[Draft] Two-Photon Workflow: Visual Cortex Population Dynamics

Introduction - Note: this tutorial is still in a drafting state

This comprehensive tutorial walks through a complete two-photon calcium imaging experiment in BrainSTEM, from subject preparation to data analysis. We’ll document a visual cortex population dynamics study with head-fixed mice, demonstrating how to integrate subjects, procedures, behavioral paradigms, data acquisition, manipulations, and collections in a realistic research workflow.

Experimental Design Overview:

Subjects: Mouse cohort for visual cortex population imaging
Setup: Head-fixed two-photon microscope with visual stimulus presentation
Sessions: Cranial window surgery → Habituation → Visual stimulus sessions
Data: Two-photon calcium imaging, visual stimuli, eye tracking
Analysis: Population dynamics, stimulus response, retinotopy mapping

This tutorial assumes you have already set up your lab infrastructure (setups, equipment, inventories) as described in the Setting Up Lab Infrastructure tutorial.

Part A: Project and Subject Setup

Step 1: Creating the Research Project

First, we’ll create a project to organize our visual cortex imaging study.

Navigate to Projects:
- Go to Projects
- Click Add project

[SCREENSHOT NEEDED: Project creation interface]

Configure Project Details:

Field	Value
Name	Visual Cortex Population Dynamics Study
Description	Investigating population-level neural dynamics in mouse visual cortex using two-photon calcium imaging during visual stimulus presentation. Study focuses on orientation selectivity, population correlations, and stimulus-response relationships in layer 2/3 neurons.
Publications	Related to ongoing visual cortex research
Tags	two-photon, visual-cortex, calcium-imaging, mice
Authenticated Groups	Select groups that should have access to this project
Public Access	No (keep project private to lab)

For two-photon imaging studies, include information about the calcium indicator, imaging parameters, and visual stimulus paradigms in the project description. Authenticated groups will get contribute permissions. You can add additional groups or change permissions in the manage page: Go to Project detail page → Manage

Step 2: Adding Individual Subjects

Now we’ll add individual mice that will participate in our imaging study.

Navigate to Add Subject:
- Go to Subjects
- Click Add subject

[SCREENSHOT NEEDED: Subject creation interface]

Subject Configuration Example:

Field	Value
Name	VC_M001
Projects	Select: Visual Cortex Population Dynamics Study
Sex	Male
Species	Select: Mus musculus
Strain	Select: C57BL/6J
Description	Male C57BL/6J mouse for visual cortex two-photon calcium imaging study. Express GCaMP6f via viral injection.
Genetic Line	Wild type
Birth Date	2024-06-15
Subject Identifier	Ear punch: Left ear, position 2

Repeat this process to create additional subjects:

VC_M002, VC_M003, VC_M004 (for a cohort of 4 mice)
Use similar configurations but update IDs and ear punch patterns

The additional subjects can be duplicated from the first subject. Go to the Subject detail page of the first subject → Duplicate Fill in the new subject name and click Duplicate. After this you can alter other fields if necessary.

Step 2b: Document Initial Housing and Weight

After creating subjects, document their housing conditions and initial weights using subject logs.

Create Housing Log:
- Go to Modules → Subject logs
- Click Add subject log

Create Initial Weighing Log:

Type-specific Details: | Field | Value | |——-|——-| | Weight (grams) | 24.5 |

Step 3: Creating the Experimental Cohort

After creating individual subjects, group them into a cohort for experimental organization.

Navigate to Cohorts:
- Go to Personal Attributes → Cohorts
- Click Add cohort

[SCREENSHOT NEEDED: Cohort creation interface]

Configure Cohort Details:

Field	Value
Name	VC_Imaging_Cohort_1
Project	Select: Visual Cortex Population Dynamics Study
Subjects	Select: VC_M001, VC_M002, VC_M003, VC_M004
Description	First experimental cohort for visual cortex two-photon imaging study. 4 male C57BL/6J mice, age-matched, with viral GCaMP6f expression. Single housed post-surgery, 12:12 light cycle, standard chow ad libitum.
Tags	visual-cortex, two-photon, gcamp6f, male-mice

[ILLUSTRATION NEEDED: Flowchart showing project → subjects → cohort → experiment relationship]

Step 4: Equipment and Setup Verification

Verify your two-photon imaging setup includes all necessary equipment:

[SCREENSHOT NEEDED: Equipment list view filtered by imaging setup]

Required Equipment:

Two-photon microscope system (Bruker/Thorlabs)
Ti:Sapphire laser (Mai Tai, Chameleon)
High-speed scanning system (resonant/galvo)
Photo-multiplier tubes (PMTs)
Head-fixation apparatus
Visual stimulus presentation system
Eye tracking camera
Temperature control system

Part B: Surgical Procedures and Preparation

Step 5: Viral Injection Procedure

Document the viral injection for calcium indicator expression.

Navigate to Procedures:
- Go to Modules → Procedures
- Click Add procedure

[SCREENSHOT NEEDED: Procedure creation interface]

Viral Injection Configuration:

Step 6: Cranial Window Surgery

Document the cranial window implantation procedure.

Cranial Window Procedure Configuration:

Allow 1-2 weeks recovery before starting imaging sessions. Monitor for inflammation or coverslip clarity issues.

[ILLUSTRATION NEEDED: Cranial window placement diagram showing V1 location]

Part C: Two-Photon Imaging Sessions

Step 7: Habituation Session

Start with habituation to head-fixation and imaging setup.

Navigate to Sessions:
- Go to Modules → Sessions
- Click Add session

[SCREENSHOT NEEDED: Session creation form for imaging]

Habituation Session Configuration:

Habituation sessions are crucial for reducing stress and motion artifacts in subsequent imaging sessions.

Step 8: Visual Stimulus Imaging Session

The main experimental session combining visual stimulation with two-photon imaging.

[SCREENSHOT NEEDED: Two-photon imaging session interface]

Visual Paradigm Setup:

Field	Value
Name	Drifting Gratings Visual Stimuli
Environment Type	Head-fixed visual display
Authenticated Groups	Select your lab groups
Description	Drifting sine-wave gratings presented at 8 orientations (0°-315°, 45° steps). Each stimulus 2s duration, 4s inter-stimulus interval. 10 repeats per orientation, randomized presentation order. Screen distance 25cm, stimulus size 20° visual angle.
Public Access	No

Complete Session Configuration:

Type-specific Details: | Field | Value | |———|——-| | Format | TIFF | | Compression | LZW | | Vertical resolution | 512 | | Horizontal resolution | 512 | | Frame rate (Hz) | 15.5 | | Number of frames | 46500 | | Laser power (mW) | 25 | | Excitation wavelength (nm) | 920 | | Imaging depth (μm) | 150 | | Voxel size (X dimension) | 0.65 | | Voxel size (Y dimension) | 0.65 | | Voxel size (Z dimension) | N/A |

Type-specific Details: | Field | Value | |———|——-| | Format | AVI | | Compression | H.264 | | Frame rate (Hz) | 60 | | Number of frames | 108000 | | Vertical resolution | 480 | | Horizontal resolution | 640 |

Session Epochs: | Epoch Name | Start (min) | Duration (min) | Description | |————|————-|—————-|————-| | Baseline | 0 | 5 | Gray screen baseline recording | | Stimulus_presentation | 5 | 25 | Drifting grating stimuli (8 orientations × 10 repeats) | | Post_stimulus | 30 | 5 | Post-stimulus baseline recording |

[SCREENSHOT NEEDED: Two-photon imaging interface showing real-time calcium signals]

Step 9: Retinotopy Mapping Session

Create a session for visual field mapping.

Retinotopy Session Configuration:

Type-specific Details: | Field | Value | |———|——-| | Format | TIFF | | Compression | LZW | | Vertical resolution | 512 | | Horizontal resolution | 512 | | Frame rate (Hz) | 15.5 | | Number of frames | 31000 | | Laser power (mW) | 28 | | Excitation wavelength (nm) | 920 | | Imaging depth (μm) | 150 | | Voxel size (X dimension) | 0.65 | | Voxel size (Y dimension) | 0.65 | | Voxel size (Z dimension) | N/A |

Part D: Advanced Imaging Features

Step 10: Multi-Depth Imaging Session

Document sessions with z-stack or multiple imaging planes.

Multi-Depth Session Configuration:

Type-specific Details (Layer 2/3): | Field | Value | |———|——-| | Format | TIFF | | Compression | LZW | | Vertical resolution | 512 | | Horizontal resolution | 512 | | Frame rate (Hz) | 15.5 | | Number of frames | 46500 | | Laser power (mW) | 25 | | Excitation wavelength (nm) | 920 | | Imaging depth (μm) | 150 | | Voxel size (X dimension) | 0.65 | | Voxel size (Y dimension) | 0.65 | | Voxel size (Z dimension) | N/A |

Type-specific Details (Layer 4): | Field | Value | |———|——-| | Format | TIFF | | Compression | LZW | | Vertical resolution | 512 | | Horizontal resolution | 512 | | Frame rate (Hz) | 15.5 | | Number of frames | 46500 | | Laser power (mW) | 35 | | Excitation wavelength (nm) | 920 | | Imaging depth (μm) | 250 | | Voxel size (X dimension) | 0.65 | | Voxel size (Y dimension) | 0.65 | | Voxel size (Z dimension) | N/A |

Step 11: Adding Optogenetic Manipulations (Optional)

For experiments combining imaging with optogenetic perturbations.

[SCREENSHOT NEEDED: Manipulation configuration for optogenetics]

Optogenetic Manipulation Configuration:

Part E: Data Organization and Analysis Integration

Step 12: Subject Health Monitoring

Track post-surgical recovery and imaging session performance.

[SCREENSHOT NEEDED: Subject log for imaging studies]

Step 13: Creating Collections for Analysis

Group related imaging sessions for comprehensive analysis.

[SCREENSHOT NEEDED: Collection creation for imaging data]

Collection Configuration:

Field	Value
Name	VC_M001_Orientation_Tuning_Week1
Project	Select: Visual Cortex Population Dynamics Study
Sessions	Select: VC_M001_Day1_DriftingGratings, VC_M001_Day2_RetinotopyMapping, VC_M001_Day3_MultiDepth_Orientation
Description	First week orientation tuning and retinotopy sessions for VC_M001. Includes single-plane and multi-depth imaging with drifting gratings and retinotopic mapping stimuli.
Tags	orientation-tuning, retinotopy, calcium-imaging, week1

Step 14: API Data Access for Analysis

Access your imaging data programmatically for analysis.

from brainstem_api_client import BrainstemClient
import numpy as np
import matplotlib.pyplot as plt

client = BrainstemClient()

# Get all imaging sessions for a subject
imaging_sessions = client.load_model('session', 
                                   filters={'projects__name': 'Visual Cortex Population Dynamics Study'}).json()

# Get two-photon data acquisition details
imaging_data = client.load_model('dataacquisition',
                               filters={'session__projects__name': 'Visual Cortex Population Dynamics Study',
                                      'type': 'Two-Photon Microscopy'}).json()

# Download specific imaging session data
session_data = client.download_session_data('vc_m001_day1_gratings_id')

Next Steps

After completing this comprehensive two-photon imaging workflow, consider these logical progressions:

Cross-Modal Experiments: Review the Electrophysiology Workflow tutorial to learn how to combine two-photon imaging with simultaneous electrophysiology recordings for comprehensive neural circuit analysis
Data Storage Optimization: Learn about Managing Data Storage to efficiently organize and access your large imaging datasets
API Integration: Master the Python API tool or MATLAB API tool tutorials to programmatically access your imaging data for automated analysis workflows
Behavioral Integration: Explore Behavioral Paradigms to understand how to combine imaging with more complex behavioral tasks
Data Sharing and Collaboration: Learn about Sharing Project Publicly to make your imaging datasets available to the research community and enable collaborative analysis