Lecture 10: Optical Sensors UNFINSIHED

Question 1

ADVANTAGES of MICROSCOPY to STUDY NEURAL ACTIVITY…4

Answer 1

1 * Non-invasive*

2 * DEPENDING on the magnification and
resolution, can study THOUSANDS OF CELLS DOWN TO INDIVIDUAL SYNAPSES OR AXONS.

3 * Can IMAGE MULTIPLE FLUROPHORES with EACH
fluorophore used to MEASURE SOMETHING DIFFERENT

4 * Can be COMBINED WITH OPTOGENETICS, ELECTROPHYSIOLOGY AND BEHAVIOUR.

Question 2

DIAADVANTAGES of MICROSCOPY to STUDY NEURAL ACTIVITY…4

Answer 2

1 * It is a PROXY MEASURE

2 * Requires a DYE OR FLUORESCENT PROTEIN to be INSERTED INTO THE CELLS which can AFFECT THIER FUNCTION AND IN SOMES CASES IS TOXIC.

3 * Range of TECHNICAL DIFFICULTIES from getting
LIGHTIN/OUT OF THE BRAUN TO IMAGING FAST WITH ENOUGH RESOLUTION TO MEASURE NEURAL SIGNALS.

4 * EXPENSIVE and requires STRONG TECHNICAL SKILLS and SKILLS FOR ACQUISITION AND ANALYSIS.

Question 3

Fluorescence:

Answer 3

Fluorescence = EXCITED (S1) STATE TO GROND STATE
- energy released as Fluorescence…energy wave released

From incoming light — ABSORPTION = Ground state energy to excited state S1’

DIAGRAM IN SLIDE 4

Question 4

UNDERSTANDING CALCIUM IMAGING: Calcium imaging

6

Answer 4

1 * Calcium is an important INTRACELLULAR MESSENGER IN MAMMALIAN NEURONS

2 * During NEURONAL FIRING, the INTRACELLULAR CONCENTRAION OF CALCUM INCREASES BY ~10 -
100 times

3 * IMAGING the increase in intracellular calcium
is used as a PROXY MEASURE OF NEURONAL ACTIVITY.

4 * When CALCIUM ENTERS OR IS RELEASED INTO THE CELL, CALCIUM CELL BUFFERS BIND TO THE FREE CALCIUM

5 * CALCIUM INDICATORS = are CALCIUM BUFFERS that
FLUORESCE WHEN THEY BIND FREE CALCIUM

6 * The OUTCOME MEASURE IS THE ‘CHANGE’ IN FLUORESCENCE (DELTA F/F)

Question 5

Calcium imaging- genetically encoded calcium indicators (GECIs)

Answer 5

1 * Dye loaded calcium indicators are
simple to use but they can’t be targeted
to specific cells or neuronal
compartments (e.g., just the soma or
axon)
* GECIs inserted into the DNA allow the
calcium indicator proteins to be
expressed in specific cell types and/or
neuronal compartments
* GECIs consist of a calcium binding
domain fused to one or two fluorescent
proteins