Neuroscience Tools

Question 1

STED (Stimulated-Emission-depleted Microscopy)

Answer 1

Point-scanning of specimen
Two lasers → 1x excitation laser & 1x depletion laser (STED-beam, donut-shaped)
Detection of fluorophores in focal center

Question 2

SMLM (Single-molecule localization Microscopy)

Answer 2

Several image cycles → 1. Photoactivation of molecules stochastically; 2. Detection & Localization of activated molecules 3. Photobleaching
STORM (Stochastic optical reconstruction microscopy) & PACM (Photoactivation localization microscopy)

Question 3

STED vs. STORM

Answer 3

Advantages:
STORM: Less laser intensities (in vivo imaging), highest spatial resolution
STED: Fast acquisition, increased temporal resoltuion

Disadvantages:
STORM: Extensive image processing, 15-30min for single-color image
STED: Increased photobleaching

Question 4

Miniflux

Answer 4

Combination of STED & STORM → Up to 1nm precision
Switching on/off individual molecules randomly
Position determination with STED beam

Question 5

Super resolution microscopy

Answer 5

Techniques applied to microscopes to overcome the 200 nm resolution limit & enabling live cell imaging to 10-5 nm
Nobel prize in chemistry 2014

Question 6

Resolution

Answer 6

Minimum distance between two distinct objects to be perceived separate
Determied by wave nature of light, numerical aperture & sample properties

Question 7

Multiphoton microscopy

Answer 7

> = 2 photons to excite a fluorophore → Deep tissue imaging with less photobleaching (Excites fluorophore on focal plane)
Two low energy photons can excite a fluorophore → Higher energy emission → Red light possible (Scatters not as much as blue)
Multi-photons have to simultaneously strike the fluorophore
High-energy pulsed laser needed but no pinhole

Question 8

Tissue clearing

Answer 8

Make biological samples transparent → Homogenizing the refractive index
Hydrophobic - Use different organic solvents
Hydrophilic - Use water-soluble reagents
Hydrogel-based - Use acrylamid gel & water soluble reagents

Question 9

Transcriptome

Answer 9

All RNA molecules/transcripts present in a cell
Transcriptomic investigation used to characterize brain cell types, gene regulatory mechanisms & development, evolution of brain cell populations

Question 10

Transcriptomic investigation

Answer 10

Bulk-RNA-seq: Sequencing of RNA content of a population of cells/tissues
Sc-RNA-seq: Transcriptome of a single cell → Identification of subpopulations based on DEGs & co-transcribed genes & gene-regulatory networks
Spatial transcriptomics: Mapping of transcriptome in spatial context

Question 11

Intracellular eletrophysiological recordings

Answer 11

Recording electrode inside the neuron, ground electrode outside

Question 12

Extracellular electrophysiological recordings

Answer 12

Local field fluctuations due to synaptic transmission. Local/Field Potential
Single-Unit recordings
Tetrode → 4 isolated microelectrodes → Increased spatial resolution
Polytrode: Multiple isolated electrodes bundeled together → Increased resolution, less tissue damage but larger size, increased single-unit isolation
Microdirve: Chronic electrophysiology → Precise position & stabalized electrodes

Question 13

In vivo Ca2+ imaging

Answer 13

Visualize neuronal activity & neuronal circuit function in vivo via Ca+ sensor
GCamP: GFP-calmodulin-M13 peptide → Calcium sensor
Imaging via Miniscope or head-fixed 2 Photon microscopy
Cannot measure currents or channel activation

Question 14

Optogenetics

Answer 14

Channel coupled to opsin → Channelorhodopsin
Light leads to conformational change in the channel = Opening = Neuron activation or inhibition
Can be expressed selectively → Possibility to study neuronal circuits, brain functions & diseases