimulation.
First peak: increased oxygen consumption. Second peak, functional hyperemia.
Near-Infrared 700-800 Light scattering changes
n.a. Cellular swelling
Table 2: The three light spectra and their signal patterns used in optical imaging of the brain.
29 7. Practical aspects
7.1 Context
The methodology relies on the paradigm that cortical light reflectance changes are related to the neuronal activity and to the neurovascular coupling [43]. In fact, as the light reflectance varies with the relative concentrations of HbO and HbR, detection of stimulation-related optical signals changes can be used for functional brain mapping. The measurement of the reflected light and the interpretation of data by a specific algorithm hence determine the activated cortical surface.
In practice, the cortical surface is exposed to the white light of the regular microscope (e.g. OPMI Pentero 900, Zeiss Medical). The reflected light passes through a bandpass filter set at λ= 610 [nm] (isosbestic points of hemoglobin). A sensitive CCD camera (optical Δ 0.5 - 5%) is also mounted on the microscope.
Images taken during the activity and images taken at rest are compared. The results obtained have high spatial (10-5 [m]) and temporal (0,02 [s]) resolutions [16]. Figure 5 shows the basic setup of IOI.
Figure 6: IOI setup. First, the cerebral cortex is surgically exposed. A white light microscope is disposed over the surgical field, illuminating the cortical surface..
Reflected light passes through a bandpass filter at λ=570/610 nm. Sensitive CCD camera (optical Δ 0.5 - 5%) mounted on the microscope. Images are taken during neuronal activity and at rest and then compared. Taken from [21].
30 In general, the measurement is made at wavelengths sensitive to the extraction of oxygen (λ= 600-630 [nm]), because it produces maps with better spatial correlation with the underlying neural activity, as opposed to measures of wavelengths sensitive to blood volume. Indeed, there is a tighter coupling between electrical activity and rapid changes in the oxidative metabolism, rather than with changes in local perfusion, which are slower. Thus, the measurement at λ=610 [nm] offers the largest SNR; it is also less sensitive to vessel artifacts when focused 2 [mm] below the cortical surface [43].
The indirect cortical stimulation consists in the sensory stimulation of a body part (the stimulation of the median nerve being the most used) or the performance of a motor or cognitive task. In the case of median nerve stimulation, it allows the visualization of the corresponding somato-sensory area of the hand and, by extension, the localization of the central sulcus, primary sensory cortex and primary motor cortex.
Figure 7 shows an example of median nerve stimulation, with the corresponding primary sensory area highlighted by intrinsic optical signals.
Figure 7: Electrical stimulation of the left median nerve at a frequency of 2 [Hz], four times (ON: 24 [S], OFF: 24[S]). Handknob and corresponding IOI signal is highlited (yellow ring).
31 7.2 Temporal and spatial evolution of intrinsic signals
At a wavelength of λ= 610 nm (HbR), the time evolution of the signal is biphasic (see figure 8): in first instance, the light absorption is increased and the reflection is hence reduced. This phenomenon corresponds to an increase of focal concentration of HbR. As there is time latency between neuronal activation and secondary blood flow variations, HbR increases first; this is due to the immediate supply in oxygen to the neurons. The net result is the initial dip seen on the figure 8, followed by a stronger increase in the light intensity measured at λ=610[nm], because the light absorption decreases with the reduction of HbR and the reflection increases; this signal reflects a widest decrease HbR. This evolution is similar to the fMRI BOLD signal. Figure 8 shows the temporal evolution of the signal at λ= 550 and λ= 610 nm in a rat model after a 2 [s] whiskers’ stimulus [43]. These two signals are analogous to the regional blood flow and the BOLD signal measured in functional magnetic resonance imaging [23].
Although the initial dip of the cortical signal at λ=610[nm] induced by the early increase in HbR is expected to give the strongest correlation with neuronal activity, both signals can be used in detecting activity-related neuronal activation.
Figure 8: temporal evolution of the optical signals in a rat model after stimulus of a whisker (grey area) at an isobestic point of λ=550 [nm] (thick black line) and λ= 610 nm (thick grey line). Thin lines correspond to the error margins [24]. Reproduced with the permission of Drr. Bouillot and Momjian [43].
32 7.3 Applications
Various human applications have been reported, for brain mapping and surgical monitoring purposes. Here, we bring some major reported applications.
7.3.1 Mapping the somatosensory cortex
During the surgery, visual control doesn’t allow to differentiate the motor strip from the sensory cortex. Intrinsic optical imaging can assist the surgeon to precisely identify those two functional areas and therefore limit the risk of eloquent area injury and postoperative sequel. To do so, the stimulation of the median nerve is the most reported technique [25-27]. The stimulation creates an intrinsic signal seated in the sensory cortex (hand-wristle area) which is easily identifiable with the above- described setup. By analyzing the spatio-temporal evolution of intrinsic signals combined with evoked potential, in response to transcutaneous electrical stimulation of the median or ulnar nerves in patients undergoing surgical resection of brain tumors, Toga et al. report obtaining signals co-localized with evoked potentials in both motor and sensory regions. Together with the currently used electrophysiological monitoring and the “phase-reversal” identification, intrinsic optical imaging is reported effective.
Figure 9: Phase inversion from positive (sensory cortex) to negative (motor cortex) of the recorded intra-operative (Courtesy: Dr. C. Boëx).
33 Cannestra et al. [27] report clearly demarcated peak optical responses for each finger stimulated in patients under general anesthesia. However, the highly demarcated phase corresponds to the peak of the stimulation only, and this phase was followed by an overlapping phase, corresponding to the fading of the signal, and data were not interpretable at this stage. Probably, this was due to the “point- spread” phenomenon, generally observed in the somato-sensory cortex. This concept will be developed further in the discussion. As there is an overlap in the second phase of the signal, data are partially interpretable and the reliability of the entire signal cannot be guaranteed. Nevertheless, this limitation doesn’t impede the localization of the somatosensory cortex; it only limits the clear localization of each finger in primary sensory area.
Four year after the report of Cannestra, Sato et al (2002) [28] identified a neuronal response area in the post-central gyrus differentially activated depending on the stimulated finger, and results suggested a hierarchical organization of the primary somato-sensory cortex, corroborating the non-equal representation of the human body in the sensory area.
7.3.2. Mapping Broca’s and Wernicke’s areas
In 1992, Haglund et al. [29] observed that functional imaging yelded significant activation in both essential and secondary languages regions, in contrast with electrocortical stimulation, which only identified the essential language cortex during awake surgery. Cannestra et al (2000) [30] used imaging coupled with electrocortical stimulation and studied cortical activation in response to different language tasks in non-sedated patients. Distinct spatial and temporal response patterns, dependent on tasks and performance, were characterized both within Broca and Wernicke areas, consistent with the existence of task-specific semantic
34 and phonologic regions within these areas; the differing temporal patterns were proposed to reflect unique processing performed by receptive (Wernicke) and productive (Broca) language centers. Moreover, common overlapping areas and distinct areas (specific to different languages in bilingual patients) have been reported by Pouratian [31].
The theoretical representation of the cortical representation of language in Broca and Wernicke doesn’t conform to the biological reality. Instead of being clearly separated, language functions are rather distributed into various functionally distinct areas that may also overlap. Intrinsic optical imaging enabled to understand better the location and distribution of language functions, as reported by above- mentioned authors.
7.3.3 Functional and interventional study of epilepsy
Ictal foci can be viewed as hyper-functional areas of neuronal firing, which renders intrinsic optical imaging very pertinent in the study of epileptic disease. In 2013, Noordmans [32] described the identification of seizure focus in the sensorimotor area of the left hand during a surgical procedure, using an hyperspectral camera. In theory, intrinsic optical imaging could lead to a better intra-operative visualization of seizure activity and, consequently, improve the efficacy of the epilepsy surgery.
7.3.4 Cortical stimulation mapping and intrinsic optical imaging; comparison is not reason
Compared with pre-operative mapping (functional magnetic resonance imaging, positron emission tomography, magnetoencephalograpy), intra-operative mapping has essentially no limitations due to the brainshift. Besides cortical stimulation mapping, intrinsic optical imaging applied has remained so far very marginal and still not used on a daily basis. This might be surprising because this technique is harmless,
35 rapid and inexpensive. Intrinsic optical imaging doesn’t require major modifications of technical installations. Probably, the amount of information given by cortical stimulation mapping, the reliability, sensitivity and specificity are high enough to impede the emergence of a similar technique. Before interpreting results in cortical stimulation mapping, a threshold must be pre-defined. Nonetheless, despite accurate thresholding and reliable interpretation of the results, cortical stimulation mapping can induce an interruption of the function of a remote cortex by stimulation of projections fibers, rendering the validity of some results debatable. By opposition, intrinsic optical imaging is easier to interpret, because only the activated cortex is detected. It also has a better spatial resolution (50-100 [µm] versus 1 [cm] for cortical stimulation mapping; when the resection extends to less than 1 [cm] of identified areas involves a risk of postoperative deficit. Furthermore, intrinsic optical imaging allows the rapid assessment of the activity of widespread cortical areas;
faster than with cortical stimulation mapping, especially when multitasking, e.g.
language functions.
Even so, intrinsic optical imaging entails heavy disadvantages, mostly due to a limited signal-to-noise ratio. Moreover, brain movements (pulsation, respiration are also limiting the value of the technique, together with the necessity of a computational step and the increased time of acquisition, compared to cortical stimulation mapping. To deal with brain movements, there is a need for increasing the numbers of activation clusters, in order to average activated areas and obtain a detectable map, which in turn augments the time necessary to the mapping, a cluster needing several minutes of recording. Another issue is that the signal doesn’t take directly origin in the electrical activity of the neurons, but indirectly via the neurovascular coupling. Also because of the indirect signal, there is a diffusion of the signal that may produce false positive results, leading to a sub-optimal resection of
36 pathologic processes. Finally, there is no 3D mapping possible with intrinsic optical imaging, mostly due to restricted depth of penetration and to the limitation of the reconstruction algorithm, so far.
37 8. Review of the literature
A total of 43 publications were reviewed, based on PUBMED-MEDLINE research (keywords: “IOI”, “optical imaging of the brain”, “optical mapping”, “Intrinsic optical imaging”). All those publications were treating exclusively or partially of IOI. Nine publications (21%) were published between the late 80s and in the 90s, n = 23 (55%) were published between 2000 and 2010 and n = 10 (24%) were published since 2010, indicating that research in IOI technology was still present, but maybe with a decreasing trend.
Publications were focused on fundamental research (n=21), clinical research (n=20) or both (n=2). 5 publications were reviews and 2 publications were in the fields of technology in medical bioengineering.
The first publications dates from 1986 [1]. The late 80’s and the first half of the 90’s were devoted to technical description and to the biophysical basis of IOI [1, 33-36].
Three of the publications cited above also dealt with the application of IOI in functional mapping [33, 35, 36]. Only two studies were published in the field of functional mapping at that time [2, 37]. Finally, Haglund et al. treats exclusively of visual mapping in the monkey using IOI [38].
To our knowledge, the first publication on a clinical application in humans is by Cannestra in 1998 [27]. Clinical applications were first intended to functional mapping and subsequently, architectural description (referred to as
"neuromonitoring" and intraoperative mapping).
38 Figure 10: Distribution of intrinsic optical imaging dedicated articles (n=42) found in the literature.
Figure 11: Rate of publication with distribution according to the specific topic.
0 5 10 15 20
0 1 2 3 4 5 6
1986 1993 1995 1997 2000 2002 2004 2006 2008 2010 2012
Technote Review ClinRes FundRes
39 9. Technical note: The use of a circular illumination device to improve the signal to noise ratio
9.1 Background and purpose
A major limitation to the spread use of intrinsic optical imaging is the noise due to glare effects. As only a fraction of the light arriving on the cortical surface is absorbed, the rest is reflected and diffused. The reflected light generates noise and leads to non-interpretable information and finally reduces the signal to noise ratio.
The use of a circular illuminator has been advocated as a possible tool to augment the signal to noise ratio by diminishing light scattering and overall glare effects [39].
In this technical section, we report our experience with a circular illumination device designed and elaborated in house. This device consists in two rows of circularly disposed light-emitting diodes (LED) adapted and fixed on a circular ring of plastic, which is adapted to a microscope (Pentero 900, Carl Zeiss, Oberkochen, Germany).
The design of the mounting is shown in Figure 12.
9.2 Methods
Two fresh sheeps’ brains were used for the experiment, in two separate sessions. In each session, two conditions were tested; the first one with the microscope positioned 50 cm above the brain, with an angle of 0 ° with the table. The second one with the microscope positioned 50 cm above the brain, with an angle of 75 ° with the table. Each condition was performed twice and hence two series of measurements were obtained (2 x 2 series of results). The experiment was carried out in the darkest possible environment.
The encephalon was wrapped in a green surgical towel and a cortical surface of circa 10 [cm2] was directly exposed to the light. Saline was regularly used to maintain brain tissue as much hydrated as possible, in order to avoid retraction. In both
40 sessions, we started with the regular illuminator of the microscope (Pentero 900, Carl Zeiss, Oberkochen, Germany) and then the circular illuminator was mounted on the microscope and tested.
With the regular illuminator, image acquisition was started at 5% of light intensity from the microscope, incrementing by 5% of light intensity at each measure, until reaching a light intensity of 100% (one image acquisition per light intensity). The second setting was with the circular illuminator and three specific settings are tested: 9, 10 and 12 Volts, corresponding to three progressively increasing light intensities. The threshold of 9 [V] has been chosen because this setting generates the lowest amount of light that the camera is able to detect. The superior limit of 12 [V] was defined because the image was saturated beyond this limit. The image acquisition is done through a bandpass filter, then a CCD camera (ANDOR LucaTM) mounted on the microscope.
Images are converted and analyzed by a dedicated program (NeuroCam Andor Solis®). The post-procedural processing and analysis of the images was performed using ImageJ / FIJI 1.46®.
During post-procedural processing, the images have been converted to grey scales;
completely white pixels (grey scale defined value of 0) represented glare effects and were considered as non-interpretable, hence increasing the signal to noise ratio. For each image, the software calculated the total amount of pixels and the number of white pixels. A separate analysis was performed with data obtained with the regular illuminator and the circular illuminator, respectively. In the case where there was a circular band of white pixels, the whole surface of the circle was considered as non- interpretable because the surface of the few remaining non-white pixels at the center of the circular glare effect was too small to be eventually considered as a significative functional area. The center pixels were therefore replaced with white
41 pixels. As an example, Figure 13 shows raw and processed images obtained with the circular illuminator set at 9 [V]. Figure 14 shows the visual difference between the images obtained with the regular illuminator and the circular illuminator. For each image, the total amount of white pixels (Grey scale =0) was counted.
9.3 Results
Circular illuminator
In condition 1/measure 1, the illumination with the circular device produced 1995, 2287 and 2294 white pixels at 9, 10 and 12 [V] respectively (mean: 2192, standard deviation (SD): 170). Results obtained in condition 1/measure 2 were similar (2047, 2175, 2248; mean: 2156, SD: 101). In condition 2, measures 1 and 2 were also similar, with an increased amount of white pixels from 9, 10 to 12 [V]: 3470, 3358, 5030 (mean:
3952, SD: 934) and 4099, 4326, 5186 (mean: 4537, SD: 573), respectively. Results are summarized in Table 3, left part.
Standard illuminator
White pixels obtained between 5% and 100% of light intensity ranged from 231 to 77906 (mean: 25004, SD: 30302) and 196 to 77963 (mean: 25717, SD: 30525) in condition 1/measure 1 and condition 1/measure 2, respectively. In condition 2, results were 608 to 68095 (mean: 19825, SD: 24851) and 641 to 75618 (mean: 23087, SD: 28475) in measure 1 and 2, respectively. White pixels obtained between 5% and 50% of light intensity ranged from 231 to 478 (mean: 312, SD: 107) and 196 to 468 (mean: 301, SD: 111) in condition 1/measure 1 and condition 1/measure 2, respectively. Higher values were obtained with 75° angulation, as results are from 608 to 1478 (mean: 1200, SD: 333) and 641 to 1372 (mean: 1272, SD: 317) in condition 2/measure 1 and condition 2/measure 2. Results are summarized in Table 3, right
42 part. As a result, light intensity beyond 50% produced most of glare effects in all conditions and measures, as seen in Figure 15.
9.4 Discussion
In this experiment, the use of a circular illuminator did not allow to improve the signal to noise ratio. The standard illuminator, used at lower settings (5%-50%), with the microscope set in neutral position (light beam perpendicular to the ground) is the most favorable configuration for the reduction of noise associated to light reflection or scattering. The experiment was carried out on sheeps’ brains, with two series of repeated measurements; this allowed to show the relative consistency and reproducibility of the results obtained with both standard and circular illuminators.
The use of sheeps’ brains was chosen because of the very similar optical properties with the human brain and the presence of sulci and gyri, reproducing as closely as possible the in vivo conditions of the daily practice. In vivo human experimentation was not possible because the available circular illuminator is not approved for human application.
The angulation at 75 ° of the microscope introduces greater amount of glare effects, expressed by the amount of the white pixels, with both illuminators. Also, the variations of results are more important, showing a certain unpredictability of the results, even if globally the trend is the similar with the results of condition 1. This unpredictability renders this condition of use less reliable; Figure 16 illustrates the variations of the measurements obtained with the standard illuminator under conditions 1 and 2.
We observe three factors which induce glare effects and decrease of the signal to noise ratio. First, the angle at which light comes to the surface of the brain, regardless
43 of the illuminator used, influences the amount of light scattering and glare effects. In our experiment, the angulation increased glare effects. For example, the white pixel values obtained in condition 1/ measure 1 and in condition 2/measure 1 increased by a factor of 2.6 (at 5%, 231 vs. 608 pixels) to 3.1 at (50%, 478 vs. 1487). However, beyond 50%, it is the perpendicular light that seems to generate more glare effects.
Thus, the angle of the illuminator is a determining factor of glare effects.
The second factor is the circular illumination. As shown in Figure 13, the circular illuminator produces rings of light reflection, as shown in Figure 13. Although the whole surface of the circle is not itself a source of light scattering and glare effects, the area within the ring is considered useless because it is too small to be eventually taken into account as a real functional area. The circular illuminator therefore possibly produces less light reflection –circles instead of blobs – but the circles are too small and the whole area has to be discarded. Overall, the total interpretable area of the image is smaller. Circular illumination appears therefore, in practice, counterproductive.
Finally, the third factor, as mentioned above, seems to be the luminous intensity itself.
Indeed, as shown in Figure 16, the number of white pixels related to the glare effect increases significantly beyond a brightness of 50%. Figure 16bis shows all the values obtained during the four measurements (two with the standard illuminator, two with the circular illuminator) and, in both cases, there is a steep increase beyond a luminosity of 50%.
44 9.5 Conclusion
In practice, the use of the circular illuminator did not increase the signal to noise ratio, mainly because the ring of reflected light renders the surface into the circle useless to detect any relevant optical information inside. With the standard illuminator, the angulation of the microscope and a light intensity beyond 50% are major contributors to glare effects and reflected light. Thus, a regular illuminator positioned perpendicular to the operating field and, by extension, to the cortical surface with a mild light intensity, is the most effective setting for the procedure.
45
Condition 1 Condition 2
Microscope perpendicular to the table Angle of 75° with the table ImgName
Value (Greyscale)
Total of pixels
Value (Greyscale)
Total of pixels
Measure 1 Measure 2 Measure 1 Measure 2
0 255 0 255 0 255 0 255
Circular illuminator 9 Volts 1995 324373 2047 324321 326368 3470 322898 4099 322269 326368
10 Volts 2287 324081 2175 324193 326368 3358 323010 4326 322042 326368
12 Volts 2294 324074 2248 324120 326368 5030 321338 5186 321182 326368
Standard illuminator
Lum_5 % 231 326137 196 326172 326368 608 325760 641 325727 326368
Lum_10 % 271 326097 235 326133 326368 755 325613 1071 325297 326368
Lum_15 % 203 326165 207 326161 326368 1109 325259 1332 325036 326368
Lum_20 % 154 326214 139 326229 326368 1448 324920 1700 324668 326368
Lum_25 % 264 326104 261 326107 326368 1362 325006 1397 324971 326368
Lum_30 % 302 326066 323 326045 326368 1601 324767 1675 324693 326368
Lum_35 % 370 325998 354 326014 326368 950 325418 971 325397 326368
Lum_40 % 404 325964 399 325969 326368 1289 325079 1251 325117 326368
Lum_45 % 442 325926 434 325934 326368 1397 324971 1345 325023 326368
Lum_50 % 478 325890 468 325900 326368 1487 324881 1342 325026 326368
Lum_55 % 9415 316953 16429 309939 326368 2122 324246 3097 323271 326368
Lum_60 % 24605 301763 26621 299747 326368 9229 317139 9989 316379 326368
Lum_65 % 41949 284419 42715 283653 326368 28789 297579 42158 284210 326368
Lum_70 % 50408 275960 50724 275644 326368 37884 288484 47698 278670 326368
Lum_75 % 53931 272437 54205 272163 326368 41549 284819 56190 270178 326368
Lum_80 % 58226 268142 58466 267902 326368 46245 280123 46229 280139 326368
Lum_85 % 63557 262811 66493 259875 326368 51702 274666 59765 266603 326368
Lum_90 % 68544 257824 69911 256457 326368 56586 269782 62604 263764 326368
Lum_95 % 73435 252933 73512 252856 326368 62130 264238 68754 257614 326368
Lum_100 % 77906 248462 77963 248405 326368 68095 258273 75618 250750 326368
Table 3: The total amount of white (grey scale value = 0) and black (grey scale value = 255) pixels were counted, in order to obtain the value of the non-interpretable surface (light reflection). Images were obtained twice for each setting.
