ABSTRACT
Background Correlation of fluorescence signals from functional changes in live cells with those from immunocytochemical indicators of their morphology following chemical fixation can be highly informative with regard to function-structure relationship. Such analyses can be technically challenging because they need consistently aligning the images between imaging sessions. Existing solutions include introducing artificial spatial landmarks and modifying the microscopes. However, these methods can require extensive changes to the experimental systems.
New method Here we introduce a simple approach for aligning images. It is based on two procedures: performing immunocytochemistry while a specimen stays on a microscope stage (on-stage), and aligning images using biological structures as landmarks after they are observed with transmitted-light optics in combination with fluorescence-filter sets.
Results We imaged a transient functional signal from a fluorescent Ca2+ indicator, and mapped it to neurites based on immunocytochemical staining of a structural marker. In the same preparation, we could identify presynaptically silent synapses, based on a lack of labeling with an indicator for synaptic vesicle recycling and on positive immunocytochemical staining for a structural marker of nerve terminals. On-stage immunocytochemistry minimized lateral translations and eliminated rotations, and transmitted-light images of neurites were sufficiently clear to enable spatial registration, effective at a single-pixel level.
Comparison with existing methods This method aligned images with minimal change or investment in the experimental systems.
Conclusions This method facilitates information retrieval across multiple imaging sessions, even when functional signals are transient or local, and when fluorescent signals in multiple imaging sessions do not match spatially.
Competing Interest Statement
The authors have declared no competing interest.
ABBREVIATIONS
- [Ca2+]c
- cytosolic Ca2+ concentration
- CCD
- charge-coupled device
- CLEM
- correlative light and electron microscopy
- DIC
- differential interference contrast
- DoG
- difference of Gaussian(s)
- EMCCD
- electron-multiplying CCD
- F(t)
- time course of fluorescence intensity
- F0
- pre-stimulus fluorescence baseline level
- ΔF(t)/F0
- time course of fold-change in fluorescence intensity
- ICC
- immunocytochemistry
- LED
- light-emitting diode
- MAP2
- microtubule-associated protein 2
- NGS
- normal goal serum
- PALM
- photoactivated localization microscopy
- PBS
- phosphate-buffered saline
- PSF
- point-spread function
- SEM
- scanning electron microscopy
- STED
- stimulated emission depletion
- TEM
- transmission electron microscopy
- VGAT
- vesicular GABA transporter
- VGLUT1
- vesicular glutamate transporter 1
- (Δx, Δy)
- coordinate translation required to correct an image displacement.