Integration of Optical Manipulation and Electrophysiological Tools to Modulate and Record Activity in Neural Networks

Figures & data

Figure 1 Schematic optical layout of the instrument (color figure available online).

Figure 2 Schematic and physical optical layout of the instrument (color figure available online).

Figure 3 Double objective holographic tweezers.

Figure 4 Holographic trapping on non-transparent substrates.

Figure 5 Simultaneous manipulation of beads in the vicinity of a neuron by a pipette and OTs.

Figure 6 Concerted and independent optical trapping of silica beads on MEAs.

Table 1. Feature comparison of different water-dipping objectives for optical trapping

Company	M	NA	Pupil Ø (mm)	WD (mm)	M/NA	3D Trap	Trap Area Ø (µm)
Olympus	20	0.95	22	2.1	21	no	148
Olympus	40	0.8	8	3.3	50	no	135
Olympus	60	0.9	7	2	66	yes	131
Olympus	60	1.1	8	1.5	54	yes	128
Nikon	60	1	7.2	2.8	60	yes	120
Nikon	100	1.1	5.2	2.5	90	yes	58
Zeiss	40	1	8	2.5	40	no	137
Zeiss	60	0.95	8	2	63	yes	132

Figure 7 Force spectroscopy measurement on an individual neuron (color figure available online).

Figure 8 Arranging cells with optical tweezers.

Figure 9 Calcium imaging of shock wave propagation induced by laser dissection on a random neural network plated on a MEA (color figure available online).

Table 2. Pros and cons of distinct optical microscope systems. Shaded boxes indicate the available components in the setup in Figure 1

OPTICAL MICROSCOPE
		PROS	CONS
FRAME TYPE	Upright	• Direct access to the sample for transparent and non-transparent substrates alike (i.e., diverse MEA types)• No optical aberration due to objective-sample interface-• Suited for in vivo experimentation	• Lower mechanical stability of the stage with respect to inverted microscopes• Water dipping objectives have NA < 1.1• Difficult to keep cell cultures sterile• Difficulty of accessing the sample with a glass pipette for patch-clamp recordings
	Inverted	• Greater mechanical stability of the stage• Availability of high NA oil objectives. No objective obstruction to pipette for patch clamp recording.• Working with closed culture supports warrants sterile conditions	• Due to their limited working distance (WD), high NA objectives (NA > 0.9) cannot be used on thick culture substrates (i.e., standard MEA chip thickness: 1 mm)• Objective-sample interface causes spherical aberrations which increase with imaging depth• Incompatible with opaque supports (i.e., electrodes of a MEA chip)• Not suited for in vivo experimentation
EXCITATION LIGHT SOURCE	TTL modulable laser	• Control of exposure time and light intensity without need for mechanical shutters or neutral density (ND) filters: suitable for long term fluorescence imaging• No need for excitation filters	• Lack of commercially available systems for widefield microscopes requires the modification of the epi-illuminator• Several lasers are needed to excite distinct fluorochromes• More expensive
	Mercury or xenon lamp	• Commercially available and easy to integrate into the microscope• Less expensive• Excitation of diverse fluorochromes by selecting the appropriate excitation filter	• Need for excitation filters• Shutters for exposure time regulation introduce mechanical noise into the system• Less precise light intensity regulation
DETECTOR	EM CCD	• High sensitivity allows the use of low excitation light intensity resulting in less photodamage and a wider time window for imaging living samples	• Expensive
	Gigabit Ethernet CCD	• Less expensive	• High frame rates (2 kHz with partial scanning and binning)
	High-res. CCD	• High res. video tracking (small pixel dimension)	• Not as sensitive as an EM CCD

Table 3. Pros and cons of distinct optical tweezers systems. Shaded boxes indicate the available components in the setup in Figure 1

OPTICAL TWEEZERS
		PROS	CONS
LASER	CW-1064 nm	• Cheap• Easy to integrate	• No possibility of tuning the laser wavelength to reduce photodamage• No possibility of two-photon excitation
	Tunable ultra-short pulse source	• Choose the laser wavelength to reduce the photodamage• Simultaneous trapping and two-photon excitation: detection of probe position in the focus or of short range interactions between the sample and the probe (i.e., in a fluorescence resonance energy transfer (FRET) assay)	• Expensive• More complex to integrate into an optical system
MULTITRAP	Acousto-optic deflector (AOD)	• High temporal resolution	• Difficult alignment• High Group Velocity Dispersion (for pulsed laser sources)• Higher phototoxicity due to the continuous laser light delivery while the time-shared laser beam travels through the sample• Only 2-D spot array
	Spatial light modulator (SLM)	• Easy integration into the optical path• Possibility to create 3-D spot array or arbitrary spot shapes on the sample (i.e., Bessel beam)• Less expensive	• Low temporal resolution• Requires software development for the calculation of diffractive optical elements
BROWNIAN MOTION DETECTOR	Interferometer (QPD)	• Higher temporal and spatial resolution (hundreds of kHz and sub-nanometer resolution)	• Critical alignment• Sensitive to sample artifacts
	Video	• Easy to implement• It is not sensitive to sample artifacts	• Lower temporal and spatial resolution

Table 4. Pros and cons of distinct laser microdissector systems. Shaded boxes indicate the available components in the setup in Figure 1

LASER MICRO-DISSECTOR
		PROS	CONS
LASER	Sub-ns pulsed UVA	• Cheap• Easy to integrate• Low average power at the sample: negligible thermal effects	• No possibility of two-photon excitation• No possibility of laser dissection deep in tissue
	Tunable ultra-short pulse source	• Laser dissection deep in tissue	• Expensive• More complex system integration• High average power at the sample: unwanted thermal effects
SCANNING SYSTEM	Laser scanning	• Laser ablation with no motion of the sample	• The laser spot can be moved on a limited portion of the field of view without change its optical properties (introduction of spherical aberrations).• More complex integration on an optical system
	Stage motion	• The laser spot properties are unchanged during the ablation protocol	• It is necessary to move the sample during the ablation protocol

Table 5. Pros and cons of distinct electrophysiological recording systems. Shaded boxes indicate the available components in the setup in Figure 1

ELECTROPHYSIOLOGY
		PROS	CONS
TECHNIQUES	Multielectrode arrays (MEAs)	• Simultaneous extracellular capture and elicitation of neural activity• Several tens to thousands of different recording sites suitable to study activity propagation in neural networks• High temporal resolution (kHz)	• Pre-determined recording and stimulation sites• Signal amplitudes are on the order of tens of µV only• Non-transparent conductors partially obstruct transparency in concurrent microscopy studies
	Patch-clamping	• Membrane potential or ionic currents of arbitrarily chosen neurons can be recorded or altered• Signal amplitudes are on the order of tens of mV or several pA• High temporal resolution (kHz)	• Addresses phenomena at the level of single neurons and their direct interconnection partners• Difficult to implement multiplicity
	Calcium imaging	• High spatial resolution to study activity propagation in neural networks• Sufficiently high signal amplitudes	• Low temporal resolution (tens of Hz)• Prone to photobleaching and phototoxicity
	Potential-sensitive dyes (PSDs)	• High spatio-temporal resolution to study activity propagation in neural networks	• Very low signal amplitudes• Prone to photobleaching and phototoxicity