Dec 11, 2025

Holographic calibration

DOI
https://dx.doi.org/10.17504/protocols.io.ewov11rqpvr2/v1
  • 1Zuckerman Mind Brain Behavior Institute, Departments of Neuroscience and Neurology, Columbia University;
  • 2Champalimaud Neuroscience Programme, Champalimaud Centre for the Unknown;
  • 3Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network
Ines Rodrigues-Vaz
Icon indicating open access to content
QR code linking to this content
DOI: https://dx.doi.org/10.17504/protocols.io.ewov11rqpvr2/v1
Protocol Citation: Ines Rodrigues-Vaz 2025. Holographic calibration. protocols.io https://dx.doi.org/10.17504/protocols.io.ewov11rqpvr2/v1
License: This is an open access  protocol  distributed under the terms of the  Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: December 02, 2025
Last Modified: December 11, 2025
Protocol  Integer ID: 234021
Keywords: ASAPCRN, holographic calibration, bruker microscope, vivo ultima bruker microscope, photon, grin lens
Funders Acknowledgements:
Aligning Science Across Parkinson’s
Grant ID: ASAP-020551
Abstract
This is the standard protocol for 2-photon holographic calibration with In vivo Ultima Bruker microscope through GRIN lens used for Rodrigues-Vaz and Athalye et al, 2025.
Materials
• Fluorescent slide
• Distilled water
• 2-photon laser scanning microscope with spatial light modulator module (Ultima In Vivo, Bruker)
• Femtosecond Ti:saphire laser (Chameleon Vision-S, Coherent)
• Low repetition photostimulation laser (Monaco 1035-40, Coherent)
Safety warnings
-Wear appropriate PPE as required by your institution.
Before start
-2-photon lasers (imaging and stimulation) and Pockels cell controller were turned on.
General calibration
Confirm imaging laser and photostimulation laser are aligned with target at objective output.
Place fluorescent sample under 16X water-immersion Nikon objective.
Focus on top of the sample.
Follow PrairieView software’s calibration routine to ensure alignment between photostimulation and imaging lasers: burn spots
Burn single spot and confirm calibration worked. If not, repeat step 4 as many times as necessary.
Power calibration
Measure power of stimulation laser at the 16X water-immersion Nikon objective output.
This was done at least once at beginning of each cohort.