Dear Busra,

Sorry for the late reply. The committee members concerns are valid here, there are downsides with using new analytical methods that have not been as extensively validated as more traditional methods: sources of error may differ and it can make comparison across studies more complicated. However, traditional methods such as stereology also have their limitations, with many upsides associated with use of standardised reference atlases. Semi-automated methods such as the QUINT workflow are becoming more common, we have written about this in our recent review article:

-Bjerke IE, Yates SC, Carey H, Bjaalie JG, Leergaard TB. Scaling up cell-counting efforts in neuroscience through semi-automated methods. iScience. 2023 Aug 7;26(9):107562. doi: 10.1016/j.isci.2023.107562. 

We have done our best to validate the QUINT workflow from different perspectives. This is documented in the following articles. The Gurdon et al article in particular focuses on validating registration to atlas regions. 

-Yates et al. QUINT: Workflow for Quantification and Spatial Analysis of Features in Histological Images From Rodent Brain. Front Neuroinform. 2019 Dec 3;13:75. doi: 10.3389/fninf.2019.00075. 

-Groeneboom et al. Nutil: A Pre- and Post-processing Toolbox for Histological Rodent Brain Section Images. Front Neuroinform. 2020 Aug 21;14:37. doi: 10.3389/fninf.2020.00037.

-Gurdon et al. Detecting the effect of genetic diversity on brain composition in an Alzheimer's disease mouse model. Commun Biol. 2024 May 20;7(1):605. doi: 10.1038/s42003-024-06242-1.

Good luck with your PhD.

Best regards,

Sharon Yates

Originally posted by busra:

Dear Dr. Yates,
Firstly, I would like to express my excitement in following your work closely. In my doctoral thesis, I conducted morphometric analyses on rats with Alzheimer's using the Quint workflow. However, due to concerns raised by my committee members regarding this workflow, I received feedback for revisions. I aligned images with an atlas template using QUICKNII and VisuAlign and performed volumetric analyses stereologically. Nonetheless, my committee members pointed out potential shrinkage or displacement during the histological preparation of samples, questioning the validity of manually fitting them to the template.

They particularly expressed concerns about errors that could arise when placing small neuroanatomical areas onto the template. They questioned how the program distinguishes local features in areas where histological boundaries are not clearly defined, even in stained sections. They inquired about the methods used in Quint workflow to validate measurement accuracy and requested information on error coefficients or standard deviations in these measurements. Furthermore, they highlighted that tissue blocks may not perfectly align with the x, y axes during sectioning by a microtome, raising questions about how axial shifts affect the placement of atlas templates and subsequent measurements.

They also asked about integrating 2D histological images into a 3D atlas and how this process ensures accuracy. Despite referencing your team’s research and providing explanations, they remained unconvinced.

Could I please ask for your assistance in addressing these concerns? Your guidance in this matter would be greatly appreciated.

Best regards,