Two-Photon Imaging Notes

Overview

• Principles of laser scanning microscopy.

• Structural imaging.

• Functional imaging.

• Chronic imaging.

• Mini scopes, imaging & behavior.

From Single-Cell Recording to 2P Imaging

• The transition from single-cell recordings to two-photon (2P) imaging is a significant advancement in neuroscience.

• Early work by Hubel and Wiesel (1962, 1977) involved recording electrical activity of individual neurons in the visual cortex (V1).

• Ohki et al. (2005) demonstrated orientation selectivity in V1 neurons using 2P imaging.

• Diagrams illustrate the mapping of orientation selectivity across cortical layers.

• The transition moves from invasive single-cell recordings to less invasive and more parallel 2P imaging techniques.

Two-Photon Imaging Principles & Setup

• Two-photon imaging involves the simultaneous absorption of two photons to excite a fluorophore.

• Uses a femtosecond laser that emits very short pulses (on the order of $10^{-15}$ seconds).

• Two-photon excitation occurs at a focal point, reducing out-of-focus photobleaching and phototoxicity.

• A comparison is made between confocal (1-photon) and two-photon imaging.

  • Confocal Microscopy: Employs a single photon excitation, resulting in fluorescence emission throughout the sample, which is then refined using a pinhole to eliminate out-of-focus light. Excitation wavelength: 488nm

  • Two-Photon Microscopy: Utilizes two photons for excitation, confining fluorescence emission to the focal point, thereby reducing background noise and photobleaching. Excitation wavelength: 960nm

• The setup includes:

  • Femtosecond laser.

  • Scanning mirrors (xy-scan mirrors).

  • Scan lens and tube lens.

  • Dichroic mirror.

  • Objective lens.

  • Photomultiplier tube (PMT) to detect the emitted light.

In Vivo Two-Photon Imaging of the Intact Cortex

• Helmchen & Denk (2005) and Sur lab have demonstrated in vivo two-photon imaging of the intact cortex in mouse V1.

• This technique allows for the observation of neuronal structure and function in a living animal.

Example of Two-Photon Time-Lapse Structural Imaging

• Majewska & Sur (2003) showed dendritic spine motility in the visual cortex of a P28 mouse expressing GFP in layer 5 neurons.

• Arrows indicate structural plasticity: red (retraction), blue (elongation), and green (spine head movement/shape change).

• Frames are 2D projected z-stacks taken $1 \,µm$ apart, with 24 frames taken every 5 minutes.

Longitudinal In Vivo Two-Photon Structural Imaging

• A fundamental challenge in imaging is detecting a signal against background noise.

• Transgenically fluorescent mice provide bright labeling, allowing for low laser energy and clear visualization of synapses.

• Over the past 10 years, researchers have studied:

  1. Homeostatic stability of dendrites and spines.

  2. How experience changes spines.

  3. How learning changes spines.

  4. How pathophysiology changes spines.

  5. How drug treatment changes spines.

• Genetically encoded calcium indicators (GECIs) have enabled the study of how learning and experience are functionally encoded in synaptic activity (Crowe & Ellis-Davies, 2014).

Long-Term In Vivo Imaging of Experience-Dependent Synaptic Plasticity in Adult Cortex

• Trachtenberg et al. (2002) investigated synaptic plasticity over several days in the adult cortex.

Calcium Indicators for Functional Imaging

• Acute Imaging with Fluorescent Dyes:

  • Examples include Oregon Green, injected into the target area before imaging.

  • Strong signal from all cells.

  • High temporal resolution.

  • Link between $[Ca^{2+}]$ and fluorescence is fairly linear.

  • Short-lived and toxic.

• Chronic Imaging with Genetically Encoded Calcium Indicators (GECIs):

  • Expressed in cells using viral vectors (typically AAV) injected into the target area 2-3 weeks before imaging.

  • Expression lasts a few weeks, allowing repeated imaging.

  • Variety of GECIs available (e.g., GCaMP3, 5, 6) with different signal strengths and temporal resolution, RCaMP, R-GECO.

• Germline Encoded GECIs:

  • Eliminate the need for viral transfection.

  • Expressed in a cell-type specific manner and from an early age.

  • Lower signal strength compared to virally expressed GECIs.

Structure and Function of GCaMP

• GCaMP is a genetically encoded calcium indicator consisting of cpEGFP, CaM, and M13.

• Binding of $Ca^{2+}$ to CaM induces a conformational change that enhances EGFP fluorescence (Sun et al. 2013, Akerboom et al. 2009).

Surgery for Imaging and Virus Injection

• Involves a craniotomy using a 3 mm biopsy punch.

Long-Term, High-Resolution Imaging in the Mouse Neocortex Through a Chronic Cranial Window

• Demonstrated by Holtmaat et al. (2009).

• Allows for repetitive deep tissue imaging (Crowe & Ellis-Davies, 2014).

• Images taken at $\sim 770-830 \,µm$ below the pia mater over several months.

• Myelinated axons and layer 6 neurons are visible.

Imaging During Performance of Orientation Discrimination Task

• Imaging is performed while the mouse is engaged in an orientation discrimination task.

• Uses GCaMK6f AAV in C57BL/6 mouse or GCaMK6s x CaMKII-Cre mouse.

Multi-Color Two-Photon Calcium Imaging

• Virally-encoded RCaMP (or RGECO, red) is injected into the postsynaptic region to image somata calcium activity.

• Virally-encoded GCaMP (green) is targeted to the presynaptic input neurons, enabling simultaneous imaging of terminal calcium activity (Jennings & Stuber 2014).

Simultaneous Imaging of Cell Bodies and Layer 1 Axonal Terminals in V1

• RGECO1a is used to image Layer 2/3 cell bodies, while GCaMP6 is used to image Layer 1 Axons.

• This method allows for the study of top-down modulation of visual cortex.

Cingulate Signals in a Visually-Guided Discrimination Task

• Broom et al. (2022) linked cingulate cortex activity to performance in a visual discrimination task.

• Functional imaging of an axon projecting into visual cortex from cingulate cortex.

Visually-Guided Discrimination Behavior

• ACC to V1 axons are recruited during go/no-go visual discrimination behavior (Broom et al., 2022).

• Example boutons are positively (green) or negatively (red) modulated by whether the animal was within a trial or in the intertrial period.

Choice of Task for Chronic Imaging of Mouse Visual Cortex During Operant Behavior

• Go-Nogo visual discrimination in head-fixed mice recapitulates many advantages of head-fixed behaviors in primates (Andermann et al. 2010).

• Mice perform hundreds of trials per session for several months to a year.

• Motivation depends on water scheduling (limit weight loss to 20%).

• Provides well-controlled recordings of visual responses in awake/behaving animals.

• Relatively easy for mice to learn compared to two-alternative forced-choice (2-AFC) tasks.

Chronic Cellular Imaging of Mouse Visual Cortex During Operant Behavior and Passive Viewing

• Mice expressing genetically-encoded calcium indicators (transgenic lines, virus injection, or in utero electroporation) and/or anatomical labels.

• Handling and habituation to headpost are critical steps (1-2 weeks).

• Cranial window implant followed by recovery (1 day - 1 week).

• Training on visual discrimination task (2 weeks - 2 months until stable behavior).

• Chronic imaging during behavior for months (up to 20+ daily sessions).

• Optional synthetic dye injection and imaging during behavior.

Operant Discrimination Task

• Discrimination trials, sorted by stimulus type (Target vs. Non-Targets).

• Lick responses indicate choice: Hit (water reward), Miss, False alarm (mild air puff & time-out), Correct reject.

• Performance Metrics:

  • Hit rate.

  • Correct reject rate.

  • False alarm rate.

  • $d'$ (d-prime) value.

Pros and Cons of Two-Photon Imaging

• Pros:

  • Unprecedented information on neuronal structure and activity in terms of spatial resolution, cell types.

  • Statistical power of observing identifiable neurons repeatedly.

  • Ability to correlate changes in neural activity with behavioral changes (e.g., learning).

  • Combination with optogenetics and behavioral tasks allows elucidation of functional brain circuits.

• Cons:

  • Risks associated with craniotomy and virus injection.

  • Cumulative burden on individual animals imaged repeatedly.

  • Water restriction for behavioral training (limit weight loss to 20%).

  • Rats do not adapt well to head restraint.

Calcium Imaging with Miniscopes

• Miniature microscopes allow for calcium imaging in freely behaving animals.

• Key components:

  • Miniature microscope objective.

  • CMOS camera.

  • LED for excitation.

  • Dichroic mirror and filters.

  • Baseplate for attachment to the skull.

Miniscope Procedure

• Virus injection (Week 0).

• Prism probe insertion (Week 1).

• Baseplate installation (Week 5-7).

• Miniscope installation and in vivo imaging.

Example Miniscope Recording

• Data includes behavior recording, raw data, processed data, spatial and temporal downsampling, spatial bandpass filter, motion correction, and $\Delta F / F$ (change in fluorescence over baseline).

Radial Arm Maze Task and RSC Neuronal Activity

• Animals spend longer in the novel arm than in the familiar arm after a short delay (up to 30 min).

• No difference in exploration time after a longer delay.

• Questions addressed:

  • Are RSC neurons as active in familiar arms as in novel arms?

  • Does RSC neuronal activity change when re-exposed to the same location after a delay?

  • Does neuronal firing in RSC change during a spatial memory task?

• Task phases:

  • Habituation.

  • Sample phase (exploration in two available arms).

  • Delay (3 min / 30 min / 6 hr / 24 hr).

  • Test phase (exploration in two familiar arms and a novel arm).

• Exploration time compared between novel and familiar arms.

Calcium Event Rate and Neuronal Activity

• Calcium event rate (events/seconds) used as a measure of neuronal activity.

• No difference in rate of calcium events between novel and familiar arm after 3 min delay (0.078 +/- 0.045 familiar, 0.081 +/- 0.036 novel).

• Higher event rate in novel arm compared to familiar arm after 30 min (0.060 +/- 0.033 familiar, 0.093 +/- 0.049 novel) and 6 hr delay (0.090 +/- 0.052 familiar, 0.108 +/- 0.090 novel).

Two-Photon Multi-Color Calcium Imaging

• Two-photon: $960 \,nm$.

• Two-photon multi-color $40x/0.80$ W calcium imaging.

• Presynaptic GCaMP6.0 neurons and Postsynaptic RCaMP neurons.