Key Questions to Ask When Ordering Micro Prisms for Medical Imaging

Continuing Education Activity

A prism is a triangular refracting surface with an apex and a base. The incident light ray passing through the prism is refracted so that it is bent towards the base. Thus the image is shifted towards the apex. The amount of light refracted through the prism depends on the power of the prism defined in prism diopters. Prisms play an essential role in orthoptics and are invaluable in an ophthalmologist's clinic as they have definitive optical, therapeutic, and diagnostic uses. Prisms are used in many ophthalmic devices such as slit lamp biomicroscope, applanation tonometer, gonioscope, and operating microscope. The diagnostic uses include measurement of squint by prism cover test (Krimsky, modified-Krimsky method), simultaneous prism cover test, Maddox rod, measurement of fusional reserve amplitudes, tests for microtropia, and abnormal retinal correspondence. Therapeutic uses include treatment for convergence insufficiency, divergence insufficiency, to relieve diplopia, and use in nystagmus patients. This activity reviews the importance of prisms in evaluating and treating patients with squint and diplopia. It highlights the interprofessional team's approach of the interprofessional team in assessing and treating these conditions.

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Objectives:

• Describe the principle of prisms in ophthalmology.

• Summarize the clinical applications of prisms.

• Outline the different types of prisms and the basics of prisms dispending.

• Review the precautions and complications associated with prisms.

Introduction

A prism is a transparent, triangular refracting surface with an apex and a base.[1] The two nonparallel surfaces intersect at an angle called the apex, and the surface opposite to the apex forms the bottom of the prism. The light rays refracted through the prism bend towards the base. The amount of deviation of the path of refracted light from the incident light depends on the power of the prism measured in "prism diopters."[2]

Charles Prentice was the first to introduce the term prism diopters to describe the intensity of prism. One prism diopter represents the deviation of light by 1 centimeter and perpendicular to the initial direction of the light ray on a plane placed 1 meter away from the prism. The power of the prism in prism diopters is represented by the symbol D. Thus, a prism of 2 prism diopters would deviate a light ray by 2 centimeters, perpendicular to the direction of the initial light ray, measured 100 cm beyond the prism.[3]

Another unit of measurement of prism power is centrad. This is less frequently used as compared to prism diopters. Centrad unit is represented by the symbol Ñ. One centrad represents the deviation of light by 1 centimeter and perpendicular to the initial direction of the light ray on an arc of a circle 1 meter away from the prism.[4] Further, the deflection of the light ray after passing through the prism also depends on the refractive index of the material and the position in which the prism is held. It is essential to understand that the light ray passing through the prism deviates towards its base, but the image appears to be displaced towards the apex. Thus, the eye being tested will deviate towards the apex of the prism.[5]

Anatomy and Physiology

A light ray passing through the prism obeys Snell's law.[6] The light ray deviates towards the base, and this causes the image displacement away from the base of the prism, i.e., towards the apex. The change of the direction of the ray is called the angle of deviation.[7] When a prism is placed in the air, the angle of deviation of the light ray is determined by three factors angle of incidence, refracting angle of the prism, and refractive index of the prism material.[8]

The characteristics of the light deviated through prism include:

• No magnification/minification of the image

• No change in vergence of the rays

• Disperses incident pencil rays into component colors

• A virtual, erect image is formed

• The image should deviate through the apex of the prism [9]

Prentice's rule measures the deviation produced by the prism.[10]

• D = cF

• c = image displacement in cm

• F = lens power

The deviation of a light ray through prisms depends on not only the power of the prism but also the refractive index of the material it is made up of and the position in which the prism is held. Prisms can be held in three ways &#; position of minimum deviation, prentice position, and frontal position.[8]

• The position of minimum deviation is defined as the position in which the angle of incidence is equal to the angle of refraction. Plastic prisms are preferably used in the position of minimum deviation, but it is challenging to obtain this in clinical practice.

• Prentice position is defined as the position in which the prism is held perpendicular to the visual axis. This position is used for ophthalmic glass prisms.

• The frontal position is defined as the placement of prism parallel to the frontal plane of the patient.

Indications

The prisms are used in ophthalmology for diagnostic as well as therapeutic purposes. Prisms are made of glass or plastic material. These are available in different models such as loose prisms, prism bar, trial set prisms, or Fresnel prism. Prisms of different powers are available in different models. Prisms in a trial set range from ½ to 12 D. Prism bars range from 1 to 40 D. Fresnel prisms range from 1 to 40 D. Loose prisms range from 1 to 60 D.[11]

Diagnostic Indications

• Prisms are used in many ophthalmic devices such as slit lamp biomicroscope, applanation tonometer, gonioscope, keratometer, pupillometer, phoropter, ophthalmoscopes, operating microscope [12]

• Objective measurement of squint by prism cover test (Krimsky method), simultaneous prism cover test, modified Krimsky method [13]

• Subjective measurement of squint by Maddox rod [14]

• Measurement of fusional reserve amplitudes [15]

• Assessment of torsion [16]

• Four prism diopter test for microtropia [17]

• To detect abnormal retinal correspondence [18]

• To assess the likelihood of diplopia after proposed squint surgery [19]

• Prism adaptation test [20]

• To assess head posture after nystagmus surgery [21]

Therapeutic Indications

• Building up fusional reserve in patients with convergence insufficiency [22]

• Building up divergence capacity in patients with divergence insufficiency [23]

• To relieve diplopia in patients with small vertical squints, decompensated phorias, or paralytic squints to relieve diplopia in primary or reading positions. [24]

• To decrease the velocity of nystagmus by simulating convergence in nystagmus patients [25]

• To increase the field of vision in patients with hemianopia [26]

• Fresnel prisms are prescribed in patients with bitemporal hemianopia, glaucoma, retinitis pigmentosa, brain injury, and stroke [27]

• As for reading glasses for bedridden patients [28]

Contraindications

Though prisms have found a wide place in orthoptics' diagnostic and therapeutic world, there are no strict contraindications for prism prescription.[29] Still, we need to be careful about adaptation issues cost factors involved and check for suitability in the clinic before the final prescription. It is crucial to avoid or take extra precautions when prescribing prisms in the following situations.

• Prism adaptation &#; If prism adaptation occurs, there occurs an increase in underlying deviation and thus needs to be closely observed by the prescribing orthoptist. [30]

• If the underlying disease/mechanism causing original deviation is still progressive, the patient can adapt to the added prism and can redevelop the deviation. [31]

• If the prism is added continuously, the deviation might increase over time and become permanent. [32]

• Dragged fovea syndrome &#; In patients with pathology at the fovea like an epiretinal membrane, the patient's fovea might get displaced. This leads to a spatial disparity between the two foveae leading to central binocular diplopia. The prisms might reduce the central diplopia temporarily, but the diplopia reoccurs as the peripheral fusion takes over the central fusion. [33]

Equipment

Prisms used in ophthalmology are of different types. These include:

• Dispersive prisms &#; Abbes, triangular

  &#; Abbes, triangular [34]

• Polarizing prisms &#; Nicol, Wollaston

  &#; Nicol, Wollaston [35]

• Reflective prisms &#; Penta, Porro, Dove prisms

  &#; Penta, Porro, Dove prisms [36]

Nicol Prisms

These are made up of calcite crystal cut diagonally, and the two halves are cemented with Canada balsam or an optical cement with a low refractive index. The incident light is split into ordinary and extra-ordinary linearly polarized rays. These prisms are used in Haidinger brushes.[37]

Wollaston Prisms 

These two right-angled prisms composed of double refracting surfaces like quartz or calcite are cemented to form a rectangular unit. An incident beam of unpolarized light emerges as two oppositely polarized diverging beams from the opposite end. This type of prism is used in keratometers.[38]

Porro Prisms

This is a type of reflection prism used to alter the orientation of the image, i.e., the image traveling through the prism is rotated by 180 degrees. These prisms are used in slit lamps. The net effect of a beam passing through this prism is a parallel displaced image rotated by 180 degrees.[39] 

The prisms used in orthoptics are available in different forms. These include:

• Loose prisms [40]

• Prisms in a trial frame

• Prism bar [41]

• Fresnel prisms [42]

• Rotating prisms [43]

• Risley double prisms&#;2 rotating prisms of the same strength on a rotating frame [44]

• Prism flippers [45]

• Vari prisms &#; prisms power can be changed by rotating two glasses [46]

Fresnel Prisms

These are made of polyvinyl chloride material. Parallel tiny prisms are stacked with an apex of one adjacent to the base of the previous prism, which can be struck on the base surface of the spectacles. This provides an overall prismatic effect of a single prism. The Fresnel prism is placed so that the base of prisms is directed towards the side of the defect.[29]

Personnel

The orthoptists, optometrists, and ophthalmologists are all actively involved and expected to understand the basics of prisms, the effects of prism in glasses, and the prismatic effect created by glass displacement in spectacles. After a detailed evaluation, it is essential to give the correct prescription and dispense the prisms with or without refractive correction.[47] There are two different notations used while ordering prisms.

One way to order prisms is by specifying the amount of prism required along with the direction of the prism's base. Example:

• Right - prism 3 UP 2out

• Left &#; prism 2 DN 2 out

An alternative method is to mention the direction of the prism using a 360-degree notation, where 0 is positioned towards the left of the lens, 90 superiorly, 180 right, and 270 inferiorly. So, the same prescription can be mentioned as:

• Right - prism 3 base 90 2 base 180

• Left &#; prism 2 base 270 2 base 0

Any refractive add for near or distance needs to be mentioned separately in the usual way of dispensing the spectacles.[48]

Preparation

While preparing glasses, it is essential to understand the prismatic effects of spherical lenses and Prentice's rule for prisms. The prismatic effect of the spherical lens is essential whenever the patient being evaluated has an underlying refractive error. A plus or a hyperopic lens behaves like two prism lenses stacked base to base.[49]

A minus or a myopic lens behaves like two prism lenses stacked apex to apex. Thus, the refractive correction affects the measured deviation and must be born in the mind. The measured deviation will be lesser with a plus lens and more when measured with a minus lens in situ.

Prentice's Rule 

This law is named after a famous optician Charles F. Prentice. As per this rule, the prismatic power of the lens at any point on its surface equals the distance from its optical center, measured in centimeters multiplied by the power of the lens in diopters. There is no prismatic power at the center of the lens. Thus, it is vital that to avoid any prismatic effect, the lens's optical center should be fitted directly in front of the pupil.

Formula

Prismatic effect = power of the lens (DD) 'Distance off from the optic center in mm

Technique or Treatment

The technique of dispensing prisms depends on the disparity of single binocular vision. It is advisable to prescribe the smallest amount of relieving prism that neutralizes the distinction. The prism base should be oriented based on the deviation being corrected. The bottom of the prism is placed in the direction opposite to the deviation.[50]

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The Direction of Prism in the Spectacles or for Neutralizing

Table

S. No Deviation

There are essential guidelines that need to be followed when considering the prescription of prism glasses to a patient. A few of the important ones include:

• Split the amount of correction equally between two eyes

• The base of the prism should be oriented opposite to the direction of the deviation of the eye

• Prisms of the range varying from 0.5D to 10D can be advised in patients with phoria.

• Both vertical or horizontal prisms can be prescribed individually or in combination in an oblique axis.

• Prisms of up to 6D can be tolerated in one eye and half in the other eye.

• Prisms can be prescribed in the form of glass prisms or Fresnel prisms stuck onto the glasses. [51]

Complications

Prisms are an effective way to relieve diplopia and improve vergence facilities. But some patients may experience side effects with prism glasses themselves.[51] A few fundamental problems associated with the use of prism glasses include:

• Headache

• Eyestrain

• Nausea/vomiting

• Double vision

• Confusion

• Deterioration of vision

Reasons for the discomfort experienced with prism correction glasses: 

• Misalignment of lenses- There can be errors in the initial alignment of the axis or the optical center while fitting the prisms. Sometimes, they can get misaligned due to the regular use of prism correction glasses.

• Incorrect or expired prescription: A wrong or expired prescription for prism correction can lead to discomfort. It is essential to give the patient an adequate adaptation time in the clinic before providing the final prescription.

Clinical Significance

Optical Uses

Prisms are an essential part of ophthalmology. These are the basics behind many instruments used routinely in ophthalmic practice, from basic investigations to the outpatient department to the operating theatre.[52] Prims are part of ophthalmic instruments like slit lamp biomicroscope, applanation tonometer, gonioscope, keratometer, pupillometer, phoropter, Haidinger brushes, ophthalmoscopes, operating microscope.[53] In the sub-specialty of strabismus and neuro-ophthalmology, prisms find their role as part of diagnostic and therapeutic interventions.[54]

Prism Adaptation Test

This test is helpful in patients with partially accommodative esotropia. Patients wearing full hyperopic correction are advised to press on base out prisms and review every two weeks. If the esotropia has increased further, additional power prisms are prescribed till a stable angle is achieved. The surgeon then operates on the full prism-adapted angle; this helps in reducing the chances of under correction.[55]

Prism Alternate Cover Test 

This test measures the total deviation, including the latent phoria. The first Hirschberg test estimates the tropia, followed by an alternate cover test to estimate the total deviation, i.e., tropia plus phoria. A prism of the estimated amount by an alternate cover test is placed over one eye to neutralize the deviation. An alternate cover test is repeated, and prism power increases or decreases until no refixation movement is noted.[56][57]

Simultaneous Prism Cover Test 

This test helps in small-angle strabismus to measure the tropia without dissociating the phoria. The Hirschberg test measures the size of tropia, and a prism of the estimated amount is placed in front of the non-fixing eye to neutralize the tropia. The fixing eye is covered simultaneously with an occluder to prevent fusion. The process is repeated with increasing or decreasing prisms' powers until no refixation movement is noted on removing the occlude.[58]

Fusional Vergence Amplitudes 

These are measured using a prism bar. Fusional convergence amplitudes are measured by placing the prism bar with the base in front of one eye in increasing steps until the patient reports double vision or inability to fuse. Similarly, fusional divergence amplitudes are measured with base in prism bar by increasing prism powers in steps till the patient reports double vision or failure to fuse.[59]

Vertical Prism Test 

This test assesses fixation preference. A 10 to 15 D base up or down prism is placed over one eye, inducing vertical strabismus. For example, when a 12 D base-up prism is placed in front of the fixing eye, both the eyes will show an infraduction. But, when the same prism is placed in front of the non-fixating or the amblyopic eye, there will be no deviation of either eye.[60]

4D Prism Test

This test is used to diagnose microtropia.[17]

To Measure the AC/A Ratio

Prisms are used to measure the AC/A ratio by the fixation disparity method. Changes in fixation disparity induced by prisms and that induced by spherical lenses are noted, which indicates the AC/A ratio. The advantage is that fusion is maintained throughout the test.[61]

Prisms as Low Vision Aids 

These are convex spherical lenses of powers ranging from +5 to +16D are prescribed as reading aids. These work on providing a magnified image, thus useful as low vision aids.[62]

Fresnel Prisms

These prisms have advantages over loose prisms as they are lighter, more comfortable to wear, cosmetically better acceptable by the patient, higher power can be prescribed compared to loose prisms.[42]

Field Expansion Lenses 

They are composed of two 12 D lateral prisms and one 8D inferior prism. The apex of lenses is placed towards the central non-channel. The lens system is designed for various degrees of peripheral field loss. It is recommended for patients with glaucoma retinitis pigmentosa.[63]

Prisms in Age-Related Macular Degeneration (ARMD)

These are based on the principle of image relocation. Prisms are added to the glass prescription to produce image relocation to the presumed retinal locus. The effect is probably created by the facilitation of oculomotor function resulting from the reduction of fixation instability.[64]

Ankylosing Spondylitis 

Patients with head or neck problems, such as severe ankylosing spondylitis, may benefit from prisms. For example, any patient with chin down posture bilateral equal power base up yoke prisms can improve straight-ahead vision and thus facilitate mobility.[65]

Prism as Reading Glasses

These are 15-30D base-down prisms in the form of recumbent spectacles, which allow bedridden patients to read or watch television comfortably in a lying-down position.[66]

Prisms in Nystagmus 

Prisms are used to move the image towards the null point, thus helping by dampening the nystagmus. Examples include:

• Base out prisms stimulate fusional convergence, thus improving visual acuity by dampening the nystagmus

• In patients with left face turn, the null position is in dextroversion. Thus placing a base in prism in front of the right eye and base-out prism in front of the left eye will shift the image towards the right, thus correcting the abnormal head posture.

• Prisms in contact lens- prisms are used to stabilize the near vision portion in a segmental bifocal contact lens and stabilize a toric contact lens using prism ballast. [57]

Enhancing Healthcare Team Outcomes

The prescription of prisms is complex and requires expertise. Optometrists, orthoptics, and ophthalmologists need to work in coordination and understand the underlying pathology causing symptoms to treat it. Patients may present with complaints of headache, eye strain, or other asthenopic symptoms to anyone involved in eye healthcare. Thus it is essential to understand the basics for evaluation and prescription of prims.

The optometrists and orthoptists should check the fusional vergences in any patient complaining of asthenopic symptoms despite wearing correct refractive power. Vision therapy exercises should be prescribed to patients with any underlying fusional weakness. Patients complaining of double vision need a complete detailed evaluation by the ophthalmologist. Appropriate referrals should be made to a physician or neurologist to rule out underlying associations.

Systemic conditions like myasthenia gravis Graves disease can often present to the ophthalmologist first, and thus detailed examination and a high index of suspicion can lead to the correct diagnosis. Neurological lesions may present with gaze palsy, skew deviation, or internuclear ophthalmoplegia. Thus it is essential to evaluate the patient thoroughly and refer the patient to the radiologist for necessary investigations. Opinions from neurologists or neurosurgeons can be lifesaving in some emergency conditions, and a lower threshold should be used for the same.[3]

Nursing, Allied Health, and Interprofessional Team Monitoring

Patients prescribed with prisms need to be followed up closely for compliance. Those prescribed prisms for exercise might experience an initial exacerbation of asthenopic symptoms and thus require the motivation to continue the exercise till the fusional reserves improve. Patients might experience confusion and practical difficulties with the use of prisms. Therefore, it is essential to review them closely and attend to the patients' issues.

A few patients might show an increased angle of deviation with the prescribed prisms, and thus close monitoring is again vital in these cases. In patients presenting with long-standing palsies or partially accommodative esotropias, a decision might be taken to operate the residual squints. Thus, nurses and counselors need to motivate the patients and follow them closely after surgical intervention.[4]

Figure

Digital image depicting prism trial box containing prisms from 0.5 D to 50 D Contributed by Dr. Kirandeep Kaur, MBBS, DNB, FPOS, FICO, MRCS Ed, MNAMS

Figure

Digital image showing prism bars showing prisms of increasing power with base out and base down Contributed by Dr. Kirandeep Kaur, MBBS, DNB, FPOS, FICO, MRCS Ed, MNAMS

Figure

Digital image showing a Fresnel prism of power 40 PD Contributed by Dr. Kirandeep Kaur, MBBS, DNB, FPOS, FICO, MRCS Ed, MNAMS

Figure

Digital image showing a loose prism of 40 PD Contributed by Dr. Kirandeep Kaur, MBBS, DNB, FPOS, FICO, MRCS Ed, MNAMS

Disclosure: Kirandeep Kaur declares no relevant financial relationships with ineligible companies.

Disclosure: Bharat Gurnani declares no relevant financial relationships with ineligible companies.

Two-photon imaging of cortical neurons in vivo has provided unique insights into the structure, function, and plasticity of cortical networks, but this method does not currently allow simultaneous imaging of neurons in the superficial and deepest cortical layers. Here, we describe a simple modification that enables simultaneous, long-term imaging of all cortical layers. Using a chronically implanted glass microprism in barrel cortex, we could image the same fluorescently labeled deeplayer pyramidal neurons across their entire somatodendritic axis for several months. We could also image visually evoked and endogenous calcium activity in hundreds of cell bodies or long-range axon terminals, across all six layers in visual cortex of awake mice. Electrophysiology and calcium imaging of evoked and endogenous activity near the prism face were consistent across days and comparable with previous observations. These experiments extend the reach of in vivo two-photon imaging to chronic, simultaneous monitoring of entire cortical columns.

To rapidly obtain two-photon imaging data from a larger range of depths, we have previously shown that insertion of a sharp, 1-mm glass microprism into the neocortex of an anesthetized mouse can be used for acute, single-session two-photon imaging of anatomical structures across all six cortical layers, including the soma and dendrites of cortical layer 5 pyramidal cells, in a single field-of-view ( Chia and Levene, a , b , ). Here, we describe an improved approach that has enabled chronic anatomical and functional imaging of hundreds of individual neurons and neuronal processes simultaneously across all cortical layers. The structure and function of neurons at distances greater than 150 µm from the prism face were not qualitatively different after prism insertion, and remained stable for months after prism insertion. We also demonstrate that microprisms can be used for simultaneous, high-speed calcium imaging from neurons in layers 2 to 6 during locomotion, and for imaging visual responses in long-range axon terminals in deep cortical layers. This approach complements traditional in vivo electrophysiological methods by enabling high-yield, simultaneous chronic monitoring of subcellular structure and neural activity in superficial and deep-layer cortical neurons in behaving mice.

Two-photon microscopy has become a key tool for monitoring the structure, function and plasticity of neurons, glia, and vasculature in vivo. For all its strengths, this method suffers from two important limitations: (1) high-speed imaging is often confined to a single focal plane parallel to the cortical surface, and (2) light scattering makes it difficult to image deep cortical layers. While deep layers of cortex such as layer 6 play a major role in regulating response amplitudes in superficial layers ( Olsen et al., ) and in distributing information to a variety of cortical and subcortical targets ( Thomson, ), existing methods for two-photon imaging are more effective in imaging superficial as opposed to deeper cortical layers. Further, methods currently do not exist for cellular or subcellular imaging across multiple cortical layers simultaneously.

Results

Two parallel approaches have dominated the study of neocortical circuits. One approach involves recordings in living coronal brain slices (typically ~400 µm thick). While many long-range axonal inputs to the cortical columns within each slice are severed, this reductionist approach has provided a wealth of insights regarding the layer-specific physiological properties of neurons and the interlaminar flow of neural impulses, using multiple intracellular and extracellular recordings (e.g. Adesnik and Scanziani, ; Sanchez-Vives and McCormick, ; Thomson, ), two-photon calcium imaging (MacLean et al., ), and voltage-sensitive dye imaging (Petersen and Sakmann, ).

A second common approach involves neuronal recordings from the intact brain, where it is possible to correlate neural activity with sensory perception and with behavior. Two-photon imaging has provided a means for monitoring neural activity and structural plasticity across days and weeks in awake animals (e.g. Dombeck et al., ; Andermann et al., ; Mank et al., ; Trachtenberg et al., ). However, many aspects of cortical processing remain out of reach because of the challenges in imaging deep-layer neurons, and in simultaneous in vivo imaging across all layers. While such laminar recordings are possible using electrophysiological approaches (Adesnik and Scanziani, ; Sakata and Harris, ; Niell and Stryker, ), these methods lack fine spatial resolution and typically provide lower yield, reduced recording durations, and greater difficulty identifying cell types.

Microprism imaging: a synthesis of in vivo and ex vivo approaches

Chronic two-photon imaging through a microprism combines the optical access of ex vivo brain slice preparations with in vivo behavioral context. This procedure involves insertion of a microprism attached to a cranial window ( and S1). The hypotenuse of the microprism is coated with aluminum and thus serves as a right-angled mirror or &#;micro-periscope,&#; with a vertical field-of-view parallel to the prism face. In different experiments, we implanted a microprism into either mouse somatosensory barrel cortex or visual cortex. As described in detail below (see Experimental Procedures and Figure S1A&#;D), a microprism (barrel cortex: 1.5 × 1.5 mm2 imaging face; visual cortex: 1 × 1 mm2 imaging face) was glued to a coverslip. A craniotomy and durotomy were performed under sterile conditions, a small incision was made orthogonal to the cortical surface, and the microprism assembly was carefully inserted into cortex. Wide-field epifluorescence and two-photon images parallel to the cortical surface showed a vertical field-of-view across cortical layers 2&#;6 through the prism, revealing radial blood vessels and the expected laminar pattern of GCaMP3 expression ( ) or YFP expression ( ). The procedure for microprism insertion in V1 ( ) involved an approximately 20% vertical compression of cortex (to ~675 µm in area V1) to decrease brain motion and prevent dural regrowth at the cortical surface, as in previous studies (Andermann et al., ; Dombeck et al., ).

Chronic translaminar snapshots of cortical neurons via a microprism implant

We first used microprisms for chronic two-photon structural imaging of genetically labeled cortical neurons across the depth of cortex. Somata and dendrites of layer 5 neurons in barrel cortex of anesthetized Thy1-YFP-H mice were imaged immediately following and for up to two months after prism insertion (n=5; ). Large field-of-view imaging with a 4x objective immediately following prism insertion revealed labeled neurons in layers 2/3 and 5 ( , left panel). Consistent with our earlier studies (Chia and Levene, b), images included dendrites of hundreds of neurons up to depths ~900 µm below the pial surface. Imaging with a 40x objective, 29 and 68 days following prism insertion, yielded progressively clearer images, allowing visualization of fine structural details in proximal portions of layer 5 pyramidal neuron basal dendrites ( , middle and right panels, and Movie S1). The population of labeled neurons was stable over time, as demonstrated by tracking of over forty neurons in one field-of-view across imaging sessions spaced 13 days apart (Figure S1E&#;F).

We found that when the surface of the cortex around the prism was unobstructed, the fluorescence collection efficiency was improved and &#;shadow&#; effects of radial vessels located between the image plane and the prism face were reduced. This is likely due to the diffuse nature of emitted fluorescent light, which can reach the objective through a clear cranial window surrounding the prism as well as through the prism (Figure S1G&#;H).

Anatomical and electrophysiological evidence of cortical health following microprism implant

The above results indicate that neurons near the microprism face (100&#;300 µm away) survived prism insertion and maintained their structural integrity for months. In addition, histological evaluation with staining for hematoxylyn and eosin (H&E, ), Nissl ( ) and DAPI (not shown) indicated that the imaged regions were comparable to more distant brain tissue (400&#;500 µm away) and to neurons from non-implanted mice (not shown). Small but significant increases in cell density were observed within the first 50 µm from the prism face (p < .05, N = 7 samples from 5 mice), followed by small but significant decreases at 50&#;100 and 100&#;150 µm away (p&#;s < .05), and a return to normal density beyond 150 µm from the prism ( , all p&#;s > .05). Staining for astrocytes (anti-GFAP) and microglia (anti-CD11b), indicators of brain trauma, did not show evidence of chronic tissue scarring at 27 days after surgery ( ). These data are consistent with studies showing a persistent macrophage response <50 µm from chronically implanted electrodes with decreased neuronal density at 0&#;150 µm from the electrode (Biran et al., ). These data are also consistent with our previous studies using acute microprism implants, in which propidium iodide staining demonstrated that neuronal damage was limited to <150 µm from the prism face (Chia and Levene, b). Nevertheless, a key question is whether the prism implant causes spreading depression, silencing or other major changes in activity of local cortical neurons at distances >150 µm from the microprism face.

We confirmed the sustained presence of generally normal spontaneous activity at distances of ~100&#;200 µm from a chronically implanted prism using multi-unit recordings of endogenous and stimulus-evoked activity in cortical layer 5 using repeated penetrations with tungsten microelectrodes in ketamine/xylazine anesthetized mice (see Figure S2A and Supplementary Experimental Procedures; n=3 animals). Neurons showed similar characteristic fluctuations between Up and Down states of spontaneous activity (e.g. Ros et al., ), before prism implantation, as little as 10 minutes after prism implantation, as well as 3 days and 120 days after implantation (Figure S2B&#;E).

Multi-unit responses to air-puff stimulation of facial vibrissae revealed that tactile sensory inputs to neurons near the prism face also remained largely intact, demonstrating localized and spatially specific responses with similar response latency (~15&#;20 ms following air-puff onset) and dynamics between recordings prior to and immediately following implantation (Figure S2F&#;G).

Together, these data suggest that the thalamic and cortico-cortical input that generates the short and long latency components of the sensory evoked responses were generally intact in neurons >100&#;150 µm from the prism face. These results provided the impetus for determining whether two-photon imaging of calcium activity in neurons via a microprism could be achieved in the visual cortex of awake behaving mice.

Microprism imaging of long-range axonal boutons

A unique advantage of two-photon imaging is the ability to monitor subcellular structures, such as dendrites ( ) and axons. Recently, we and others have described functional imaging of long-range projection axons using GCaMP3 in awake mice (Glickfeld et al., ; Petreanu et al., ). Because of the small size of individual axons and synaptic boutons, functional imaging of axons has been restricted to superficial depths in cortex (~0&#;150 µm deep). However, many classes of projection neurons selectively innervate deep cortical layers (e.g. Petreanu et al., ). To determine whether use of a microprism could enable monitoring of long-range axonal activity deep within the cortex, we made a small injection of GCaMP3 into area V1 (Glickfeld et al., ), and inserted the prism into the posteromedial secondary visual cortical area (PM), an area densely innervated by V1 axons, with the prism oriented to face area V1. We could visualize characteristic patterns of axons and putative boutons in a 75 µm × 75 µm field of view, located 100 µm in front of the prism face and 200&#;275 µm below the cortical surface ( ), at 10 days following prism implant. Endogenous co-activation of multiple boutons along each of two axonal arbors is shown in . We also observed robust visual responses of individual boutons at depths of 480&#;510 µm below the cortical surface (putative layer 5) during presentation of stimuli at multiple temporal frequencies (1&#;15 Hz; cf. Glickfeld et al., ) and spatial frequencies (0.02&#;0.16 cpd) at 1 day post-implant (see Movie S3, and Experimental Procedures). Recording quality was sufficient to obtain spatiotemporal frequency response tuning estimates for individual boutons ( ; cf. colored arrows in and single-trial responses in ).

High-speed cellular imaging of endogenous activity across all cortical depths

Simultaneous imaging of all cortical layers allowed direct examination of interlaminar neural dynamics, on a trial-by-trial basis, in awake mice that stood or ran on a linear trackball, as shown in and Movie S4. Using a fast, resonance scanning two-photon microscope (~32 Hz frame rate, 720 µm × 720 µm, 256 × 240 pixels / frame, see Experimental Procedures and Bonin et al., ), we were able to measure endogenous neural activity simultaneously in 208 neurons spanning all six cortical layers of V1. Data were collected in near-complete darkness from the same mouse as in , 38 days after prism implant, ~140 µm from the prism face.

This dataset provided a proof of principle demonstration of the capacity of chronic microprism imaging for examining changes in neural activity across layers following the onset of running bouts ( and Movie S5; average of all 53 running onsets from the twenty minute recording session, each preceded >2 s of immobility and followed by sustained locomotion >2 s; see Experimental Procedures). We observed neurons whose average endogenous activity increased at running onset, and other neurons whose activity consistently decreased at running onset (black dots in , paired t-test, p < .05/208; see also ). Intriguingly, the strongest suppression of endogenous activity was observed in several layer 6 neurons ( and Movie S5).

To better understand these changes in neuronal activity across layers, we also examined changes in activity across individual running onsets ( ). While such activity changes typically had the same sign for each neuron, different neurons demonstrated different degrees of trial-to-trial variability (compare reliability of neurons at depths of 288, 554 and 581 µm in ). In particular, most pairs of neurons had relatively low trial-to-trial co-variability ( ). For example, of the pairs of simultaneously recorded neurons in this dataset, only 11% had a correlation of magnitude greater than 0.2. These data illustrate the rich repertoire of inter- and intra-laminar neural dynamics accessible using microprism-based columnar recordings in behaving animals.

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