Investigating biocompatibility of super-resolution compatible fluorescent DNA probes in mouse living tissues

by Shalini Pradhan
Doctoral thesis
Date of Examination:2024-12-09
Date of issue:2025-12-08
Advisor:Dr. Gražvydas Lukinavičius
Referee:Dr. Gražvydas Lukinavičiu
Referee:Prof. Dr. Ivo Feussner
Referee:Dr. Peter Lenart
Persistent Address: http://dx.doi.org/10.53846/goediss-11683

 

 

Files in this item

Name:Shalini Pradhan_Thesis.pdf
Size:15.3Mb
Format:PDF

The following license files are associated with this item:

Abstract

English

Fluorescence nanoscopy allows the observation of living biological structures at the nanometer scale. However, choosing the best fluorophore for in-vivo imaging often is a challenging task as systematic studies investigating the biocompatibility of these molecules are lacking. In-vivo imaging of thick tissues benefits from the use of red or far-red fluorescent probes as tissues show reduced scattering at longer wavelengths. SiR-Hoechst and 610CP-Hoechst are one of the first far-red fluorescent probes compatible with live-cell STimulated Emission Depletion (STED) nanos copy targeting DNA in living cells. Here, we study the carboxyrhodamine 4', 5' and 6' regioisomers of 610CP-Hoechst and SiR-Hoechst. The probes efficiently accumulate in the tissues allowing acquisition of images after injection into the mouse bloodstream (dosage: 10mg/kg of mouse body weight). The staining is stable and clearly visible for a few hours after tissue extraction. Probes containing 5' regioisomers were the best performing and yielded bright nuclear staining. This goes in line with the previously described experiments on the monolayer of cultivated cells (Bucevicius, Keller-Findeisen et al. 2019). Fluorescence measurements of the urine samples collected from injected animals indicate that the renal clearance of the probes is almost complete after 24 h. Due to their low cytotoxicity, excellent specificity and superb photophysical properties, the probes enable confocal and 2-Photon microscopy in living liver, kidney, lung, and heart tissue. This study extends the use of biocompatibile rhodamine-based DNA probes into living animals.
Keywords: DNA labeling, Hoechst dyes, in vivo labelling, super-resolution microscopy, two-photon fluorescence microscopy, serum albumin
 
Statistik